Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy

PRINCIPLES of PHARMACOLOGY T H E PAT H O P H Y S I O L O G I C B A S I S O F D R U G T H E R A P Y

Third Edition

PRINCIPLES of PHARMACOLOGY T H E PAT H O P H Y S I O L O G I C B A S I S O F D R U G T H E R A P Y

Third Edition

David E. Golan, MD, PhD Editor in Chief Armen H. Tashjian, Jr., MD Deputy Editor Ehrin J. Armstrong, MD, MSc April W. Armstrong, MD, MPH Associate Editors

Acquisitions Editor: Susan Rhyner Product Manager: Stacey Sebring Vendor Manager: Bridgett Dougherty Senior Manufacturing Manager: Margie Orzech Marketing Manager: Joy Fisher-Williams Design Coordinator: Teresa Mallon Production Service: Absolute Service, Inc./Maryland Composition © 2012 by LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business Two Commerce Square 2001 Market Street - 4th Floor Philadelphia, PA 19103 All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form by any means, including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. Printed in The People’s Republic Of China Library of Congress Cataloging-in-Publication Data Principles of pharmacology : the pathophysiologic basis of drug therapy / David E. Golan, editor in chief ; Armen H. Tashjian Jr., deputy editor. — 3rd ed. p. ; cm. Includes bibliographical references and index. ISBN 978-1-60831-270-2 (alk. paper) 1. Pharmacology. 2. Physiology, Pathological. I. Golan, David E. II. Tashjian, Armen H. [DNLM: 1. Pharmacological Phenomena. 2. Drug Therapy. QV 38] RM301.P65 2011 615'.1—dc22 2011008453

Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of the information in a particular situation remains the professional responsibility of the practitioner. The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in the publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in their clinical practice. To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. International customers should call (301) 223-2300. Visit Lippincott Williams & Wilkins on the Internet: at LWW.com. Lippincott Williams & Wilkins customer service representatives are available from 8:30 AM to 6 PM, EST. 10 9 8 7 6 5 4 3 2 1

To Armen H. Tashjian, Jr. (1932–2009) Friend, mentor, colleague The pharmacologist’s pharmacologist Your spirit lives on within us and within this textbook The Editors

Contents Foreword ................................................................................... ix

Section IIB

Preface ..................................................................................... xi

Principles of Autonomic and Peripheral Nervous System Pharmacology

Preface to the First Edition .........................................................xiii Acknowledgments..................................................................... xv Contributors.............................................................................xvii

Section I Fundamental Principles of Pharmacology

1

1 Drug-Receptor Interactions ................................................2 Zachary S. Morris and David E. Golan 2 Pharmacodynamics..........................................................17 Quentin J. Baca and David E. Golan 3 Pharmacokinetics ............................................................27 Quentin J. Baca and David E. Golan 4 Drug Metabolism..............................................................43 Cullen Taniguchi and F. Peter Guengerich 5 Drug Toxicity ....................................................................56 Michael W. Conner, Catherine Dorian-Conner, Laura C. Green, Sarah R. Armstrong, Cullen Taniguchi, Armen H. Tashjian, Jr., and David E. Golan 6 Pharmacogenomics .........................................................71 Liewei Wang and Richard M. Weinshilboum

Section II Principles of Neuropharmacology

80

Section IIA Fundamental Principles of Neuropharmacology

81

7 Principles of Cellular Excitability and Electrochemical Transmission....................................................................82 Lauren K. Buhl, John Dekker, and Gary R. Strichartz 8 Principles of Nervous System Physiology and Pharmacology ...........................................................93 Joshua M. Galanter, Susannah B. Cornes, and Daniel H. Lowenstein

109

9 Cholinergic Pharmacology ..............................................110 Alireza Atri, Michael S. Chang, and Gary R. Strichartz 10 Adrenergic Pharmacology ..............................................132 Brian B. Hoffman and Freddie M. Williams 11 Local Anesthetic Pharmacology ......................................147 Joshua M. Schulman and Gary R. Strichartz

Section IIC Principles of Central Nervous System Pharmacology

163

12 Pharmacology of GABAergic and Glutamatergic Neurotransmission .........................................................164 Stuart A. Forman, Janet Chou, Gary R. Strichartz, and Eng H. Lo 13 Pharmacology of Dopaminergic Neurotransmission ........186 David G. Standaert and Ryan R. Walsh 14 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission .........................................................207 Miles Berger and Bryan Roth 15 Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System .........225 Susannah B. Cornes, Edmund A. Griffin, Jr., and Daniel H. Lowenstein 16 General Anesthetic Pharmacology ..................................240 Jacob Wouden and Keith W. Miller 17 Pharmacology of Analgesia ............................................264 Robert S. Griffin and Clifford J. Woolf 18 Pharmacology of Drugs of Abuse ....................................284 Peter R. Martin, Sachin Patel, and Robert M. Swift

Section III Principles of Cardiovascular Pharmacology

310

19 Pharmacology of Cholesterol and Lipoprotein Metabolism ..................................................311 David E. Cohen and Ehrin J. Armstrong 20 Pharmacology of Volume Regulation...............................332 Mallar Bhattacharya and Seth L. Alper vii

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21 Pharmacology of Vascular Tone ......................................353 Deborah Yeh Chong and Thomas Michel 22 Pharmacology of Hemostasis and Thrombosis ................372 April W. Armstrong and David E. Golan 23 Pharmacology of Cardiac Rhythm...................................401 Ehrin J. Armstrong, April W. Armstrong, and David E. Clapham 24 Pharmacology of Cardiac Contractility ............................422 Ehrin J. Armstrong and Thomas P. Rocco 25 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure............................................................437 Ehrin J. Armstrong, April W. Armstrong, and Thomas P. Rocco

Section IV Principles of Endocrine Pharmacology

464

26 Pharmacology of the Hypothalamus and Pituitary Gland .........................................................465 Anand Vaidya and Ursula B. Kaiser 27 Pharmacology of the Thyroid Gland ................................480 Ehrin J. Armstrong, Armen H. Tashjian, Jr., and William W. Chin 28 Pharmacology of the Adrenal Cortex ...............................489 Rajesh Garg and Gail K. Adler 29 Pharmacology of Reproduction .......................................505 Ehrin J. Armstrong and Robert L. Barbieri 30 Pharmacology of the Endocrine Pancreas and Glucose Homeostasis .....................................................524 Aimee D. Shu and Steven E. Shoelson 31 Pharmacology of Bone Mineral Homeostasis ..................541 Robert M. Neer, Ehrin J. Armstrong, and Armen H. Tashjian, Jr.

Section V Principles of Chemotherapy

562

32 Principles of Antimicrobial and Antineoplastic Pharmacology ................................................................563 Quentin J. Baca, Donald M. Coen, and David E. Golan 33 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation .........................................581 Marvin Ryou and Donald M. Coen 34 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis ........................................599 Tania Lupoli, David C. Hooper, Ramy A. Arnaout, Daniel Kahne, and Suzanne Walker 35 Pharmacology of Fungal Infections .................................618 Ali Alikhan, Charles R. Taylor, and April W. Armstrong 36 Pharmacology of Parasitic Infections ..............................629 Louise C. Ivers and Edward T. Ryan 37 Pharmacology of Viral Infections.....................................649 Robert W. Yeh and Donald M. Coen 38 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance .............................................674 David A. Barbie and David A. Frank

39 Pharmacology of Cancer: Signal Transduction ................699 David A. Barbie and David A. Frank 40 Principles of Combination Chemotherapy .......................716 Quentin J. Baca, Donald M. Coen, and David E. Golan

Section VI Principles of Inflammation and Immune Pharmacology 728 41 Principles of Inflammation and the Immune System .......729 Ehrin J. Armstrong and Lloyd B. Klickstein 42 Pharmacology of Eicosanoids .........................................740 David M. Dudzinski and Charles N. Serhan 43 Histamine Pharmacology................................................765 Cindy Chambers, Joseph C. Kvedar, and April W. Armstrong 44 Pharmacology of Hematopoiesis and Immunomodulation .................................................776 Andrew J. Wagner, Ramy A. Arnaout, and George D. Demetri 45 Pharmacology of Immunosuppression ............................790 April W. Armstrong, Ehrin J. Armstrong, and Lloyd B. Klickstein 46 Integrative Inflammation Pharmacology: Peptic Ulcer Disease..........................................................807 Dalia S. Nagel and Helen M. Shields 47 Integrative Inflammation Pharmacology: Asthma ............820 Joshua M. Galanter and Stephen Lazarus 48 Integrative Inflammation Pharmacology: Gout.................837 Ehrin J. Armstrong and Lloyd B. Klickstein

Section VII Fundamentals of Drug Development and Regulation

846

49 Drug Discovery and Preclinical Development ..................847 John L. Vahle, David L. Hutto, Daniel M. Scott, and Armen H. Tashjian, Jr. 50 Clinical Drug Evaluation and Regulatory Approval ...........860 Mark A. Goldberg, Alexander E. Kuta, and John L. Vahle 51 Systematic Detection of Adverse Drug Events.................872 Jerry Avorn

Section VIII Environmental Toxicology

880

52 Environmental Toxicology ...............................................881 Laura C. Green, Sarah R. Armstrong, Joshua M. Galanter, and Armen H. Tashjian, Jr.

Section IX Frontiers in Pharmacology

894

53 Protein Therapeutics ......................................................895 Quentin J. Baca, Benjamin Leader, and David E. Golan 54 Drug Delivery Modalities ................................................917 Joshua D. Moss and Robert S. Langer Credit List...............................................................................924 Index ......................................................................................928

Foreword “In such a night, Medea gather’d the enchanted herbs That did renew old Aeson.” William Shakespeare The Merchant of Venice (Act 5; Scene 1) The human need for medicines to alleviate suffering, cure disease, and even delay aging is both timeless and personal. Indeed, the critical goal of personalized medicine is to address this need for each individual. Identifying the right drug for the right patient at the right dose and time promises to revolutionize the treatment of disease while also improving drug safety. Pharmacology—the scientific discipline that seeks to describe the actions of drugs on living systems—has begun to provide us with answers to these age-old questions. My own career as a medical researcher and teacher at Harvard Medical School and as a leader of a drug discovery group in a pharmaceutical company has focused on the fundamental role that pharmacology plays in all fields of medicine. The study of pharmacology is essential to understand not only the mechanisms of action of drugs that treat diseases, but also important properties of drugs that may vary from individual to individual. Such properties include variations in drug absorption, tissue distribution, metabolism and excretion, as well as the synergistic, antagonistic, and other interactions that may occur when drugs are administered in combinations (as is increasingly the case). This third edition of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy refreshes substantively the previous editions, which have already drawn justifiable acclaim. Born from the needs of students of medicine and other health care professions, and created by the inspiration and collaborative efforts of students and faculty at Harvard Medical School, this textbook serves the purpose

well. The chapters are all clearly organized and written, with many major updates and revisions. Of note, there are new sections in the third edition on pharmacogenomics and protein therapeutics. In total, the textbook embodies a seamless and clear exposition of the principles of pharmacology. The book’s emphasis on pharmacologic, physiologic, and pathophysiologic mechanisms makes this text indispensable for students, practicing scientists, and health care professionals. The chapters add practical texture to the didactic material by providing clinical case studies relevant to the physiologic and pathophysiologic system under discussion, and the illustrations and tables are clear and displayed in multiple brilliant hues. It is with considerable sadness that I note the recent passing of Armen H. Tashjian, Jr., MD, one of the original authors and editors of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. Armen’s legacy of care, enthusiasm, and critical thought, all brought to serve the needs of students and teachers, is established in these volumes. He will be missed. This contribution by Dr. Golan and colleagues will undoubtedly provide future generations of teachers and students a strong foundation for the therapeutic practice of medicine and allied fields. In this sense, Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy will contribute to the care of countless patients both now and in the future. William W. Chin, MD Bertarelli Professor in Translational Medical Science Executive Dean for Research Harvard Medical School Professor of Medicine Brigham and Women’s Hospital Boston, Massachusetts

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Preface The editors are grateful for many helpful suggestions from readers of the first and second editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. The third edition features many changes to reflect the rapidly evolving nature of pharmacology and drug development. We believe that these updates will continue to contribute to the learning and teaching of pharmacology both nationally and internationally: • Creation of full-color figures throughout the textbook— about 450 in all. Every figure has been updated and colorized and over 50 figures are new or substantially modified to highlight advances in our understanding of physiologic, pathophysiologic, and pharmacologic mechanisms. As in the first two editions, our collaboration with a single illustrator creates a uniform “look and feel” among the figures that facilitates understanding and helps the reader make connections across broad areas of pharmacology. • Addition of new pedagogical elements to enhance learning, including icons placed within the text to indicate answers to the introductory case questions in each chapter. • Reorganization of chapters in the fundamentals of pharmacology. Along with drug-receptor interactions, pharmacodynamics, pharmacokinetics, drug metabolism, and drug toxicity, pharmacogenomics is now discussed in the first section of the textbook to complete a conceptual framework for the fundamental principles of pharmacology that serve as the foundation for material in all subsequent chapters. • Comprehensive update of all 37 drug summary tables. These tables, which have been particularly popular with readers, group drugs and drug classes according to mechanism of action and list clinical applications, serious and common adverse effects, contraindications, and therapeutic considerations for each drug discussed in the chapter. • Comprehensive update of all chapters, including new drugs approved through 2010. We have focused especially on newly discovered and revised mechanisms that sharpen our understanding of the physiology, pathophysiology, and pharmacology of the relevant system. Sections

throughout the book contain substantial amounts of new and updated material, especially the chapters on drug toxicity, pharmacogenomics, adrenergic pharmacology, the pharmacology of analgesia, the pharmacology of addiction, the pharmacology of the endocrine pancreas, the pharmacology of bone mineral metabolism, the pharmacology of bacterial and mycobacterial cell wall synthesis, the pharmacology of eicosanoids, the pharmacology of immunosuppression, the fundamentals of drug development and regulation, and protein therapeutics. As with the second edition, we have recruited a panel of new, expert chapter authors who have added tremendous strength and depth to the existing panel of authors, and the editorial team has reviewed each chapter in detail to achieve uniformity of style, presentation, and currency across the entire text. We note with great sadness the passing of Armen H. Tashjian, Jr., MD, the most senior author and editor of the first and second editions of this text. Armen’s career was long and distinguished, with research and teaching accomplishments and recognition across the spectrum of pharmacology, toxicology, endocrinology, and cell biology. His laboratory made a great many contributions to our fundamental understanding of pituitary hormone regulation and calcium homeostasis. Of equal importance was his guidance and mentorship of two generations of scientists and physicians. Armen brought to our shared enterprise a love of science and medicine, an encyclopedic knowledge base, a voracious appetite for the literature, an infectious enthusiasm for drug discovery, a keen appreciation for analytic rigor, a prodigious work ethic, and a genuine warmth and excitement for people. His spirit lives on in the hearts and memories of his family, friends, students, and colleagues and in this and future editions of this textbook. David E. Golan, MD, PhD Ehrin J. Armstrong, MD, MSc April W. Armstrong, MD, MPH

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Preface to the First Edition This book represents a new approach to the teaching of a first or second year medical school pharmacology course. The book, titled Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, departs from standard pharmacology textbooks in several ways. Principles of Pharmacology provides an understanding of drug action in the framework of human physiology, biochemistry, and pathophysiology. Each section of the book presents the pharmacology of a particular physiologic or biochemical system, such as the cardiovascular system or the inflammation cascade. Chapters within each section present the pharmacology of a particular aspect of that system, such as vascular tone or eicosanoids. Each chapter presents a clinical vignette, illustrating the relevance of the system under consideration; then discusses the biochemistry, physiology, and pathophysiology of the system; and, finally, presents the drugs and drug classes that activate or inhibit the system by interacting with specific molecular and cellular targets. In this scheme, the therapeutic and adverse actions of drugs are understood in the framework of the drug’s mechanism of action. The physiology, biochemistry, and pathophysiology are illustrated using clear and concise figures, and the pharmacology is depicted by displaying the targets in the system on which various drugs and drug classes act. Material from the clinical vignette is referenced at appropriate points in the discussion of the system. Contemporary directions in molecular and human pharmacology are introduced in chapters on modern methods of drug discovery and drug delivery and in a chapter on pharmacogenomics. This approach has several advantages. We anticipate that students will use the text not only to learn pharmacology, but

also to review essential aspects of physiology, biochemistry, and pathophysiology. Students will learn pharmacology in a conceptual framework that fosters mechanism-based learning rather than rote memorization, and that allows for ready incorporation of new drugs and drug classes into the student’s fund of knowledge. Finally, students will learn pharmacology in a format that integrates the actions of drugs from the level of an individual molecular target to the level of the human patient. The writing and editing of this textbook have employed a close collaboration among Harvard Medical School students and faculty in all aspects of book production, from student–faculty co-authorship of individual chapters to student–faculty editing of the final manuscript. In all, 43 HMS students and 39 HMS faculty have collaborated on the writing of the book’s 52 chapters. This development plan has blended the enthusiasm and perspective of student authors with the experience and expertise of faculty authors to provide a comprehensive and consistent presentation of modern, mechanism-based pharmacology. David E. Golan, MD, PhD Armen H. Tashjian, Jr., MD Ehrin J. Armstrong, MD, MSc Joshua M. Galanter, MD April W. Armstrong, MD, MPH Ramy A. Arnaout, MD, DPhil Harris S. Rose, MD FOUNDING EDITORS

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Acknowledgments The editors are grateful for the support of students and faculty from around the world who have provided encouragement and helpful suggestions. Stuart Ferguson continued his exemplary work as an executive assistant by managing all aspects of project coordination, including submission of chapter manuscripts, multiple layers of editorial revisions, coordination of figure generation and revision, and delivery of the final manuscript. We are extraordinarily grateful for his unwavering dedication to this project. Rob Duckwall did a superb job to create the full-color figures. Rob’s standardization and coloration of the figures in this textbook reflect his creativity and expertise as a leading medical illustrator. His artwork is a major asset and highlight of this textbook. Liz Allison provided constant support and guidance with all aspects of this project. Her timely and insightful advice was vital to the successful completion of this edition. Quentin Baca and Sylvan Baca electronically rendered the striking images on the cover and inside front cover of this textbook. We are most grateful for their creativity and expertise. The editors would like to thank profusely the publication, editorial, and production staff at LWW. Susan Rhyner provided leadership in the development and execution of this new edition. Keith Donnellan was a highly effective project manager; with good humor, attention to detail, and tremendous organization, he kept a complicated text and art program running smoothly. Stacey Sebring and Kelley Squazzo expertly managed the production of this handsome volume.

David Golan would like to thank the many faculty, student, and administrative colleagues whose support and understanding were critical for the successful completion of this project. Members of the Golan laboratory and faculty and staff in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and in the Hematology Division at Brigham and Women’s Hospital and the Dana-Farber Cancer Institute were gracious and supportive throughout. Deans Jeffrey Flier and Richard Mills were especially supportive and encouraging. Laura, Liza, and Sarah provided valuable insights at many critical stages of this project and were constant sources of support and love. Ehrin Armstrong would like to thank April for making life meaningful and fun. He is also grateful to the cardiology divisions at the University of California, San Francisco and Davis for providing research time during fellowship to complete this edition. April Armstrong would like to thank Ehrin for being her best friend and bringing her joy every day. April is grateful for unwavering support and mentorship from Dr. Fu-Tong Liu, whose dedication to research and exemplary work ethic are a source of inspiration. She also thanks her sister Amy, mom Susan, and grandma Chen Xiao Chun for their affection and support. Credit lines identifying the original source of a figure or table borrowed or adopted from copyrighted material, and acknowledging the use of noncopyrighted material, are gathered together in a list at the end of the book. We thank all of these sources for permission to use this material.

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Contributors Gail K. Adler, MD, PhD Associate Professor of Medicine Harvard Medical School Associate Physician Division of Endocrinology, Diabetes and Hypertension Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts

Ramy A. Arnaout, MD, DPhil Instructor in Pathology Harvard Medical School Associate Director, Clinical Microbiology Department of Pathology Beth Israel Deaconess Medical Center Boston, Massachusetts

Ali Alikhan, MD Resident, Department of Dermatology Mayo Clinic Rochester, Minnesota

Alireza Atri, MD, PhD Clinical Instructor in Neurology Harvard Medical School Assistant in Neurology Massachusetts General Hospital Boston, Massachusetts Deputy Director Geriatric Research, Education and Clinical Center Bedford, Massachusetts

Seth L. Alper, MD, PhD Professor of Medicine Harvard Medical School Renal Division and Molecular and Vascular Medicine Division Department of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts April W. Armstrong, MD, MPH Assistant Professor of Dermatology Director, Dermatology Clinical Research Unit Director, Teledermatology Program University of California Davis Health System Davis, California Ehrin J. Armstrong, MD, MSc Clinical Fellow in Cardiology University of California, San Francisco San Francisco, California Sarah R. Armstrong, MS, DABT Senior Scientist Cambridge Environmental, Inc. Cambridge, Massachusetts

Jerry Avorn, MD Professor of Medicine Harvard Medical School Chief, Division of Pharmacoepidemiology Brigham and Women’s Hospital Boston, Massachusetts Quentin J. Baca, PhD MD Candidate, Harvard-MIT MD-PhD Program Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts David A. Barbie, MD Assistant Professor of Medicine Harvard Medical School Associate Physician Department of Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts

Robert L. Barbieri, MD Kate Macy Ladd Professor of Obstetrics, Gynecology and Reproductive Biology Department of Obstetrics, Gynecology and Reproductive Biology Harvard Medical School Chairman, Department of Obstetrics and Gynecology Brigham and Women’s Hospital Boston, Massachusetts Miles Berger, MD, PhD Resident, Department of Anesthesiology Duke University Medical Center Durham, North Carolina Mallar Bhattacharya, MD, MSc Clinical Instructor of Medicine University of California, San Francisco San Francisco, California Lauren K. Buhl, MB PhD Candidate Department of Brain and Cognitive Sciences Massachusetts Institute of Technology Cambridge, Massachusetts MD Candidate Division of Health Sciences and Technology Harvard Medical School Boston, Massachusetts Cindy Chambers, MAS, MPH MD Candidate University of California, Davis Sacramento, California Michael S. Chang, MD Fellowship Director Adult and Pediatric Spine Surgery Sonoran Spine Center Phoenix, Arizona

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xviii Contributors

Lily Cheng, BS Co-author for Drug Summary Tables MD Candidate University of California, Davis Sacramento, California

Michael W. Conner, DVM Vice President Safety Assessment Theravance, Inc. South San Francisco, California

Joshua M. Galanter, MD Fellow, Department of Medicine University of California, San Francisco San Francisco, California

William W. Chin, MD Bertarelli Professor in Translational Medical Science Executive Dean for Research Harvard Medical School Professor of Medicine Brigham and Women’s Hospital Boston, Massachusetts

Susannah B. Cornes, MD Assistant Professor, Department of Neurology University of California, San Francisco Department of Neurology UCSF Medical Center San Francisco, California

Rajesh Garg, MD Assistant Professor of Medicine Harvard Medical School Associate Physician Division of Endocrinology, Diabetes and Hypertension Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts

Deborah Yeh Chong, MD Assistant Professor Department of Ophthalmology University of Texas Southwestern Medical School Dallas, Texas

John P. Dekker, MD, PhD Resident, Department of Pathology Massachusetts General Hospital Boston, Massachusetts

Janet Chou, MD Instructor, Department of Pediatrics Harvard Medical School Assistant in Medicine Department of Immunology Children’s Hospital Boston Boston, Massachusetts David E. Clapham, MD, PhD Aldo R. Castañeda Professor of Cardiovascular Research Professor of Neurobiology Harvard Medical School Chief, Basic Cardiovascular Research Department of Cardiology Children’s Hospital Boston Boston, Massachusetts Donald M. Coen, PhD Professor of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts David E. Cohen, MD, PhD Robert H. Ebert Associate Professor of Medicine and Health Sciences and Technology Director, Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology Harvard Medical School Director of Hepatology Division of Gastroenterology, Hepatology and Endoscopy Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts

George D. Demetri, MD Associate Professor of Medicine Department of Medical Oncology Harvard Medical School Director, Ludwig Center at Dana-Farber/Harvard Cancer Center Department of Experimental Therapeutics and Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts Catherine Dorian-Conner, PharmD, PhD Consultant in Toxicology Half Moon Bay, California David M. Dudzinski, MD, JD Clinical Fellow in Medicine Harvard Medical School Fellow, Department of Cardiology Massachusetts General Hospital Boston, Massachusetts Stuart A. Forman, MD, PhD Associate Professor of Anaesthesia Harvard Medical School Boston, Massachusetts David A. Frank, MD, PhD Associate Professor of Medicine Harvard Medical School Associate Professor Departments of Medicine and Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts

David E. Golan, MD, PhD Professor of Biological Chemistry and Molecular Pharmacology Professor of Medicine Dean for Graduate Education Special Advisor for Global Programs Harvard Medical School Scholar and Founding Member, The Academy at Harvard Medical School Physician, Hematology Division, Brigham and Women’s Hospital and Dana-Farber Cancer Institute Department of Biological Chemistry and Molecular Pharmacology, Department of Medicine Harvard Medical School Boston, Massachusetts Mark A. Goldberg, MD Associate Professor of Medicine Harvard Medical School Boston, Massachusetts Senior Vice President Clinical Development Genzyme Corporation Cambridge, Massachusetts Laura C. Green, PhD, DABT Senior Scientist and President Cambridge Environmental, Inc. Cambridge, Massachusetts Edmund A. Griffin, Jr Resident Physician Department of Psychiatry Columbia University New York State Psychiatric Institute New York, New York Robert S. Griffin, MD Resident, Department of Anesthesia, Critical Care, and Pain Medicine Massachusetts General Hospital Boston, Massachusetts

Contributors xix

F. Peter Guengerich, PhD Professor, Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee Brian B. Hoffman, MD Professor of Medicine Harvard Medical School Physician, Department of Medicine VA Boston Healthcare System Boston, Massachusetts David C. Hooper, MD Professor of Medicine Harvard Medical School Chief, Infection Control Unit Massachusetts General Hospital Boston, Massachusetts David L. Hutto, DVM, PhD, DACVP Senior Director, Drug Safety Eisai, Inc. Andover, Massachusetts Louise C. Ivers, MD, MPH, DTM&H Assistant Professor of Medicine Harvard Medical School Associate Physician Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts Daniel Kahne, PhD Professor of Chemistry and Chemical Biology Harvard University Cambridge, Massachusetts Ursula B. Kaiser, MD Associate Professor of Medicine Harvard Medical School Chief, Division of Endocrinology, Diabetes and Hypertension Brigham and Women’s Hospital Boston, Massachusetts Lloyd B. Klickstein, MD, PhD Head of Translational Medicine New Indication Discovery Unit Novartis Institutes for Biomedical Research Cambridge, Massachusetts Alexander E. Kuta, PhD Vice President, Regulatory Affairs AMAG Pharmaceuticals Lexington, Massachusetts

Joseph C. Kvedar, MD Associate Professor Department of Dermatology Harvard Medical School Dermatologist Department of Dermatology Massachusetts General Hospital Boston, Massachusetts Robert S. Langer, ScD David H. Koch Institute Professor Departments of Chemical Engineering and Bioengineering Massachusetts Institute of Technology Cambridge, Massachusetts Senior Research Associate Children’s Hospital Boston Boston, Massachusetts Stephen C. Lazarus, MD Professor of Medicine Division of Pulmonary and Critical Care Medicine Director, Training Program in Pulmonary and Critical Care Medicine University of California, San Francisco San Francisco, California Benjamin Leader, MD, PhD Chief Executive Officer ReproSource Woburn, Massachusetts Eng H. Lo, PhD Professor of Neuroscience Harvard Medical School Director, Neuroprotection Research Laboratory Departments of Radiology and Neurology Massachusetts General Hospital Boston, Massachusetts Daniel H. Lowenstein, MD Professor, Department of Neurology University of California, San Francisco Director, UCSF Epilepsy Center UCSF Medical Center San Francisco, California Tania Lupoli, AM PhD Candidate Department of Chemistry and Chemical Biology Harvard University Cambridge, Massachusetts

Peter R. Martin, MD Professor, Departments of Psychiatry and Pharmacology Vanderbilt University Director, Division of Addiction Psychiatry and Vanderbilt Addiction Center Vanderbilt University Medical Center Nashville, Tennessee Thomas Michel, MD, PhD Professor of Medicine (Biochemistry) Harvard Medical School Senior Physician in Cardiovascular Medicine Department of Medicine Brigham and Women’s Hospital Boston, Massachusetts Keith W. Miller, MA, DPhil Edward Mallinckrodt Professor of Pharmacology Department of Anaesthesia Harvard Medical School Pharmacologist, Department of Anesthesia, Critical Care and Pain Medicine Massachusetts General Hospital Boston, Massachusetts Zachary S. Morris, PhD MD Candidate, Harvard-MIT MD-PhD Program Department of Pathology Harvard Medical School Boston, Massachusetts Joshua D. Moss, MD Assistant Professor of Medicine Heart Rhythm Center University of Chicago Medical Center Chicago, Illinois Dalia S. Nagel, MD Clinical Instructor, Department of Ophthalmology Mount Sinai School of Medicine Attending Physician Department of Ophthalmology Mount Sinai Hospital New York, New York Robert M. Neer, MD Associate Professor of Medicine Harvard Medical School Endocrine Unit, Department of Medicine Massachusetts General Hospital Boston, Massachusetts

xx Contributors

Sachin Patel, MD, PhD Assistant Professor, Departments of Psychiatry and Molecular Physiology and Biophysics Vanderbilt University Medical Center Nashville, Tennessee Thomas P. Rocco, MD Associate Professor of Medicine Harvard Medical School Cardiovascular Division, Brigham and Women’s Hospital Boston, Massachusetts Cardiology Section, VA Boston Healthcare System West Roxbury, Massachusetts Bryan L. Roth, MD, PhD Michael Hooker Distinguished Professor Department of Pharmacology University of North Carolina Chapel Hill Medical School Chapel Hill, North Carolina Edward T. Ryan, MD Associate Professor of Medicine Harvard Medical School Associate Professor of Immunology and Infectious Diseases Harvard School of Public Health Director, Tropical Medicine Massachusetts General Hospital Boston, Massachusetts Marvin Ryou, MD Instructor in Medicine Harvard Medical School Advanced Endoscopy / Gastrointestinal Interventional Fellow Division of Gastroenterology Brigham and Women’s Hospital Gastrointestinal Unit Massachusetts General Hospital Boston, Massachusetts Joshua M. Schulman, MD Resident, Department of Dermatology University of California, San Francisco San Francisco, California Daniel M. Scott, PhD Director of Chemistry Pharmaceutical CMC Management Biogen Idec, Inc. Cambridge, Massachusetts

Charles N. Serhan, PhD Simon Gelman Professor of Anaesthesia (Biochemistry and Molecular Pharmacology) Department of Anesthesiology, Perioperative and Pain Medicine Harvard Medical School Director, Center for Experimental Therapeutics and Reperfusion Injury Brigham and Women’s Hospital Boston, Massachusetts Helen Marie Shields, MD Professor of Medicine Harvard Medical School Physician, Department of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts Steven E. Shoelson, MD, PhD Professor of Medicine Harvard Medical School Associate Director of Research, Section Head, Cellular and Molecular Physiology Joslin Diabetes Center Boston, Massachusetts Aimee Der-Huey Shu, MD Assistant Professor, Departments of Medicine and Obstetrics and Gynecology Division of Endocrinology Columbia University Medical Center New York, New York David G. Standaert, MD, PhD Professor, Department of Neurology University of Alabama at Birmingham Director, Division of Movement Disorders University Hospital Birmingham, Alabama Gary R. Strichartz, PhD Professor of Biological Chemistry and Molecular Pharmacology Harvard Medical School Vice-Chairman for Research, Department of Anesthesiology Brigham and Women’s Hospital Boston, Massachusetts Robert M. Swift, MD, PhD Professor of Psychiatry and Human Behavior Center for Alcohol and Addiction Studies Brown University Associate Chief of Staff for Research Providence Veterans Administration Medical Center Providence, Rhode Island

Cullen Taniguchi, MD, PhD Resident, Department of Radiation Oncology Stanford University Stanford, California *Armen H. Tashjian, Jr., MD Professor of Biological Chemistry and Molecular Pharmacology, emeritus Harvard Medical School Professor of Toxicology, emeritus Harvard School of Public Health Department of Genetics and Complex Diseases Harvard School of Public Health Boston, Massachusetts *deceased Charles Russell Taylor, MD Associate Professor of Dermatology Harvard Medical School Director of Phototherapy and Staff Dermatologist Department of Dermatology Massachusetts General Hospital Boston, Massachusetts John L. Vahle, DVM, PhD, DACVP Research Fellow, Department of Toxicology and Pathology Lilly Research Laboratories Indianapolis, Indiana Anand Vaidya, MD Research Fellow in Medicine (Endocrinology) Harvard Medical School Division of Endocrinology, Diabetes, and Hypertension Brigham and Women’s Hospital Boston, Massachusetts Andrew J. Wagner, MD, PhD Instructor, Department of Medicine Harvard Medical School Medical Oncologist Center for Sarcoma and Bone Oncology Dana-Farber Cancer Institute Boston, Massachusetts Suzanne Walker, PhD Professor of Microbiology and Molecular Genetics Harvard Medical School Boston, Massachusetts Ryan R. Walsh, MD, PhD Instructor, Department of Neurology University of Alabama at Birmingham University of Alabama at Birmingham Hospital Birmingham, Alabama

Contributors xxi

Liewei Wang, MD, PhD Associate Professor, Department of Molecular Pharmacology and Experimental Therapeutics Mayo Clinic College of Medicine Rochester, Minnesota Richard M. Weinshilboum, MD Professor, Department of Molecular Pharmacology and Experimental Therapeutics Mayo Clinic College of Medicine Rochester, Minnesota

Freddie M. Williams, MD Senior Cardiologist Wellmont CVA Heart Institute Kingsport, Tennessee Clifford J. Woolf, MD, BCh, PhD Professor of Neurology and Neurobiology Harvard Medical School Director, F.M. Kirby Neurobiology Center Children’s Hospital Boston Boston, Massachusetts

Jacob Wouden, MD Radiologist, Washington Hospital Medical Staff Washington Hospital Healthcare Group Fremont, California Robert W. Yeh, MD, MSc Instructor in Medicine Harvard Medical School Interventional Cardiologist Department of Medicine Massachusetts General Hospital Boston, Massachusetts

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I Fundamental Principles of Pharmacology

1 Drug–Receptor Interactions Zachary S. Morris and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 CONFORMATION AND CHEMISTRY OF DRUGS AND RECEPTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Impact of Drug Binding on the Receptor . . . . . . . . . . . . . . . . 4 Membrane Effects on Drug–Receptor Interactions . . . . . . . . 5 MOLECULAR AND CELLULAR DETERMINANTS OF DRUG SELECTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 MAJOR TYPES OF DRUG RECEPTORS . . . . . . . . . . . . . . . . . . . 6 Transmembrane Ion Channels . . . . . . . . . . . . . . . . . . . . . . . 7 Transmembrane G Protein-Coupled Receptors . . . . . . . . . . . 9 Transmembrane Receptors with Enzymatic Cytosolic Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Receptor Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . 11 Receptor Tyrosine Phosphatases . . . . . . . . . . . . . . . . . . 12

Tyrosine Kinase-Associated Receptors . . . . . . . . . . . . . 12 Receptor Serine/ Threonine Kinases . . . . . . . . . . . . . . . 12 Receptor Guanylyl Cyclases . . . . . . . . . . . . . . . . . . . . . 12 Intracellular Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Extracellular Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Cell Surface Adhesion Receptors . . . . . . . . . . . . . . . . . . . . 13 PROCESSING OF SIGNALS RESULTING FROM DRUG–RECEPTOR INTERACTIONS. . . . . . . . . . . . . . . . . . . . . 13 CELLULAR REGULATION OF DRUG–RECEPTOR INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 DRUGS THAT DO NOT FIT THE DRUG–RECEPTOR MODEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 15 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

 INTRODUCTION

that, upon binding to a drug, mediate those biochemical and physiologic changes.

Why is it that one drug affects cardiac function and another alters the transport of specific ions in the kidney? Why does ciprofloxacin effectively kill bacteria, but rarely harms a patient? These questions can be answered by first examining the interaction between a drug and its specific molecular target and then considering the role of that action in a broader physiologic context. This chapter focuses on the molecular details of drug–receptor interactions, emphasizing the variety of receptors and their molecular mechanisms. This discussion provides a conceptual basis for the action of the many drugs and drug classes discussed in this book. It also serves as a background for Chapter 2, Pharmacodynamics, which discusses the quantitative relationships between drug–receptor interactions and pharmacologic effect. Although drugs can theoretically bind to almost any three-dimensional target, most drugs achieve their desired (therapeutic) effects by interacting selectively with target molecules that play important roles in physiologic or pathophysiologic functioning. In many cases, selectivity of drug binding to receptors also determines the undesired (adverse) effects of a drug. In general, drugs are molecules that interact with specific molecular components of an organism to cause biochemical and physiologic changes within that organism. Drug receptors are macromolecules

2

 CONFORMATION AND CHEMISTRY OF DRUGS AND RECEPTORS Why does imatinib act specifically on the BCR-Abl receptor tyrosine kinase and not on other molecules? The answer to this question, and an understanding of why any drug binds to a particular receptor, can be found in the structure and chemical properties of the two molecules. This section discusses the basic determinants of receptor structure and the chemistry of drug–receptor binding. The discussion here focuses primarily on the interactions of drugs that are small organic molecules with target receptors that are mainly macromolecules (especially proteins), but many of these principles also apply to the interactions of protein-based therapeutics with their molecular targets (see Chapter 53, Protein Therapeutics). Because many human and microbial drug receptors are proteins, it is useful to review the four major levels of protein structure (Fig. 1-1). At the most basic level, proteins consist of long chains of amino acids, the sequences of which are determined by the sequences of the DNA that code for the proteins. A protein’s amino acid sequence is referred to as its primary structure. Once a long chain of amino acids has been synthesized on a ribosome, many of the amino acids begin to interact with nearby

Primary

Amino acids Secondary

Beta pleated sheet

Alpha helix

Tertiary

Beta sheet

Alpha helix

Quaternary

A

B

C

Glu 286

Imatinib

Met 290

Ile 313

Imatinib

Phe 382

Ala 269

Gly 383 Asp 381

Activation loop of kinase

Asn 368 Thr 315 Phe 382

Leu 248

Lys 271

Asp 363

Phe 317 Asp 381 Tyr 253

Met 318 Gly 321 Leu 370

Arg 367 Val 256

Tyr 393

A

B

C

α β GDP

γ

D

A α

α

γ

Ligand binding sites

B

α

α

O Receptor gate closed

N+ O Acetylcholine

Na+ C

α

α

Na+ Receptor gate open

CHAPTER 1 / Drug–Receptor Interactions 9

increase the conductance of chloride ions across neuronal membranes, thereby driving the membrane potential farther away from its threshold for activation.

Transmembrane G Protein-Coupled Receptors G protein-coupled receptors are the most abundant class of receptors in the human body. These receptors are exposed at the extracellular surface of the plasma membrane, traverse the membrane, and possess intracellular regions that activate a unique class of signaling molecules called G proteins. (G proteins are so named because they bind the guanine nucleotides GTP and GDP.) G protein-coupled signaling mechanisms are involved in many important processes, including vision, olfaction, and neurotransmission. G protein-coupled receptors have seven transmembrane regions within a single polypeptide chain. Each transmembrane region consists of a single ␣ helix, and the ␣ helices are arranged in a characteristic structural motif that is similar in all members of this receptor class. The extracellular domain of this class of proteins usually contains the ligand-binding region, although some G protein-coupled receptors bind ligands within the transmembrane domain of the receptor. G proteins have ␣ and ␤␥ subunits that are noncovalently linked in the resting state. Stimulation of a G protein-coupled receptor causes its cytoplasmic domain to bind and activate a nearby G protein, whereupon the ␣ subunit of the G protein exchanges GDP for GTP. The ␣GTP subunit then dissociates from the ␤␥ subunit, and the ␣ or ␤␥ subunit diffuses along the inner leaflet of the plasma membrane to interact with a number of different effectors. These effectors include adenylyl cyclase, phospholipase C, various ion channels, and other classes of proteins. Signals

mediated by G proteins are usually terminated by the hydrolysis of GTP to GDP, which is catalyzed by the inherent GTPase activity of the ␣ subunit (Fig. 1-5). One major role of the G proteins is to activate the production of second messengers; that is, signaling molecules that convey the input provided by the first messenger—usually an endogenous ligand or an exogenous drug—to cytoplasmic effectors (Fig. 1-6). The activation of cyclases such as adenylyl cyclase, which catalyzes the production of the second messenger cyclic adenosine-3⬘,5⬘-monophosphate (cAMP), and guanylyl cyclase, which catalyzes the production of cyclic guanosine-3⬘,5⬘-monophosphate (cGMP), constitutes the most common pathway linked to G proteins. In addition, G proteins can activate the enzyme phospholipase C (PLC), which, among other functions, plays a key role in regulating the concentration of intracellular calcium. Upon activation by a G protein, PLC cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 triggers the release of Ca2⫹ from intracellular stores, thereby dramatically increasing the cytosolic Ca2⫹ concentration and activating downstream molecular and cellular events. DAG activates protein kinase C, which then mediates other molecular and cellular events including smooth muscle contraction and transmembrane ion transport. All of these events are dynamically regulated, so that the different steps in the pathways are activated and inactivated with characteristic kinetics. A large number of G␣ protein isoforms have now been identified, each of which has unique effects on its targets. A few of these G proteins include G-stimulatory (Gs), Ginhibitory (Gi), Gq, Go, and G12/13. Examples of the effects of these isoforms are shown in Table 1-4. The differential

Receptor

Effector

A 1 Agonist binding

1 Agonist unbinding 2 GTP hydrolysis

α β

3 Heterotrimeric G protein reconstituted

2 GTP-GDP exchange γ

3 G protein activation

GDP

Effector activated

GTP

C

Agonist B

1 α-GTP diffusion to effector β

γ

α

α β

2 Effector activation

GTP

γ

GTP

GDP

FIGURE 1-5. Receptor-mediated activation of a G protein and the resultant effector interaction. A. In the resting state, the ␣ and ␤␥ subunits of a G protein are associated with one another, and GDP is bound to the ␣ subunit. B. Binding of an extracellular ligand (agonist) to a G protein-coupled receptor causes the exchange of GTP for GDP on the ␣ subunit. C. The ␤␥ subunit dissociates from the ␣ subunit, which diffuses to interact with effector proteins. Interaction of the GTP-associated ␣ subunit with an effector activates the effector. In some cases (not shown), the ␤␥ subunit can also activate effector proteins. Depending on the receptor subtype and the specific G␣ isoform, G␣ can also inhibit the activity of an effector molecule. The ␣ subunit possesses intrinsic GTPase activity, which leads to hydrolysis of GTP to GDP. This leads to reassociation of the ␣ subunit with the ␤␥ subunit, and the cycle can begin again.

A

Agonist Adenylyl cyclase

Receptor

β

αs

γ

GTP ATP

cAMP

PKA Protein phosphorylation

B PLC

PIP2

DAG PKC

β

γ

(active)

αq GTP

PKC IP3 Ca2+

Ca2+

Protein phosphorylation

CHAPTER 1 / Drug–Receptor Interactions 11

FIGURE 1-7.

A

Tyr

Tyr

P Tyr

Tyr P

Tyrosine kinase activity

Cytoplasmic protein Tyr

P Tyr

B

Tyrosine phosphatase activity P Tyr

Tyr

C

Activated kinase Inactive kinase

Tyrosine kinase activity Tyr

P Tyr

D

Ser/Thr

P Ser/Thr

Serine/threonine kinase activity Ser/Thr

P Ser/Thr

E

Guanylyl cyclase activity

GTP

cGMP

Major types of transmembrane receptors with enzymatic cytosolic domains. There are five major categories of transmembrane receptors with enzymatic cytosolic domains. A. The largest group is comprised of receptor tyrosine kinases. After ligand-induced activation, these receptors dimerize and transphosphorylate tyrosine residues in the receptor and, often, on target cytosolic proteins. Examples of receptor tyrosine kinases include the insulin receptor and the BCR-Abl protein. B. Some receptors can act as tyrosine phosphatases. These receptors dephosphorylate tyrosine residues either on other transmembrane receptors or on cytosolic proteins. Many cells of the immune system have receptor tyrosine phosphatases. C. Some tyrosine kinase-associated receptors lack a definitive enzymatic domain, but binding of ligand to the receptor triggers activation of receptor-associated protein kinases (termed nonreceptor tyrosine kinases) that then phosphorylate tyrosine residues on certain cytosolic proteins. D. Receptor serine/threonine kinases phosphorylate serine and threonine residues on certain target cytosolic proteins. Members of the TGF-␤ superfamily of receptors are in this category. E. Receptor guanylyl cyclases contain a cytosolic domain that catalyzes the formation of cGMP from GTP. The receptor for B-type natriuretic peptide is one of the receptor guanylyl cyclases that has been well characterized.

play roles in a diverse set of physiologic processes, including cell metabolism, growth, and differentiation. Receptors that have an intracellular enzymatic domain can be grouped into five major classes based on their cytoplasmic mechanism of action (Fig. 1-7). All of these receptors are single–membrane-spanning proteins, in contrast to the seven– membrane-spanning motif present in G protein-coupled receptors. Many receptors with enzymatic cytosolic domains form dimers or multisubunit complexes to transduce their signals. Many of the receptors with enzymatic cytosolic domains modify proteins by adding or removing phosphate groups to or from specific amino acid residues. Phosphorylation is a ubiquitous mechanism of protein signaling. The large negative charge of phosphate groups can dramatically alter the three-dimensional structure of a protein and thereby change that protein’s activity. In addition, phosphorylation is easily reversible, thus allowing this signaling mechanism to act specifically in time and space. Receptor Tyrosine Kinases The largest group of transmembrane receptors with enzymatic cytosolic domains is the receptor tyrosine kinase family. These receptors transduce signals from many hormones and growth factors by phosphorylating tyrosine residues on the cytoplasmic tail of the receptor. This leads to recruitment and subsequent tyrosine phosphorylation of a number of cytosolic signaling molecules. The insulin receptor is a well-characterized receptor tyrosine kinase. This receptor consists of two extracellular ␣ subunits that are covalently linked to two membrane-spanning ␤ subunits. Binding of insulin to the ␣ subunits results in a change in conformation of the adjacent ␤ subunits, causing the ␤ subunits to move closer to one another on the intracellular side of the membrane. The proximity of the two ␤ subunits promotes a transphosphorylation reaction, in which one ␤ subunit phosphorylates the other (“autophosphorylation”). The phosphorylated tyrosine residues then act to recruit other cytosolic proteins, known as insulin receptor substrate (IRS) proteins. Type 2 diabetes mellitus may, in some cases, be associated with defects in post-insulin receptor signaling; thus, understanding the insulin receptor signaling pathways is relevant for the potential design of rational therapeutics. The mechanism of insulin receptor signaling is discussed in more

CHAPTER 1 / Drug–Receptor Interactions 13

modify the expression levels of specific gene transcripts, although technical challenges in delivering such therapeutics to their targets currently limit their utility to specialized applications.

Steroid hormone

Hormone receptor Chaperone

A

Nucleus

B

DNA

C

FIGURE 1-8.

Lipophilic molecule binding to an intracellular transcription factor. A. Small lipophilic molecules can diffuse through the plasma membrane and bind to intracellular transcription factors. In this example, steroid hormone binding to a cytosolic hormone receptor is shown, although some receptors of this class may be located in the nucleus before ligand binding. B. Ligand binding triggers a conformational change in the receptor (and often, as shown here, dissociation of a chaperone repressor protein) that leads to transport of the ligand–receptor complex into the nucleus. In the nucleus, the ligand–receptor complex typically dimerizes. In the example shown, the active form of the receptor is a homodimer (two identical receptors binding to one another), but heterodimers (such as the thyroid hormone receptor and the retinoid X receptor) may also form. C. The dimerized ligand–receptor complex binds to DNA and may then recruit coactivators or corepressors (not shown). These complexes alter the rate of gene transcription, leading to a change (either up or down) in cellular protein expression.

the intracellular or extracellular concentrations of specific gene products, drugs that target transcription factors can have a profound effect on cellular function. The cellular responses to such drugs, and the effects that result from this cellular response in tissues and organ systems, provide links between the molecular drug–receptor interaction and the effects of the drug on the organism as a whole. Because gene transcription is a relatively slow (minutes to hours) and long-lasting process, drugs that target transcription factors often require a longer period of time for the onset of action to take place, and have longer-lasting effects, than do drugs that alter more transient processes such as ion conduction (seconds to minutes). Structural proteins are another important class of cytosolic drug targets. For example, the antimitotic vinca alkaloids bind to tubulin monomers and prevent the polymerization of this molecule into microtubules. This inhibition of microtubule formation arrests the affected cells in metaphase, making the vinca alkaloids useful antineoplastic drugs. Other drugs bind directly to RNA or ribosomes; such drugs are important agents in antimicrobial and antineoplastic chemotherapy. With the continued development of RNA interference (RNAi) therapeutics, such targets may become increasingly prominent. RNAi technology could someday enable physicians to routinely

Extracellular Enzymes Many important drug receptors are enzymes with active sites located outside the plasma membrane. The extracellular environment consists of a milieu of proteins and signaling molecules. Many of these proteins serve a structural role, and others are used to communicate information between cells. Enzymes that modify the molecules mediating these important signals can influence physiologic processes such as vasoconstriction and neurotransmission. One example of this class of receptor is the angiotensin converting enzyme (ACE), which converts angiotensin I to the potent vasoconstrictor angiotensin II. ACE inhibitors are drugs that inhibit this enzymatic conversion and thereby lower blood pressure (among other effects; see Chapter 20). Another example is acetylcholinesterase, which degrades acetylcholine after this neurotransmitter is released from cholinergic neurons. Acetylcholinesterase inhibitors enhance neurotransmission at cholinergic synapses by preventing neurotransmitter degradation at these sites (see Chapter 9, Cholinergic Pharmacology).

Cell Surface Adhesion Receptors Cells often interact directly with other cells to perform specific functions or to communicate information. The formation of tissues and the migration of immune cells to a site of inflammation are examples of physiologic processes that require cell–cell adhesive interactions. A region of contact between two cells is termed an adhesion, and cell–cell adhesive interactions are mediated by pairs of adhesion receptors on the surfaces of the individual cells. In many cases, several such receptor–counter-receptor pairs combine to secure a firm adhesion, and intracellular regulators control the activity of the adhesion receptors by changing their affinity or by controlling their expression and localization on the cell surface. Several adhesion receptors involved in the inflammatory response are attractive targets for selective inhibitors. Inhibitors of a specific class of adhesion receptors, known as integrins, have entered the clinic in recent years, and these drugs are being studied in the treatment of a range of conditions including coagulation, inflammation, multiple sclerosis, and cancer (see Chapter 45).

 PROCESSING OF SIGNALS RESULTING FROM DRUG–RECEPTOR INTERACTIONS Many cells in the body are continuously bombarded with multiple inputs, some stimulatory and some inhibitory. How do cells integrate these signals to produce a coherent response? G proteins and other second messengers appear to provide important points of integration. As noted above, relatively few second messengers have been identified, and it is unlikely that many more remain to be discovered. Thus, second messengers are an attractive candidate mechanism for providing cells with a set of common points upon which numerous outside stimuli could converge to generate a coordinated cellular effect (Fig. 1-9).

14 Fundamental Principles of Pharmacology Ligand 1

Adenylyl cyclase

Receptor

αs β GDP

γ

Receptor

αs

αs

αi

αi

GTP

GTP

GTP

GTP

ATP

cAMP

cAMP

γ

β

Ligand 2

αi GDP

ATP

Net result = integrated effect

FIGURE 1-9.

Signaling convergence of two receptors. A limited number of mechanisms are used to transduce intracellular signal cascades. In some cases, this allows for convergence, where two different receptors have opposite effects that tend to negate one another in the cell. In a simple example, two different G protein-coupled receptors could be stimulated by different ligands. The receptor shown on the left is coupled to G␣s, a G protein that stimulates adenylyl cyclase to catalyze the formation of cAMP. The receptor shown on the right is coupled to G␣i, a G protein that inhibits adenylyl cyclase. When both of these receptors are activated simultaneously, they can attenuate or even neutralize each other, as shown. Sometimes, signaling through a pathway may alternate as the two receptors are sequentially activated.

Ion concentrations provide another point of integration for cellular effects because the cellular concentration of a particular ion is the result of the integrated activity of multiple ionic currents that both increase and decrease the concentration of the ion within the cell. For example, the contractile state of a smooth muscle cell is a function of the intracellular calcium ion concentration, which is determined by several different Ca2⫹ conductances. These conductances include calcium ion leaks into the cell and calcium currents into and out of the cytoplasm through specialized channels in the plasma membrane and smooth endoplasmic reticulum. Because the magnitude of cellular response is often considerably greater than the magnitude of the stimulus that caused the response, cells appear to have the ability to amplify the effects of receptor binding. G proteins provide an excellent example of signal amplification. Ligand binding to a G protein-coupled receptor activates a single G protein molecule. This G protein molecule can then bind to and activate many effector molecules, such as adenylyl cyclase, which can then generate an even greater number of second messenger molecules (in this example, cAMP). Another example of signal amplification is “trigger Ca2⫹,” in which a small influx of Ca2⫹ through voltage-gated Ca2⫹ channels in the plasma membrane “triggers” the release of larger amounts of Ca2⫹ into the cytoplasm from intracellular stores.

 CELLULAR REGULATION OF DRUG–RECEPTOR INTERACTIONS Drug-induced activation or inhibition of a receptor often has a lasting impact on the receptor’s subsequent responsiveness to drug binding. Mechanisms that mediate such effects are important because they prevent overstimulation that could lead to cellular damage or adversely affect the organism as a whole. Many drugs show diminishing effects over time; this phenomenon is called tachyphylaxis. In pharmacologic terms, the receptor and the cell become desensitized to the action of the drug. Mechanisms of desensitization can be divided into two types: homologous, in which the effects of agonists at only one type of receptor are diminished; and

heterologous, in which the effects of agonists at two or more types of receptors are coordinately diminished. Heterologous desensitization is thought to be caused by druginduced alteration in a common point of convergence in the mechanisms of action of the involved receptors, such as a shared effector molecule. Many receptors exhibit desensitization. For example, the cellular response to repeated stimulation of ␤-adrenergic receptors by epinephrine diminishes steadily over time (Fig. 1-10). ␤-Adrenergic receptor desensitization is mediated by epinephrine-induced phosphorylation of the cytoplasmic tail of the receptor. This phosphorylation promotes the binding of ␤-arrestin to the receptor; in turn, ␤-arrestin inhibits the receptor’s ability to stimulate the G protein Gs. With lower levels of activated Gs present, adenylyl cyclase produces less cAMP. In this manner, repeated cycles of ligand–receptor binding result in smaller and smaller cellular effects. Other molecular mechanisms have even more profound effects, completely turning off the receptor to stimulation by ligand. The latter phenomenon, referred to as inactivation, may also result from phosphorylation of the receptor; in this case, the phosphorylation completely blocks the signaling activity of the receptor or causes removal of the receptor from the cell surface. Another mechanism that can affect the cellular response caused by drug–receptor binding is called refractoriness. Receptors that assume a refractory state following activation require a period of time to pass before they can be stimulated again. As noted above, voltage-gated sodium channels, which mediate the firing of neuronal action potentials, are subject to refractory periods. After channel opening induced by membrane depolarization, the voltagegated sodium channel spontaneously closes and cannot be reopened for some period of time (called the refractory period). This inherent property of the channel determines the maximum rate at which neurons can be stimulated and transmit information. The effect of drug–receptor binding can also be influenced by drug-induced changes in the number of receptors on or in a cell. One example of a molecular mechanism by which receptor number can be altered is called down-

A Phosphorylation by PKA and/or βARK

P P P P

β-arrestin binding

P P P P

β-arrestin

Agonist

G protein binding prevented

B Sequestration

C

Endosome

Lysosome

Degradation

16 Fundamental Principles of Pharmacology

mechanisms of action described in this chapter serve as paradigms for the principles of pharmacodynamics. The ability to classify drugs based on their mechanisms of action makes it possible to simplify the study of pharmacology, because the molecular mechanism of action of a drug can usually be linked to its cellular, tissue, organ, and system levels of action. In turn, it becomes easier to understand how a given drug mediates its therapeutic effects and its unwanted or adverse effects in a particular patient. The major aim of modern drug development is to identify drugs that are highly selective by tailoring drug molecules to unique targets responsible for disease. As knowledge of drug development and the genetic and pathophysiologic basis of disease progresses, physicians and scientists will learn to combine the molecular specificity of a drug with the genetic and pathophysiologic specificity of the drug target to provide more and more selective therapies.

Acknowledgment We thank Christopher W. Cairo and Josef B. Simon for their valuable contributions to this chapter in the First and Second

Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Alexander SP, Mathie A, Peters JA. Guide to receptors and channels. 3rd ed. Br J Pharmacol 2008;153(Suppl 2):S1–S209. (Brief overview of molecular targets for drugs, organized by types of receptors.) Berg JM, Tymoczko JL, Stryer L. Biochemistry. 6th ed. New York: WH Freeman and Company; 2006. (Contains structural information on receptors, especially G proteins.) Lagerstom MC, Schloth HB. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 2008;7:339– 357. (Discusses the five families of G protein-coupled receptors, with an eye toward future drug development.) Pratt WB, Taylor P, eds. Principles of drug action: the basis of pharmacology. 3rd ed. New York: Churchill Livingstone; 1990. (Contains a detailed discussion of drug–receptor interactions.) Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009;8:129–138. (Highlights early successes and remaining challenges in the development of RNAi therapeutics.) Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 2009;9:28–39. (Discusses the dysregulation of protein kinases in cancer and the targeting of these molecules by drugs such as imatinib.)

2 Pharmacodynamics Quentin J. Baca and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 17-18 DRUG–RECEPTOR BINDING . . . . . . . . . . . . . . . . . . . . . . . . . . 17 DOSE–RESPONSE RELATIONSHIPS . . . . . . . . . . . . . . . . . . . . 18 Graded Dose–Response Relationships . . . . . . . . . . . . . . . . 19 Quantal Dose–Response Relationships . . . . . . . . . . . . . . . 19 DRUG–RECEPTOR INTERACTIONS. . . . . . . . . . . . . . . . . . . . . 20 Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Competitive Receptor Antagonists . . . . . . . . . . . . . . . . . 21

Noncompetitive Receptor Antagonists . . . . . . . . . . . . . . 22 Nonreceptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . 23 Partial Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Inverse Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Spare Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 CONCEPTS IN THERAPEUTICS . . . . . . . . . . . . . . . . . . . . . . . . 25 Therapeutic Index and Therapeutic Window . . . . . . . . . . . . 25 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 26 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

 INTRODUCTION

Consider the simplest case, in which the receptor is either free (unoccupied) or reversibly bound to drug (occupied). We can describe this case as follows:

Pharmacodynamics is the term used to describe the effects of a drug on the body. These effects are typically described in quantitative terms. The previous chapter considered the molecular interactions by which pharmacologic agents exert their effects. The integration of these molecular actions into an effect on the organism as a whole is the subject addressed in this chapter. It is important to describe the effects of a drug quantitatively in order to determine appropriate dose ranges for patients, as well as to compare the potency, efficacy, and safety of one drug to that of another.

 DRUG–RECEPTOR BINDING The study of pharmacodynamics is based on the concept of drug–receptor binding. When either a drug or an endogenous ligand (such as a hormone or neurotransmitter) binds to its receptor, a response may result from that binding interaction. When a sufficient number of receptors are bound (or “occupied”) on or in a cell, the cumulative effect of receptor “occupancy” may become apparent in that cell. At some point, all of the receptors may be occupied, and a maximal response may be observed (an exception is the case of spare receptors; see below). When the response occurs in many cells, the effect can be seen at the level of the organ or even the patient. But this all starts with the binding of drug or ligand to a receptor (for the purpose of discussion, “drug” and “ligand” will be used interchangeably for the remainder of this chapter). A model that accurately describes the binding of drug to receptor would therefore be useful in predicting the effect of the drug at the molecular, cellular, tissue (organ), and organism (patient) levels. This section describes one such model.

kon

L  RÆ ¨ LR

Equation 2-1

koff

where L is ligand (drug), R is free receptor, and LR is bound drug–receptor complex. At equilibrium, the fraction of receptors in each state is dependent on the dissociation constant, Kd, where Kd ⫽ koff /kon. Kd is an intrinsic property of any given drug–receptor pair. Although Kd varies with temperature, the temperature of the human body is relatively constant, and it can therefore be assumed that Kd is a constant for each drug–receptor combination. According to the law of mass action, the relationship between free and bound receptor can be described as follows: Kd 

[L][R] [L][R] , rearranged to [LR]  Equation 2-2 Kd [LR]

where [L] is free ligand concentration, [R] is free receptor concentration, and [LR] is ligand–receptor complex concentration. Because Kd is a constant, some important properties of the drug–receptor interaction can be deduced from this equation. First, as ligand concentration is increased, the concentration of bound receptors increases. Second, and not so obvious, is that as free receptor concentration is increased (as may happen, for example, in disease states or upon repeated exposure to a drug), bound receptor concentration also increases. Therefore, an increase in the effect of a drug 17

[Ro]  [R]  [LR]  [R] 

[L][R] Kd

[L] ˆ  [R] Ê1  Ë Kd ¯

[LR] 

Equation 2-3

[Ro] [L] , rearranged to [L]  Kd

[L] [LR]  [L]  Kd [Ro]

Equation 2-4

response [DR] [D]   [Ro] max response [D]  Kd

Equation 2-5

A Linear 1.0 Drug A Drug B [LR] 0.5 [Ro]

0 KdA KdB

[L]

B Semilogarithmic 1.0 Drug A A Linear Drug B 1.0

[LR] 0.5 [Ro]

Drug A Drug B

E 0.5 EMAX

0 KdA KdB

[L]

0 EC50(A) EC50(B)

[L]

B Semilogarithmic 1.0 Drug A Drug B

E 0.5 EMAX

0 EC50(A) EC50(B)

[L]

Cumulative % exhibiting Therapeutic effect

Toxic effect

Lethal effect

% Individuals responding

100

50

Therapeutic effect

% Requiring dose to achieve Lethal effect Toxic effect

0 ED50

TD50

LD50

Dose

DR Æ ¨ D  R* Ø≠ Ø≠ Æ DR DR* ¨

Equation 2-6

DRÆ ¨ DR**

Equation 2-7

DR

kon Æ ¨ koff Potency

DR

k Æ ¨ k Efficacy

DR*

Equation 2-8

CHAPTER 2 / Pharmacodynamics 21 Antagonists

Receptor antagonists

Active site binding Reversible

Irreversible

Competitive antagonist

Noncompetitive active site antagonist

Nonreceptor antagonists

Allosteric binding Reversible

Irreversible

Noncompetitive allosteric antagonist

Chemical antagonist

Physiologic antagonist

FIGURE 2-4.

Antagonist classification. Antagonists can be categorized based on whether they bind to a site on the receptor for agonist (receptor antagonists) or interrupt agonist–receptor signaling by other means (nonreceptor antagonists). Receptor antagonists can bind either to the agonist (active) site or to an allosteric site on the receptor; in either case, they do not affect basal receptor activity (i.e., the activity of the receptor in the absence of agonist). Agonist (active) site receptor antagonists prevent the agonist from binding to the receptor. If the antagonist competes with the ligand for agonist site binding, it is termed a competitive antagonist; high concentrations of agonist are able to overcome competitive antagonism. Noncompetitive agonist site antagonists bind covalently or with very high affinity to the agonist site, so that even high concentrations of agonist are unable to activate the receptor. Allosteric receptor antagonists bind to the receptor at a site other than the agonist site. They do not compete directly with agonist for receptor binding, but rather alter the Kd for agonist binding or inhibit the receptor from responding to agonist binding. High concentrations of agonist are generally unable to reverse the effect of an allosteric antagonist. Nonreceptor antagonists fall into two categories. Chemical antagonists sequester agonist and thus prevent the agonist from interacting with the receptor. Physiologic antagonists induce a physiologic response opposite to that of an agonist, but by a molecular mechanism that does not involve the receptor for agonist.

Antagonists

an agonist to initiate a response. At the molecular level, this inhibition can occur by inhibiting the agonist directly (e.g., using antibodies), by inhibiting a downstream molecule in the activation pathway, or by activating a pathway that opposes the action of the agonist. Nonreceptor antagonists can be divided into chemical antagonists and physiologic antagonists. Chemical antagonists inactivate an agonist before it has the opportunity to act (e.g., by chemical neutralization); physiologic antagonists cause a physiologic effect opposite to that induced by the agonist. The following section discusses competitive receptor antagonists and noncompetitive receptor antagonists. Nonreceptor antagonists are also examined briefly.

An antagonist is a molecule that inhibits the action of an agonist but has no effect in the absence of the agonist. Figure 2-4 shows one approach to classifying the various types of antagonists. Antagonists can be divided into receptor and nonreceptor antagonists. A receptor antagonist binds to either the active site (agonist binding site) or an allosteric site on a receptor. Binding of an antagonist to the active site prevents the binding of the agonist to the receptor, whereas binding of an antagonist to an allosteric site either alters the Kd for agonist binding or prevents the conformational change required for receptor activation. Receptor antagonists can also be divided into reversible and irreversible antagonists; that is, antagonists that bind to their receptors reversibly and those that bind irreversibly. Figure 2-5 illustrates the general effects of these antagonist types on agonist binding; more detail is provided in the following sections. A nonreceptor antagonist does not bind to the same receptor as an agonist, but it nonetheless inhibits the ability of

Competitive Receptor Antagonists A competitive antagonist binds reversibly to the active site of a receptor. Unlike an agonist, which also binds to the active site of the receptor, a competitive antagonist does not stabilize the conformation required for receptor activation.

Agonist Agonist binding site

A

Agonist

Agonist Allosteric antagonist binding site

Unbound receptor

Competitive antagonist

B

C

Agonist binding

Competitive antagonist binding

D

Noncompetitive antagonist

Noncompetitive antagonist binding

FIGURE 2-5. Types of receptor antagonists. A schematic illustrating the differences between agonist (active) site and allosteric antagonists. A. The unbound inactive receptor. B. The receptor activated by agonist. Note the conformational change induced in the receptor by agonist binding, for example, the opening of a transmembrane ion channel. C. Agonist site antagonists bind to the receptor’s agonist site but do not activate the receptor; these agents block agonist binding to the receptor. D. Allosteric antagonists bind to an allosteric site (different from the agonist site) and thereby prevent receptor activation, even when the agonist is bound to the receptor.

Equation 2-10

A Competitive antagonist 100

% Response

[DR] [D]  [A]ˆ [Ro] [D]  Kd Ê1  Ë KA ¯

Equation 2-9

Agonist alone

Agonist + Antagonist 50

Antagonist alone 0

B Noncompetitive antagonist

100

% Response

Æ AR Æ ¨ A  D  R ¨ DR*

Agonist alone

Agonist + Antagonist 50

0

Antagonist alone

Agonist or antagonist concentration

CHAPTER 2 / Pharmacodynamics 23

Nonreceptor Antagonists Nonreceptor antagonists can be divided into chemical antagonists and physiologic antagonists. A chemical antagonist inactivates the agonist of interest by modifying or sequestering it, so that the agonist is no longer capable of binding to and activating the receptor. Protamine is an example of a chemical antagonist; this basic protein binds stoichiometrically to the acidic heparin class of anticoagulants and thereby inactivates these agents (see Chapter 22, Pharmacology of Hemostasis and Thrombosis). Because of this chemical antagonism, protamine can be used to terminate the effects of heparin rapidly. A physiologic antagonist most commonly activates or blocks a receptor that mediates a response physiologically opposite to that of the receptor for the agonist. For example, in the treatment of hyperthyroidism, -adrenergic antagonists are used as physiologic antagonists to counteract the tachycardic effect of endogenous thyroid hormone. Although thyroid hormone does not produce its tachycardic effect via ␤-adrenergic stimulation, blocking ␤-adrenergic stimulation can nonetheless relieve the tachycardia caused by hyperthyroidism (see Chapter 10, Adrenergic Pharmacology, and Chapter 27, Pharmacology of the Thyroid Gland).

Partial Agonists A partial agonist is a molecule that binds to a receptor at its active site but produces only a partial response, even when all of the receptors are occupied (bound) by the agonist. Figure 2-7A shows a family of dose–response curves for several full and partial agonists. Each agonist acts by binding to the same site on the muscarinic acetylcholine (ACh) receptor. Note that butyl trimethylammonium (TMA) is not only more potent than longer-chain derivatives at stimulating muscle contraction, but also more efficacious than some of the derivatives (e.g., the heptyl and octyl forms) at producing a greater maximal response. For this reason, butyl TMA is a full agonist at the muscarinic ACh receptor, whereas the octyl derivative is a partial agonist at this receptor. Because partial agonists and full agonists bind to the same site on a receptor, a partial agonist can reduce the response produced by a full agonist. In this way, the partial agonist can act as a competitive antagonist. For this reason, partial agonists are sometimes called “partial antagonists” or even “mixed agonist-antagonists.”

A 100

Butyl Hexyl

% Contraction

Heptyl

50 Octyl

0 10-7

10-6

10-5

10-4

10-3

[D] (Molar)

B 100

Morphine

% Analgesia

for receptor binding, effectively reducing the receptor’s affinity for an agonist without limiting the number of available receptors. In contrast, a noncompetitive antagonist removes functional receptors from the system, thereby limiting the number of available receptors. Figures 2-6A and 2-6B compare the effects of competitive and noncompetitive antagonists on the agonist dose–response relationship. Aspirin is one example of a noncompetitive antagonist. This agent irreversibly acetylates cyclo-oxygenase, the enzyme responsible for generating thromboxane A2 in platelets. In the absence of thromboxane A2 generation, platelet aggregation is inhibited. Because the inhibition is irreversible and platelets are not capable of synthesizing new cyclooxygenase molecules, the effects of a single dose of aspirin last for 7 to 10 days (the time required for the bone marrow to generate new platelets), even though the free drug is cleared from the body much more rapidly.

50 Buprenorphine

0 0.01

0.1

ED50(B)

ED50(M)

10

[D] (mg/kg)

FIGURE 2-7. Full and partial agonist dose–response curves. There are many instances in which drugs that all act at the agonist site on the same receptor produce different maximal effects. A. Various alkyl derivatives of trimethylammonium all stimulate muscarinic acetylcholine (ACh) receptors to cause muscle contraction in the gut, but they produce different maximal responses, even when all receptors are occupied. In this example, the butyl and hexyl trimethylammonium derivatives are full agonists—although they have different potencies, they are both capable of eliciting a maximal response. Agonists that produce only a partial response, such as the heptyl and octyl derivatives, are called partial agonists. Note that the dose–response curves of these partial agonists plateau at values less than those of full agonists. ACh acts as a full agonist in this system (not shown). B. Partial agonists may be more or less potent than full agonists. In this case, buprenorphine (ED50 ⫽ 0.3 mg/kg) is more potent than morphine (ED50 ⫽ 1.0 mg/kg), although it cannot achieve the same maximal response as the full agonist. Buprenorphine is used clinically in the treatment of opioid addiction, where it is desirable to use a partial agonist that is less efficacious than an addicting opioid such as heroin or morphine. Low concentrations of the partial agonist buprenorphine bind tightly to the opioid receptor and competitively inhibit the binding of the more efficacious opioids. Very high doses of buprenorphine show a paradoxically diminished analgesic effect that may be due to lower-affinity interactions of the drug with non–mu-opioid receptors (not shown).

24 Fundamental Principles of Pharmacology

It is interesting to consider how an agonist could produce a less-than-maximal response if a receptor can exist in only the active or the inactive state. This is an area of current investigation, for which several hypotheses have been proposed. Recall that Equation 2-6 was simplified to Equation 2-7 based on the assumption that R and DR* are much more stable than R* and DR. But what would happen if a drug (call it a partial agonist) could stabilize DR as well as DR*? In that case, addition of the partial agonist would result in stabilization of some receptors in the DR form and some receptors in the DR* form. At full receptor occupancy, some receptors would be in the active state and some in the inactive state, and the efficacy of the drug would be reduced compared to that of a full agonist (which stabilizes only DR*). In this formulation, a full agonist binds preferentially to the active state of the receptor, a partial agonist binds with comparable affinity to both the active and inactive states of the receptor, and an inverse agonist binds preferentially to the inactive state of the receptor (see below). A second hypothesis for the action of partial agonists is that a receptor may have multiple DR* conformations, each with a different intrinsic activity. Depending on the particular conformations of the receptor that are bound by the agonist, a fraction of the maximum possible effect may be observed even when a partial agonist is bound to 100% of the receptors. This may be the case with the so-called selective estrogen receptor modulators (SERMs) such as raloxifene and tamoxifen (see Chapter 29, Pharmacology of Reproduction). Raloxifene acts as a partial agonist at estrogen receptors in bone and an antagonist at estrogen receptors in breast. The crystal structure of raloxifene bound to the estrogen receptor, when compared to that of estrogen bound to the estrogen receptor, reveals that the side chain of raloxifene inhibits an ␣ helix of the estrogen receptor from aligning in the active site (see Figure 29-8). This may result in inhibition of some downstream effects of the estrogen receptor, while maintaining other effects. At a physiologic level, this would be observed as partial agonist activity in bone (see Figure 29-7). A recent study of partial agonists acting on ligand-gated ion channels has suggested a third model, in which the receptor requires a “priming” conformational change that must occur before activation of the receptor is possible. In this model, although a partial agonist may bind to the receptor with high affinity, it is less efficient than a full agonist at inducing the “primed” conformation of the receptor. Because this “primed” conformation is a prerequisite for activation of the receptor, the receptor will spend less time in the open conformation, and a partial agonist will have lower efficacy than a full agonist. The relative potency of full agonists and partial agonists may be clinically relevant (Figure 2-7B). A partial agonist with high affinity for its receptor (such as buprenorphine) may be more potent but less efficacious than a full agonist with lower affinity for the same receptor (such as morphine). This characteristic is leveraged clinically when the partial agonist buprenorphine is used to treat opioid addiction. Buprenorphine, with its high affinity for the mu-opioid receptor, can be administered to outcompete other opioids taken by a patient and can therefore help to prevent relapse of opioid addiction. Buprenorphine must be carefully administered to a patient who is currently addicted to full-agonist opioids such as heroin or morphine, because it can out-compete these opioids and cause withdrawal symptoms. Another example of a partial agonist is pindolol, a drug often classified as a ␤-adrenergic antagonist (see Chapter 10). In actuality, however, pindolol demonstrates partial agonist

properties, and this drug may be of clinical value because of the intermediate response that it produces. Although resting heart rate and blood pressure are not reduced as much by pindolol as by other pure ␤-adrenergic antagonists, pindolol does inhibit the potentially dangerous heart rate and blood pressure increases that would otherwise occur with sympathetic stimulation (e.g., with exercise) in patients with cardiovascular disease.

Inverse Agonists The action of inverse agonists can be understood by considering Equation 2-6 again. As noted above, in some cases, receptors can have inherent stability in the R* state. In these cases, there is intrinsic activity (“tone”) of the receptor system, even in the absence of an endogenous ligand or an exogenously administered agonist. An inverse agonist acts by abrogating this intrinsic (constitutive) activity of the free (unoccupied) receptor. Inverse agonists may function by binding to and stabilizing the receptor in the DR (inactive) form. This has the effect of deactivating receptors that had existed in the R* form in the absence of drug. The physiologic importance of receptors that have inherent stability in the R* state is currently under investigation; receptors with mutations that render them constitutively active may become attractive targets for inverse agonist approaches. Consider the similarities and differences between the actions of inverse agonists and competitive antagonists. Both types of drug act to reduce the activity of a receptor. In the presence of a full agonist, both competitive antagonists and inverse agonists act to reduce agonist potency. Recall, however, that a competitive antagonist has no effect in the absence of an agonist, whereas an inverse agonist deactivates receptors that are constitutively active in the absence of an agonist. Using Equations 2-6 through 2-9 as models, these concepts can be summarized by stating that full agonists stabilize DR*, partial agonists stabilize both DR and DR* (or alternate forms of DR* or “primed” forms of DR), inverse agonists stabilize DR, and competitive antagonists “stabilize” R (or AR) by preventing full, partial, and inverse agonists from binding to the receptor.

Spare Receptors Recall that the initial discussion of drug–receptor binding assumed that 100% receptor occupancy is required for an agonist to exert its maximal effect. Now, consider the possibility that a maximal response could be achieved with less than 100% receptor occupancy. Figure 2-8 shows an example of a drug–receptor binding curve and a dose–response curve that illustrate this situation. In this example, a maximal effect is achieved at a lower dose of agonist than that required for receptor saturation, that is, the EC50 is less than the Kd for this system. This type of discrepancy between the drug–receptor binding curve and the dose–response curve signifies the presence of spare receptors. At least two molecular mechanisms are thought to be responsible for the spare receptor phenomenon. First, the receptor could remain activated after the agonist departs, allowing one agonist molecule to activate several receptors. Second, the cell signaling pathways described in Chapter 1 could allow for significant amplification of a relatively small signal, and activation of only a few receptors could be sufficient to produce a maximal response. The latter is true, for example, with many G protein-coupled receptors; activation of a single

A Drug-receptor binding curve 1.0

[DR] [Ro]

0.5

0 Kd

B Dose-response curve 1.0

E EMAX

Therapeutic Index (TI)  0.5

TD50 ED50

Equation 2-11

0 EC50

Kd

[D]

1.0 Agonist only

E 0.5 EMAX

Agonist + increasing noncompetitive antagonist

0

[D]

3 Pharmacokinetics Quentin J. Baca and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 27-28 PHYSIOLOGIC BARRIERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Biological Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Traversing the Membrane . . . . . . . . . . . . . . . . . . . . . . . 28 Membrane Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Central Nervous System. . . . . . . . . . . . . . . . . . . . . . . . . . . 29 ABSORPTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Administration Routes and Rationale . . . . . . . . . . . . . . . . . 30 Enteral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Parenteral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Mucous Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Transdermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Local, Regional, and Systemic Factors Affecting Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 DISTRIBUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Volume of Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Plasma Protein Binding . . . . . . . . . . . . . . . . . . . . . . . . . 33

Modeling the Kinetics and Thermodynamics of Drug Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Oxidation/Reduction Reactions. . . . . . . . . . . . . . . . . . . . . . 34 Conjugation/Hydrolysis Reactions . . . . . . . . . . . . . . . . . . . 34 EXCRETION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Renal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Biliary Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 CLINICAL APPLICATIONS OF PHARMACOKINETICS . . . . . . . 37 Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Metabolism and Excretion Kinetics . . . . . . . . . . . . . . . . 37 Half-Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Factors Altering Half-Life. . . . . . . . . . . . . . . . . . . . . . . . 38 Therapeutic Dosing and Frequency . . . . . . . . . . . . . . . . . . 39 Loading Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Maintenance Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 42 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

 INTRODUCTION

drug is eliminated from the body (primarily by the kidneys and liver, and in the feces). This chapter presents a broad overview of the pharmacokinetic processes of absorption, distribution, metabolism, and excretion (often abbreviated as ADME; Fig. 3-1), with a conceptual emphasis on basic principles that, when applied to an unfamiliar situation, should enable the student or physician to understand the pharmacokinetic basis of drug therapy.

Even the most promising of pharmacologic therapies will fail in clinical trials if the drug is unable to reach its target organ at a concentration sufficient to have a therapeutic effect. Many of the characteristics that render the human body resistant to harm by foreign invaders and toxic substances also limit the ability of modern drugs to combat pathologic processes within a patient. An appreciation of the many factors that affect a drug’s ability to act within a patient and the dynamic nature of these factors over time is vitally important to the clinical practice of medicine. All drugs must meet certain minimal requirements to achieve clinical effectiveness. A successful drug must be able to cross the physiologic barriers that limit the access of foreign substances to the body. Drug absorption may occur by a number of mechanisms that are designed either to exploit or to breach these barriers. After absorption, the drug uses distribution systems within the body, such as the blood and lymphatic vessels, to reach its target organ in an appropriate concentration. The drug’s ability to access its target is also limited by several processes within the patient. These processes fall broadly into two categories: metabolism, in which the body inactivates the drug through enzymatic degradation (primarily in the liver), and excretion, in which the

 PHYSIOLOGIC BARRIERS A drug must overcome physical, chemical, and biological barriers to reach its molecular and cellular sites of action. The epithelial lining of the gastrointestinal tract and other mucous membranes is one sort of barrier; additional barriers are encountered after the drug enters the blood and lymphatics. Most drugs must distribute from the blood into local tissues, a process that may be impeded by structures such as the blood–brain barrier. Typically, drugs leave the intravascular compartment at the level of the postcapillary venules, where there are gaps between the endothelial cells through which the drug can pass. Drug distribution occurs mainly through passive diffusion, the rate of which is affected by local ionic and cellular conditions. This section describes the major physical, chemical, and biological barriers to drug 27

Receptors Free

Bound

Tissue reservoirs Free

Bound

Systemic circulation Absorption

Free drug

Protein-bound drug

Excretion

Metabolites (active and inactive)

Metabolism

Flux 

(C2  C1)  (Area  Permeability) Equation 3-1 Thicknessmembrane

CHAPTER 3 / Pharmacokinetics 29

factors such as ionic, pH, and charge gradients across the membrane. In vivo, however, these additional factors affect the ability of a drug to enter cells. For example, a higher concentration of drug outside the cell would ordinarily favor net drug entry into the cell, but if both the cell interior and the drug are negatively charged, then net drug entry into the cell may be impeded. In contrast, a negatively charged cell interior could favor entry of a positively charged drug. Net diffusion of acidic and basic drugs across lipid bilayer membranes may also be affected by a charge-based phenomenon known as pH trapping, which depends on the drug’s acid dissociation constant (pKa) and the pH gradient across the membrane. For weakly acidic drugs, such as phenobarbital and aspirin, the protonated, electrically neutral, form of the drug is predominant in the highly acidic environment of the stomach. The uncharged form of the drug can pass through the lipid bilayers of the gastric mucosa, speeding the drug’s absorption (Fig. 3-2). The weakly acidic drug is then effectively trapped as it is deprotonated to its electrically charged form in the more basic environment of the plasma. In quantitative terms, the pKa of a drug represents the pH value at which one-half of the drug is present in its ionic form. The Henderson–Hasselbalch Equation describes the relationship between the pKa of an acidic or basic drug A and the pH of the biological medium containing the drug: pKa  pH  logg

[HA] [A]

Equation 3-2

where HA is the protonated form of drug A. For example, consider the hypothetical case of a weakly acidic drug with

[1,000]

[1]

HA

A-

+

H+

a pKa of 4. In the stomach, which has a pH of approximately 1, Equation 3-2 becomes: pKadrug  pHstomach  log

[HA] , [A]

which simplifies to: 3  log

[HA] , [A]

and finally: 1,000 

[HA] . [A]

The protonated form of the drug is present at 1,000 times the concentration of the deprotonated form, and 99.9% of the drug is in the neutral form. Conversely, in the plasma, where the pH is approximately 7.4, more than 99.9% of the drug is in the deprotonated form (see Fig. 3-2).

Central Nervous System The central nervous system (CNS) presents special challenges to pharmacologic therapy. Unlike most other anatomic regions, the CNS is particularly well insulated from foreign substances. The blood–brain barrier uses specialized tight junctions to prevent the passive diffusion of most drugs from the systemic to the cerebral circulation. Therefore, drugs designed to act in the CNS must either be sufficiently small and hydrophobic to traverse biological membranes easily or use existing transport proteins in the blood–brain barrier to penetrate central structures. Hydrophilic drugs that fail to target facilitated or active transport proteins in the blood–brain barrier cannot penetrate the CNS. The blood–brain barrier can be bypassed using intrathecal drug infusion, in which drugs are delivered directly into the cerebrospinal fluid (CSF). Although this approach can be used to treat infectious or carcinomatous meningitis, the intrathecal route is impractical for drugs that must be taken regularly by a patient.

Stomach pH~1

 ABSORPTION Gastric Mucosal Barrier

Plasma pH~7

HA [1,000]

A-

+

H+

[1,000,000]

FIGURE 3-2. pH trapping across lipid bilayers. In the example shown, consider a hypothetical drug with pKa ⫽ 4. Although this drug is a weak acid, in the highly acidic environment of the stomach, it is largely protonated. If the stomach pH is approximately 1, then for every 1,001 molecules of drug, 1,000 molecules are protonated (and neutral) and only 1 is deprotonated (and negatively charged). The protonated, neutral form of the drug is able to diffuse across the gastric mucosal barrier into the blood. Because the blood plasma has a pH of approximately 7 (it is actually 7.4), and the drug has a pKa of 4, the vast majority of drug now exists in the deprotonated (negatively charged) form: for every 1,001 molecules of drug, only 1 molecule is protonated (and neutral), while 1,000 molecules are deprotonated (and negatively charged). The negatively charged form of the drug is no longer able to diffuse across the lipid bilayers of the gastric mucosa, and the drug is effectively trapped in the plasma.

The human body presents formidable obstacles to invasion by micro-organisms. The integument has a keratinized outer layer and defensins in the epithelium. Mucous membranes are protected by mucociliary clearance in the trachea, lysozyme secretion from lacrimal ducts, acid in the stomach, and base in the duodenum. These nonspecific defense mechanisms present barriers to drug absorption and may limit the drug’s bioavailability at target organs. Bioavailability, or the fraction of administered drug that reaches the systemic circulation, may depend on the route by which the drug is administered, the chemical form of the drug, and a number of patient-specific factors, such as gastrointestinal and hepatic transporters and enzymes. Bioavailability is defined quantitatively as: Quantity of drug reaching systemic circulation Bioavailability  Quantity of drug administered

Equation 3-3

Plasma drug concentration

Oral, subcutaneous, or intramuscular: 100% bioavailability

Intravenous

Oral, subcutaneous, or intramuscular: 50% bioavailability

Time

Plasma drug concentration

A B

C

Time

CHAPTER 3 / Pharmacokinetics 33

accounted for in designing dosing regimens that achieve therapeutic drug levels.

A

Site of pharmacologic action

Volume of Distribution

Vascular space

The volume of distribution (Vd) describes the extent to which a drug partitions between the plasma and tissue compartments. In quantitative terms, Vd represents the fluid volume that would be required to contain the total amount of absorbed drug in the body at a concentration equivalent to that in the plasma at steady state: Dose Vd  [Drug]plasma

Clearing organ

Equation 3-4

The volume of distribution is an extrapolated volume based on the concentration of drug in the plasma, not a physical volume. Thus, Vd is low for drugs that are retained primarily within the vascular compartment and high for drugs that are highly distributed into muscle, adipose, and other nonvascular compartments. For very highly distributed drugs, the volume of distribution is often much greater than the volume of total body water, reflecting the low concentration of drug in the vascular compartment at steady state. A number of drugs have very large volumes of distribution; examples include amiodarone (4,620 liters [L] for a 70-kg person), azithromycin (2,170 L), chloroquine (9,240 L), and digoxin (645 L), among others. The capacity of the blood and the various organs and tissues to take up and retain a drug depends on both the volume (mass) of the tissue and the density of specific and nonspecific binding sites for the drug within that tissue. A drug that is taken up in large quantities by tissues such as adipose and muscle will be largely removed from the circulation at steady state. In many cases, these tissues must be saturated before plasma levels of such drugs can increase sufficiently to affect the drug’s target organ. Thus, for drugs of equal potency, a drug that is more highly distributed among body tissues generally requires a higher initial dose to establish a therapeutic plasma concentration than does a drug that is less highly distributed. Plasma Protein Binding The capacity of muscle and adipose tissue to bind a drug increases the tendency of the drug to diffuse from the blood into nonvascular compartments, but this tendency can be counteracted to some extent by plasma protein binding of the drug. Albumin is the most abundant plasma protein (⬃4 g/dL) and is the protein responsible for most drug binding. Many drugs bind with low affinity to albumin through both hydrophobic and electrostatic forces. Plasma protein binding tends to reduce the availability of a drug for diffusion or transport into the drug’s target organ because, in general, only the free or unbound form of the drug is capable of diffusion across membranes (Fig. 3-5). Plasma protein binding may also reduce the transport of drugs into nonvascular compartments such as adipose tissue and muscle. Because a highly proteinbound drug tends to remain within the vasculature, such a drug often has a relatively low volume of distribution (typically, 7 to 8 L for a 70-kg person). Theoretically, plasma protein binding could be important as a mechanism for some drug–drug interactions. Coadministration of two or more drugs that bind to plasma protein could

B

Site of pharmacologic action Vascular space

Clearing organ

Drug A

Drug A bound to albumin

Drug B

Drug B bound to albumin

Albumin

FIGURE 3-5.

Protein binding and drug trapping. A drug that is bound to albumin or other plasma proteins cannot diffuse from the vascular space into surrounding tissues. A. Drugs that do not bind plasma proteins appreciably (shown here as Drug A) diffuse readily into tissues. This results in both a high level of binding to the site of pharmacologic action (usually receptors) and a high rate of elimination (represented by flux through a clearing organ). Examples of such drugs include acetaminophen, acyclovir, nicotine, and ranitidine. B. In contrast, for drugs that exhibit high levels of binding to plasma proteins (shown here as Drug B), a higher total plasma drug concentration is required to ensure an adequate concentration of free (unbound) drug in the circulation. Otherwise, only a small fraction of the drug can diffuse into the extravascular space and only a small percentage of the receptors are occupied. Examples of such drugs include amiodarone, fluoxetine, naproxen, and warfarin. It should be emphasized that plasma protein binding is only one of many variables that determine drug distribution. Drug molecule size, lipophilicity, and rate of metabolism are other important parameters that must be taken into account when considering the pharmacokinetics of a particular drug.

result in a higher-than-expected plasma concentration of the free form of either or both drugs as the coadministered drugs compete for the same binding sites. The increased free drug concentration could potentially cause increased therapeutic and/or toxic effects of the drug. In such cases, the dosing regimen of one or both of the drugs would need to be adjusted to

34 Fundamental Principles of Pharmacology

keep the free drug concentration in the therapeutic range. In practice, however, it has been difficult to demonstrate clinically significant drug–drug interactions caused by competitive binding of drugs to plasma proteins, possibly due to the increased clearance of the free drugs as they are displaced from their plasma protein binding sites (see below).

Modeling the Kinetics and Thermodynamics of Drug Distribution

Plasma drug concentration

Most drugs are distributed rapidly from the systemic circulation (intravascular compartment) to other compartments in the body. This distribution phase results in a sharp decrease in the plasma drug concentration shortly after intravenous administration of a drug bolus. Even after the drug equilibrates among its tissue reservoirs, the plasma drug concentration continues to decline because of drug elimination from the body. However, the plasma drug concentration decreases more slowly during the elimination phase, in part due to a “reservoir” of drug in the tissues that can diffuse back into the blood to replace the drug that has been eliminated (Figs. 3-6 and 3-7). The tendency for a drug to be taken up by adipose and muscle tissue during the distribution phase determines a set of dynamic equilibria among drug concentrations in the various body compartments. As shown in Figure 3-8, the rapid decline of plasma drug concentration after administration of an intravenous bolus of drug can be approximated by using a four-compartment model consisting of the blood and vessel-rich, muscle-rich, and adipose-rich tissues. The vessel-rich group is the first extravascular compartment in which the concentration of drug increases, because the high blood flow received by this group kinetically favors drug entry into this compartment. However, the muscle-rich group and adipose-rich group often have a higher capacity for taking up drug than the vessel-rich group, with the

Distribution phase

adipose-rich group accumulating the greatest amount of drug at the slowest rate. The capacity of a compartment for a drug and the rate of blood flow to the compartment also affect the rate at which the drug exits from the compartment. Drugs tend to exit first from the vessel-rich group, followed by the muscle group and then the adipose group. A complex and dynamic pattern of changing blood concentrations may develop, and the pattern is specific for each drug. The pattern may also be patient-specific, depending on factors such as the size, age, and fitness level of the patient. For example, an older patient typically has less skeletal muscle mass than a younger patient, decreasing the contribution of muscle uptake to changes in the plasma concentration of drug. An opposite effect may be seen in an elite athlete, who would be expected to have both greater muscle mass and greater proportional muscle blood flow. As a third example, an obese person typically exhibits greater capacity for drug uptake into adipose tissue.

 METABOLISM A number of organs are capable of metabolizing drugs to some extent, using enzymatic reactions that are discussed in Chapter 4, Drug Metabolism. The kidneys, gastrointestinal tract, lungs, skin, and other organs all contribute to systemic drug metabolism. However, the liver contains the greatest diversity and quantity of metabolic enzymes, and the majority of drug metabolism occurs there. The ability of the liver to modify drugs depends on the amount of drug that enters the hepatocytes. Highly hydrophobic drugs can generally enter cells readily (including liver cells), and the liver preferentially metabolizes hydrophobic drugs. However, the liver contains a multitude of transporters in the human solute-linked carrier (SLC) superfamily that allow entry of some hydrophilic drugs into hepatocytes as well. Hepatic enzymes chemically modify a variety of substituents on drug molecules, thereby either rendering the drugs inactive or facilitating their elimination. These modifications are collectively referred to as biotransformation. Biotransformation reactions are classified into two types, termed oxidation/reduction reactions and conjugation/hydrolysis reactions. (Although biotransformation reactions are often called phase I and phase II reactions, in this book, we typically use the more precise terms oxidation/reduction and conjugation/hydrolysis; see Chapter 4.)

Oxidation/Reduction Reactions Elimination phase

Time

Oxidation/reduction reactions modify the chemical structure of a drug; typically, a polar group is added or uncovered. The most common pathway, the microsomal cytochrome P450 enzyme system in the liver, mediates a large number of oxidative reactions. Some drugs may be administered in inactive (prodrug) form and are altered metabolically by oxidation/reduction reactions to the active (drug) form in the liver. This prodrug strategy can facilitate oral bioavailability, decrease gastrointestinal toxicity, and/or prolong the elimination half-life of a drug.

FIGURE 3-6.

Drug distribution and elimination after intravenous administration. Immediately after intravenous administration of a drug, the plasma drug concentration declines rapidly as the drug distributes from the vascular compartment to other body compartments. This rapid decline is followed by a slower decline as the drug is metabolized and excreted from the body. Both drug distribution and elimination display first-order kinetics, as demonstrated by linear kinetics on a semilogarithmic plot.

Conjugation/Hydrolysis Reactions Conjugation/hydrolysis reactions hydrolyze a drug or conjugate a drug to a large, polar molecule in order to inactivate the drug or, more commonly, to enhance the drug’s solubility and excretion in the urine or bile. Occasionally,

Plasma drug concentration

CHAPTER 3 / Pharmacokinetics 35

A

Blood

Time

Plasma drug concentration

Extravascular volume

B

Blood

Plasma drug concentration

Time

C

Extravascular volume

Blood

Time

FIGURE 3-7.

Schematic model of drug distribution and elimination. A two-compartment pharmacokinetic model can be used to describe drug distribution and elimination after administration of a single intravenous dose. The drug concentration rises rapidly as the drug is added to the first compartment. A. In the absence of elimination, the initial rise in drug concentration is followed by a rapid decline to a new plateau as the drug equilibrates (distributes) between the two compartments. B. If the distribution of the drug is confined to the blood volume, then the plasma drug concentration declines more slowly as the drug is eliminated from the body. In both cases, as the concentration of drug in the plasma decreases, the forces driving (A) drug distribution and (B) elimination decrease, and the absolute amount of drug distributed or eliminated per unit time decreases. Therefore, the kinetics of both distribution and elimination appear as straight lines on a semilogarithmic plot; this is the definition of first-order kinetics. Note that the half-time for drug elimination is generally longer than the half-time for drug distribution. C. When drug distribution and elimination are occurring simultaneously, the decline of plasma drug concentration with time is represented by the sum of the two processes. Note that the curve in (C) is the sum of the two first-order processes shown in (A) and (B). In the schematics on the left of the figure, the volume in the “Blood” compartment represents plasma drug concentration, the volume in the “Extravascular volume” compartment represents tissue drug concentration, the dropper above the “Blood” compartment represents absorption of drug into the systemic circulation, and the drops below the “Blood” compartment represent elimination of drug by metabolism and excretion.

hydrolysis or conjugation can result in the metabolic activation of prodrugs. The most commonly added groups include glucuronate, sulfate, glutathione, and acetate. As described in more detail in the next chapter, the effects of oxidation/reduction and conjugation/hydrolysis reactions on a particular drug also depend on the presence of other drugs that are being taken concomitantly by the patient. Certain classes of drugs, such as barbiturates, are powerful inducers of enzymes that mediate oxidation/reduction reactions; other drugs are capable of inhibiting these enzymes. An

understanding of these drug–drug interactions is an essential prerequisite to the appropriate dosing of drug combinations. Physicians and researchers have begun to elucidate the important role of genetic differences among individuals in the various transporters and enzymes responsible for drug absorption, distribution, excretion, and especially metabolism. For example, an individual’s complement of cytochrome P450 enzymes in the liver determines the rate and extent to which that individual can metabolize numerous therapeutic agents. This topic is discussed in detail in Chapter 6, Pharmacogenomics.

36 Fundamental Principles of Pharmacology

Fraction of dose in compartment

1.0 Blood

Muscle

0.8 VRG

0.6 Adipose

0.4

0.2

0.1 0.1

1.0

10

100

1000

Time (minutes)

FIGURE 3-8.

Four-compartment model of drug distribution. After administration of an intravenous bolus, the drug is delivered to various tissues via the systemic circulation. The fraction of the administered dose is initially highest in the vascular compartment (blood), but the blood fraction subsequently falls rapidly as the drug is distributed to the different tissue compartments. The most vessel-rich tissues (i.e., the tissues that are supplied by the highest fraction of the cardiac output) are generally the first to accumulate drug. However, the tissue compartments also vary in their capacity for taking up drug. Because the mass of the muscle group is larger than that of the vessel-rich group (VRG), the muscle group has a larger uptake capacity. But because the muscles are less well perfused than the vesselrich group, this effect is manifested only after the drug has begun to distribute to the VRG. The most poorly perfused group is the adipose-rich group, but this group has the highest capacity to accumulate drug. The peak level of drug in the adipose group is not as high as that in the muscle-rich group, because a significant amount of drug has been eliminated by metabolism or excretion before the adipose group begins to accumulate drug. After the administration of drug has been completed, the reverse pattern is seen—the drug leaves first from the vessel-rich group and then from the muscle and adipose groups, respectively. The drug in this example is thiopental, a barbiturate used to induce general anesthesia.

filtration rate, and drug binding to plasma protein all affect the amount of drug that enters the tubule at the glomerulus. Enhancing blood flow, increasing glomerular filtration rate, and decreasing plasma protein binding cause a drug to be excreted more rapidly. Renal excretion plays the primary role in the clearance of many drugs; examples include vancomycin, atenolol, and ampicillin. Drugs such as these can accumulate to toxic levels in patients with compromised renal function and in elderly patients (who often manifest some degree of renal compromise). Urinary drug concentration rises in the proximal tubule because of passive diffusion of uncharged drug molecules, facilitated diffusion of charged or uncharged molecules, and active secretion of anionic and cationic molecules from the blood into the urinary space. The secretory mechanisms are generally not specific for the drugs; rather, drug secretion takes advantage of molecular similarities between the drug and naturally occurring substances such as organic anions (transported by OAT family proteins) and cations (transported by OCT family proteins). Penicillin is an example of a drug that is eliminated largely by active transport in the proximal tubule. The extent of plasma protein binding appears to have a relatively small effect on drug secretion into the proximal tubule, because the highly efficient transporters that mediate active tubular secretion rapidly remove free (unbound) drug from the peritubular capillaries and thereby alter the equilibrium between free and protein-bound drug at these sites.

Proximal tubule

Peritubular capillary Tubular Secretion

Glomerular Filtration

1

2

Afferent arteriole

 EXCRETION Oxidation/reduction and conjugation/hydrolysis reactions enhance the hydrophilicity of a hydrophobic drug and its metabolites, enabling such drugs to be excreted along a final common pathway with drugs that are intrinsically hydrophilic. Most drugs and drug metabolites are eliminated from the body through renal and biliary excretion. Renal excretion is the most common mechanism of drug excretion, and it relies on the hydrophilic character of a drug or metabolite. Only a relatively small number of drugs are excreted primarily in the bile or through respiratory and dermal routes. Many orally administered drugs are incompletely absorbed from the upper gastrointestinal tract, and residual drug is eliminated by fecal excretion.

Renal Excretion Renal blood flow comprises about 25% of total systemic blood flow, ensuring that the kidneys are continuously exposed to any drug found in the blood. The rate of drug elimination through the kidneys depends on the balance of drug filtration, secretion, and reabsorption rates (Fig. 3-9). The afferent arteriole introduces both free (unbound) drug and plasma protein-bound drug into the glomerulus. Typically, however, only the free drug form is filtered into the renal tubule. Therefore, renal blood flow, glomerular

Drug in blood

3 Tubular Reabsorption

Efferent arteriole

4

Urine

FIGURE 3-9. Drug filtration, secretion, and reabsorption in the kidney. Drugs may be (1) filtered at the renal glomerulus, (2) secreted into the proximal tubule, (3) reabsorbed from the tubular lumen and transported back into the blood, and (4) excreted in the urine. The relative balance of filtration, secretion, and reabsorption rates determines the kinetics of drug elimination by the kidney. Enhancing blood flow, increasing glomerular filtration rate, and decreasing plasma protein binding all cause a drug to be excreted more rapidly, because all these changes result in increased filtration of drug at the glomerulus. Some drugs, such as penicillin, are actively secreted into the proximal tubule. Although reabsorption can decrease the elimination rate of a drug, many drugs exhibit pH trapping in the distal tubule and are therefore efficiently excreted in the urine. For drugs that are dependent on the kidney for elimination, compromised renal function can result in higher plasma drug concentrations, and the dose and frequency of drug administration must be altered accordingly.

CHAPTER 3 / Pharmacokinetics 37

The urinary concentration of a drug may fall as the drug is reabsorbed in the proximal and distal tubules. Reabsorption is limited primarily by pH trapping, as described above. The renal tubular fluid is typically acidic in and beyond the proximal tubule, which tends to favor trapping of the ionic form of weak bases. Because this region of the tubule contains transporter proteins that are different from those in preceding segments of the nephron, ionic drug forms resist facilitated diffusional reabsorption, and their excretion is thereby enhanced. Drug reabsorption in the tubule can be enhanced or inhibited by chemical adjustment of the urinary pH. Changing the rate of urine flow through the tubules can also modify the rate of drug reabsorption. An increased rate of urine output tends to dilute the drug concentration in the tubule and to decrease the amount of time during which facilitated diffusion can occur; both of these effects tend to decrease drug reabsorption. For example, aspirin is a weak acid that is excreted by the kidney. Aspirin overdose is treated by administering sodium bicarbonate to alkalinize the urine (and thus trap aspirin in the tubule) and by increasing the urine flow rate (and thus dilute the tubular concentration of the drug). Both of these clinical maneuvers result in faster elimination of the drug.

Biliary Excretion Drug reabsorption also plays an important role in biliary excretion. Some drugs are secreted from the liver into the bile by members of the ATP binding cassette (ABC) superfamily of transporters, which includes seven families of proteins such as the multidrug resistance (MDR) family. Because the bile duct enters the gastrointestinal tract in the duodenum, such drugs must pass through the length of the small and large intestine before being eliminated. In many cases, these drugs undergo enterohepatic circulation, in which they are reabsorbed in the small intestine and subsequently retained in the portal and then the systemic circulation. Drugs such as steroid hormones, digoxin, and some cancer chemotherapeutic agents are largely excreted in the bile.

 CLINICAL APPLICATIONS OF PHARMACOKINETICS The dynamic interactions among drug absorption, distribution, metabolism, and excretion determine the plasma concentration of a drug and dictate the ability of the drug to reach its target organ in an effective concentration. Often, the desired duration of drug therapy exceeds that achievable by a single dose, and multiple doses are needed to provide a relatively constant plasma concentration of drug within the limits of efficacy and toxicity. The results of clinical trials of drugs under development, as well as clinical experience using U.S. Food and Drug Administration (FDA)-approved drugs, suggest target plasma levels for a drug in the average patient. However, pharmacokinetic and other differences among patients (such as disease status and pharmacogenomic profile) must also be considered in designing a dosing regimen for a drug or drug combination in the individual patient.

Clearance The clearance of a drug is the pharmacokinetic parameter that most significantly limits the time course of action of the drug at its molecular, cellular, and organ targets. Clearance

can be conceptualized in two complementary ways. First, it is defined as the rate of elimination of the drug from the body relative to the concentration of the drug in plasma. Alternatively, clearance is the rate at which plasma would have to be cleared of the drug to account for the observed kinetics of change of the total amount of drug in the body, assuming that all the drug in the body is present at the same concentration as that in the plasma. Therefore, clearance is expressed in units of volume/time, as follows: Clearance 

Metabolism  Excretion [Drug]plasma

Equation 3-5

where metabolism and excretion are expressed as rates (amount/time). Although metabolism and excretion are distinct physiologic processes, the pharmacologic endpoint is equivalent—a reduction in circulating levels of active drug. As such, metabolism and excretion are often referred to collectively as clearance mechanisms, and the principles of clearance can be applied to both: Clearancetotal  Clearancerenal  Clearancehepatic  ClearanceOther

Equation 3-6

Metabolism and Excretion Kinetics The rate of drug metabolism and excretion by an organ is limited by the rate of blood flow to that organ. The majority of drugs demonstrate first-order kinetics when used in standard therapeutic doses; that is, the amount of drug that is metabolized or excreted in a given unit of time is directly proportional to the concentration of drug in the systemic circulation at that time. Because the clearance mechanisms for most drugs are not saturated under ordinary circumstances, increases in plasma drug concentration are matched by increases in the rate of drug metabolism and excretion (see Equation 3-5). The first-order elimination rate (where elimination includes both metabolism and excretion) follows Michaelis–Menten kinetics: E

Vmax  C Km  C

Equation 3-7

where Vmax is the maximum rate of drug elimination, Km is the drug concentration at which the rate of elimination is 1/2 Vmax, C is the concentration of drug in the plasma, and E is the elimination rate (Fig. 3-10). Because elimination is usually a first-order process, a semilogarithmic plot of plasma drug concentration versus time typically shows a straight line during the elimination phase (see Fig. 3-6). A small number of drugs (e.g., phenytoin) and recreational substances (e.g., ethanol) demonstrate saturation kinetics, in which the clearance mechanisms become saturated at or near the therapeutic concentration of the drug. Once saturation occurs, the clearance rate fails to increase with increasing plasma drug concentrations (zero-order kinetics). This can result in dangerously elevated plasma concentrations of the drug that can cause toxic (or even lethal) effects.

Drug elimination rate

Vmax

Vmax 2

Km

Plasma drug concentration

Extraction 

t1/2 

Cin  Cout Cin

0.693  Vd Clearance

Equation 3-8

Equation 3-9

40 Fundamental Principles of Pharmacology

see Chapter 2.) In contrast, a highly distributed drug—as evidenced by a high volume of distribution—necessitates higher drug dosing. The elimination rate of a drug influences its halflife and thereby determines the frequency of dosing required to maintain therapeutic plasma drug levels. In general, therapeutic dosing of a drug seeks to maintain the peak (highest) plasma drug concentration below the toxic concentration, and the trough (lowest) drug concentration above the minimally effective level (Fig. 3-11). This can be accomplished most efficiently using continuous drug delivery by intravenous (continuous infusion), subcutaneous (continuous pump or implant), oral (sustained-release tablet), and other routes of administration, as described in more detail in Chapter 54, Drug Delivery Modalities. In many cases, however, the dosing regimen must also consider patient convenience. Frequent small doses (usually oral) can be administered to achieve minimal variation in steady-state plasma drug concentration, but this strategy subjects the patient to the inconvenience of frequent drug administration. Less frequent dosing requires the use of higher doses and

leads to greater fluctuations in peak and trough drug levels; this type of regimen is more convenient for the patient but also more likely to cause problems due to excessive (toxic) or insufficient (subtherapeutic) drug levels (Fig. 3-12). Optimal dosing regimens typically maintain the steadystate plasma drug concentration within the therapeutic window for that drug. Because steady state is reached when the rate of drug input is equal to its output, the steady-state drug concentration is affected by drug bioavailability, clearance, dose, and dosing interval (the frequency of administration): Csteady state 

Plasma drug concentration

Plasma drug concentration

B Therapeutic Dosing with Loading Dose

Toxic range

1.5 1.0

Therapeutic range

0.5 Subtherapeutic range 0 0

3

6

9

Plasma drug concentration

Plasma drug concentration

2.1 1.4 Therapeutic range 0.7 Subtherapeutic range

1st dose

Therapeutic range

0.5 Subtherapeutic range

0 3

6

9

12

Days

D Subtherapeutic Dosing Toxic range

3

1.0

1st dose

2.8

0

1.5

0

C Toxic Dosing

0

Toxic range

2.0

12

Days

1st dose

Equation 3-10

where C is the plasma concentration of the drug. Immediately following the initiation of drug therapy, the rate of drug entry into the body (kin) is much greater than the elimination rate (kout); therefore, the drug concentration in the blood increases. Assuming that elimination follows

A Therapeutic Dosing

2.0

Bioavailability  Dose Intervaldosing  Clearance

6

Days

9

12

Toxic range

2.0 1.5

Therapeutic range

1.0 0.5

Subtherapeutic range 0

0

1st dose

3

6

9

12

Days

FIGURE 3-11. Therapeutic, subtherapeutic, and toxic drug dosing. From a clinical perspective, drug concentrations in plasma can be divided into subtherapeutic, therapeutic, and toxic ranges. The goal of most drug-dosing regimens is to maintain the drug at concentrations within the therapeutic range (referred to as the “therapeutic window”). A. The first several doses of a drug are typically subtherapeutic as the drug equilibrates to its steady-state concentration (approximately four elimination half-lives are required to achieve steady state). Appropriate drug dosing and dosing frequency result in steady-state drug levels that are therapeutic, and the maximal and minimal concentrations of the drug remain within the therapeutic window. B. If the initial (loading) dose is larger than the maintenance dose, the drug reaches therapeutic concentrations more rapidly. The magnitude of the loading dose is determined by the volume of distribution of the drug. C. Excessive maintenance doses or dosing frequency result in drug accumulation and toxicity. D. Insufficient maintenance doses or dosing frequency result in subtherapeutic steady-state drug concentrations. In all four panels, the drug is administered once daily, distributed very rapidly to the various body compartments, and eliminated with first-order kinetics.

Continuous infusion Infrequent large doses

Plasma drug concentration (mg/L)

Frequent small doses

Csteady state 

Therapeutic range 8

0.93  5 mg  1.01 mg/L 24 h  0.192 L/h

6

4

2

0 0

Time

Doseloading  Vd  Csteady state

Equation 3-11

Doseloading  77 L  3.5 mg/L  269.5 mg

Dosemaintenance  Clearance  Csteady state

Equation 3-12

Plasma drug concentration

Toxic range

Initial concentration

=

Therapeutic range

Steady-state concentration

=

Loading dose Volume of distribution Fraction absorbed × Maintenance dose Dosing interval × Clearance

Subtherapeutic range

Time Elimination half -life

Dosemaintenance  0.192 L/h  1.01 mg/La  0.194 mg/h  4.65 mg/day

=

0.693 × Volume of distribution Clearance

4 Drug Metabolism Cullen Taniguchi and F. Peter Guengerich

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 43-44 SITES OF DRUG METABOLISM. . . . . . . . . . . . . . . . . . . . . . . . 43 PATHWAYS OF DRUG METABOLISM . . . . . . . . . . . . . . . . . . . 44 Oxidation/Reduction Reactions. . . . . . . . . . . . . . . . . . . . . . 47 Conjugation/Hydrolysis Reactions . . . . . . . . . . . . . . . . . . . 47 Drug Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Induction and Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Active and Toxic Metabolites . . . . . . . . . . . . . . . . . . . . . . . 50

INDIVIDUAL FACTORS AFFECTING DRUG METABOLISM . . . . 50 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Race and Ethnicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Age and Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Diet and Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Metabolic Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . 54 Diseases Affecting Drug Metabolism . . . . . . . . . . . . . . . . . 54 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 54 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

 INTRODUCTION

chapter we use “oxidation/reduction” and “conjugation/ hydrolysis” to describe these processes more accurately. The chapter concludes with a discussion of the factors that can lead to differences in drug metabolism among individuals.

Our tissues are exposed on a daily basis to xenobiotics— foreign substances that are not naturally found in the body. Most drugs are xenobiotics that are used to modulate bodily functions for therapeutic ends. Drugs and other environmental chemicals that enter the body are modified by a vast array of enzymes. The biochemical transformations performed by these enzymes can alter the compound to render it beneficial, harmful, or simply ineffective. The processes by which biochemical reactions alter drugs within the body are collectively called drug metabolism or drug biotransformation. The previous chapter introduced the importance of renal clearance in the pharmacokinetics of drugs. Although the biochemical reactions that alter drugs to renally excretable forms are an essential part of drug metabolism, drug metabolism encompasses more than this one function. Drug biotransformation can alter drugs in four important ways: • An active drug may be converted to an inactive drug. • An active drug may be converted to an active or toxic metabolite. • An inactive prodrug may be converted to an active drug. • An unexcretable drug may be converted to an excretable metabolite (e.g., to enhance renal or biliary clearance). This chapter presents the major processes of drug metabolism. Following the case is an overview of the sites of drug metabolism, focusing principally on the liver. The two major types of biotransformation are then discussed; these are often termed phase I and phase II reactions, although the terminology is imprecise and it incorrectly implies a temporal order of the reactions. (In addition, “phase III” is sometimes used to describe the process of drug transport, which is even more confusing.) In this

 SITES OF DRUG METABOLISM The liver is the main organ of drug metabolism. This fact figures prominently in the phenomenon known as the firstpass effect. Orally administered drugs are often absorbed in the gastrointestinal (GI) tract and transported directly to the liver via the portal circulation (Fig. 4-1). In this manner, the liver has the opportunity to metabolize drugs before they reach the systemic circulation and, therefore, before they reach their target organs. The first-pass effect must be taken into account when designing dosing regimens because, if hepatic metabolism is extensive, the amount of drug that reaches the target tissue is much less than the amount (dose) that is administered orally (see Chapter 3, Pharmacokinetics). Certain drugs are inactivated so efficiently upon their first pass through the liver that they cannot be administered orally and must be given parenterally. One such drug is the antiarrhythmic lidocaine, which has a bioavailability of only 3% when taken orally. Although the liver is quantitatively the most important organ in metabolizing drugs, every tissue in the body is capable of drug metabolism to some degree. Particularly active sites include the skin, the lungs, the gastrointestinal tract, and the kidneys. The gastrointestinal tract deserves special mention because this organ, like the liver, can contribute to the first-pass effect by metabolizing orally administered drugs before they reach the systemic circulation. 43

PO

Subcutaneous

Transdermal

GI

IV

Portal vein Liver

Contains first-pass metabolites Systemic circulation

Other organs

OH R R

OH R O HO R

R

O

R1

N H

+

R1

R2

NH2

H

R2 O

R2

O R

+

OH

H

R

R2

O

S

S

R1

R

R2

R1

R2 H N

NH2

R

OH

S R1

O R2

R1

R2

O R1

R1

R2

R2

O

O R

OH

R

R

H

OH

O

R

+

NH2

R

H

R

OH

OH R

NH3

+ CO2

O

O2N

H2N R

R

R

R

X

O R1

H

OH

R2

R1

R2

O

O

+

R2 R1

O

R1

O R1

R2

HO

OH

O

+

R2

N H

R1

R2

H2N

OH

OH

O R1

R1

R2

R2 OH

COOH

COOH OH

+

OH

OH

O

R

OH

OH

OH

OH

O

O

+

OH

R O

O

R

O

UDP

CoA

R

S

O

O

O

O

+ R

OH

H N

R

H2N

OH

OH O

R

NH2

+

HO3S

OH

+

HO3S

R

H N

O ADP

R

ADP

R

SO3H

O

O

SO3H

O

+ HOOC

X R

NH2 H N

N H

COOH O HS

O

NH2 H N

HOOC

N H

COOH O S R

HOOC R

H N O

S

H N R1

N

R2

HO

R1

R

O

HO

HO SH

R

R2

S R

R

CHAPTER 4 / Drug Metabolism 47

Oxidation/Reduction Reactions

Conjugation/Hydrolysis Reactions

Oxidation reactions involve membrane-associated enzymes expressed within the endoplasmic reticulum (ER) of hepatocytes and, to a lesser extent, of cells in other tissues. The enzymes that catalyze these phase I reactions are typically oxidases; the majority of these enzymes are heme protein mono-oxygenases of the cytochrome P450 class. Cytochrome P450 enzymes (sometimes abbreviated CYP) are also known as microsomal mixed-function oxidases and are involved in the metabolism of approximately 75% of all drugs used today. (The term P450 refers to the 450-nm absorption peak characteristic of these heme proteins when they bind carbon monoxide.) The net result of a cytochrome P450-dependent oxidation reaction is:

Conjugation and hydrolysis reactions provide a second set of mechanisms for modifying compounds for excretion (Fig. 4-3). Although hydrolysis of ester- and amidecontaining drugs is sometimes included among the phase I reactions (in the older terminology), the biochemistry of hydrolysis is more closely related to conjugation than to oxidation/reduction. Substrates for these reactions include both metabolites of oxidation reactions (e.g., epoxides) and compounds that already contain chemical groups appropriate for conjugation, such as hydroxyl (-OH), amine (-NH2), or carboxyl (-COOH) moieties. These substrates are coupled by transfer enzymes to endogenous metabolites (e.g., glucuronic acid and its derivatives, sulfuric acid, acetic acid, amino acids, and the tripeptide glutathione) in reactions that often involve high-energy intermediates (Table 4-2). The conjugation and hydrolysis enzymes are located in both the cytosol and the endoplasmic reticulum of hepatocytes (and other tissues). In most cases, the conjugation process makes the drug more polar. Virtually all of the conjugated products are pharmacologically inactive, with some important exceptions (e.g., morphine glucuronide). Some conjugation reactions are important clinically in the case of neonates, who have not yet fully developed the capacity to carry out this set of reactions. UDP-glucuronyl transferase (UDPGT) is responsible for conjugating bilirubin in the liver and facilitating its excretion. The developmental deficiency of this enzyme at birth puts infants at risk for neonatal jaundice, which results from increased serum levels of unconjugated bilirubin. Neonatal jaundice is a problem because neonates have not only underdeveloped activity of this enzyme but also an undeveloped blood–brain barrier. Unconjugated bilirubin is water-insoluble and very lipophilic; it binds readily to the unprotected neonatal brain and is capable of causing significant damage to the central nervous system. This pathologic condition is known as bilirubin encephalopathy or kernicterus. Neonatal hyperbilirubinemia (unconjugated) can be treated with phototherapy with 450-nm light, which converts circulating bilirubin to an isomer that is more rapidly excreted. Another effective treatment is the administration of small doses of the barbiturate phenobarbital, which powerfully up-regulates the expression of the enzyme UDPGT and thereby reduces serum levels of unconjugated bilirubin. This illustrates a recurring theme: understanding drug metabolism can help predict both adverse and potentially advantageous drug–drug interactions. It is important to note that conjugation and hydrolysis reactions do not necessarily constitute the last step of biotransformation. Since the conjugation of these highly polar moieties occurs intracellularly, they often require active transport across cellular membranes to be excreted (active transport of the parent drug can also occur). Moreover, some conjugation products may be subjected to further metabolism.

Drug  O2  NADPH  H Æ Drug-OH  H2O  NADP Equation 4-1 The reaction proceeds when the drug binds to the oxidized (Fe3⫹) cytochrome P450 to form a complex, which is then reduced in two sequential oxidation/reduction steps as outlined in Figure 4-2A. Nicotinamide adenine dinucleotide phosphate (NADPH) donates the electrons in both of these steps via a flavoprotein reductase. In the first step, the donated electron reduces the cytochrome P450–drug complex. In the second step, the electron reduces molecular oxygen to form an activated oxygen–cytochrome P450–drug complex. Finally, as the complex becomes more active through rearrangement, the reactive oxygen atom is transferred to the drug, resulting in the formation of the oxidized drug product and recycling oxidized cytochrome P450 in the process. The mechanism of these reactions is illustrated in Figure 4-2B. Most liver cytochrome P450 oxidases exhibit broad substrate specificity (Table 4-1). This is due in part to the activated oxygen of the complex, which is a powerful oxidizing agent that can react with a variety of substrates. The names of the cytochrome P450 enzymes are sometimes designated by “P450” followed by the number of the P450 enzyme family, capital letter of the subfamily, and an additional number to identify the specific enzyme (e.g., P450 3A4). Many of the P450 enzymes have partially overlapping specificities that together allow the liver to recognize and metabolize a wide array of xenobiotics. Together, P450-mediated reactions account for more than 95% of oxidative biotransformations. Other pathways may also oxidize lipophilic molecules. A pertinent example of a non-P450 oxidative pathway is the alcohol dehydrogenase pathway that oxidizes alcohols to their aldehyde derivatives as part of the overall process of excretion. These enzymes are the basis for the toxicity of methanol. Methanol is oxidized by alcohol dehydrogenase to formaldehyde, which does considerable damage to some tissues. The optic nerve is particularly sensitive to formaldehyde, and methanol toxicity can cause blindness. Another important non-P450 enzyme is monoamine oxidase (MAO). This enzyme is responsible for the oxidation of amine-containing endogenous compounds such as catecholamines and tyramine (see Chapter 10, Adrenergic Pharmacology) and some xenobiotics, including drugs.

Drug Transport Although many drugs are sufficiently lipophilic to cross cell membranes passively, it is now appreciated that many drugs need to be transported actively into cells. This fact has significant consequences for oral bioavailability (transport into enterocytes or active excretion into the intestinal lumen), hepatic metabolism (transport into hepatocytes for enzymatic metabolism and for excretion into bile), and renal clearance

48 Fundamental Principles of Pharmacology NADP+

NADPH

Flavoprotein (reduced)

Flavoprotein (oxidized)

A

2 +e-

P450-Fe2+

RH RH

P450-Fe3+

+e-

3

O2 P450-Fe2+

RH H2O

O 2-

1

4

P450-Fe3+ R-H

R-OH

(parent drug)

(oxidized drug)

R-OH

B

H

(oxidized drug)

H 0

H2O

R-H (parent drug)

H 2O

Fe3+

H2O

6

0

Heme

1 R-H

R-H Fe3+

Fe3+

Flavoprotein (reduced)

NADP+

Flavoprotein (oxidized)

NADPH

e-

H2O

5

2

2H+ 0

2-

0

R-H

R-H Fe3+

4

Fe2+

0-

0 0

R-H

0

R-H

3 e(from NADPH)

Fe2+

O2

Fe3+

FIGURE 4-2. Cytochrome P450-mediated drug oxidation. Many drug metabolism reactions involve a system of hepatic P450 microsomal enzymes that catalyze the oxidation of drugs. A. An overview of the reaction involves a set of oxidation/reduction steps in which an iron moiety in the P450 enzyme acts as an electron carrier to transfer electrons from NADPH to molecular oxygen. The reduced oxygen is then transferred to the drug, resulting in an additional -OH group on the now-oxidized drug (for this reason, P450 enzymes are sometimes referred to colloquially as “oxygen guns” or even “nature’s blowtorch”). The addition of the -OH group results in increased drug hydrophilicity and an increased rate of drug excretion. B. The detailed mechanism of the P450 reaction can be divided into six steps: (1) drug complexes with oxidized cytochrome P450; (2) NADPH donates an electron to the flavoprotein reductase, which reduces the P450-drug complex; (3 and 4) oxygen joins the complex, and NADPH donates another electron, creating the activated oxygen–P450 substrate complex; (5) iron is oxidized, with the loss of water; and (6) the oxidized drug product is formed. There are multiple P450 enzymes; each has a somewhat different specificity for substrates (such as drugs). Five of the human P450s (1A2, 2C9, 2C19, 2D6, and 3A4) account for approximately 95% of the oxidative metabolism of drugs.

CHAPTER 4 / Drug Metabolism 49

D-glucuronate

D

D-acetate Drug or phase I drug metabolite OH

D

D-glycine D-sulfate

NH2

Excretion

D-glutathione

D

D-methyl

FIGURE 4-3. Conjugation reactions. In these reactions, a drug (represented by D) or drug metabolite (represented by D-OH and D-NH2) is conjugated to an endogenous moiety. Glucuronic acid, a sugar, is the most common group that is conjugated to drugs, but conjugations of acetate, glycine, sulfate, glutathione, and methyl groups are also common. The addition of one of these moieties makes the resulting drug metabolite more hydrophilic and often enhances drug excretion. (Methylation, an important exception, does not increase drug hydrophilicity.) Transport mechanisms also play a major role in the elimination of drugs and their metabolites.

(transport into proximal tubular cells and excretion into the tubular lumen). Several important molecules mediate these processes. The multidrug resistance protein 1 (MDR1), or P-glycoprotein, which is a member of the ABC family of efflux transporters, actively transports compounds back into the intestinal lumen. This process limits the oral bioavailability of several important drugs, including digoxin and HIV-1 protease inhibitors. The metabolism of drugs from the portal circulation (i.e., the first-pass effect) often requires the transport of compounds into hepatocytes via the organic anion transporting polypeptide (OATP) and the organic cation transporter (OCT) family of proteins. These transporters are particularly relevant for the metabolism of several 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), which are used in the treatment of hypercholesterolemia. For example, metabolism of

A

the HMG-CoA reductase inhibitor pravastatin is dependent on the transporter OATP1B1, which transports the drug into hepatocytes. Drug uptake into hepatocytes via OATP1B1 is thought to be the rate-limiting step in the clearance of pravastatin. The uptake of pravastatin on its first pass through the liver also provides a potential advantage by keeping the drug out of the systemic circulation, from which it could be taken up by muscle cells and thereby cause toxic effects such as rhabdomyolysis. The organic anion transporter (OAT) family of transporters is responsible for renal secretion of many clinically important anionic drugs, such as ␤-lactam antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and antiviral nucleoside analogs.

Induction and Inhibition The use of phenobarbital to prevent neonatal jaundice demonstrates that drug metabolism can be influenced by the expression levels of drug-metabolizing enzymes. Although some P450 enzymes are constitutively active, others can be induced or inhibited by different compounds. Induction or inhibition can be incidental (a side effect of a drug) or deliberate (the desired effect of therapy). The primary mechanism of P450 enzyme induction is an increase in the expression of the enzyme chiefly through increased transcription, although augmented translation and decreased degradation can also have minor roles. The induction of P450 enzymes by a wide array of drugs reflects the biology of xenobiotic receptors that act as the body’s surveillance system to metabolize potentially toxic compounds. Drugs, environmental pollutants, industrial chemicals, and even foodstuffs can enter hepatocytes and bind to several different xenobiotic receptors, such as the pregnane X receptor (PXR), constitutively active/androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) (Fig. 4-4). These molecules

D

D

D

Extracellular

OH

Cytoplasm

D

D

A P450

D

D

D

D

C

I

P450

P450

P450 enzyme

FIGURE 4-4. Nucleus

Coactivator A

RxR PXR

P450 transcription

Conceptualization of P450 induction and inhibition. Drugs can both induce the expression and inhibit the activity of P450 enzymes. Some drugs can induce the synthesis of P450 enzymes (left panel). In this example, drug A activates the pregnane X receptor (PXR), which heterodimerizes with the retinoid X receptor (RXR) to form a complex with co-activators and initiate transcription of the P450 enzyme. Induction can also occur via the constitutively active/androstane receptor (CAR) or the aryl hydrocarbon receptor (AhR) (not shown). Drug D enters the cell and is hydroxylated by a P450 enzyme (right panel). The P450 enzyme can be inhibited by a second drug acting as a competitive inhibitor (drug C) or an irreversible inhibitor (drug I). The mechanism by which a drug inhibits P450 enzymes is not necessarily predictable from the drug’s chemical structure; the mechanism can only be determined experimentally. In addition, metabolites of drugs A, C, and I can play a role in enzyme induction and inhibition (not shown).

CHAPTER 4 / Drug Metabolism 55

Ho RH, Kim RB. Transporters and drug therapy: implications for drug disposition and disease. Clin Pharmacol Ther 2005;78:260–277. (Review of the crucial role played by drug transporters in drug metabolism.) Kliewer SA, Goodwin B, Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev 2002;23:687–702. (Review of PXR induction.) Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS. Cytochrome P450 polymorphisms and response to clopidogrel. N Engl J Med 2009;360:354– 362. (Example of P450 genetic polymorphisms and clinical efficacy of clopidogrel.) Wienkers L, Pearson P, eds. Handbook of drug metabolism. 2nd ed. New York: Marcel Dekker; 2009. (Collection of articles on aspects of drug metabolism.)

Wilke RA, Lin DW, Roden DW, Watkins PB, Folckhart D, Zineh I, Giacomini KM, Krauss RM. Identifying genetic risk factors for serious adverse reactions: current progress and challenges. Nat Rev Drug Discov 2007;6:904-916. (Review of current status of use of genetics for predicting adverse reactions.) Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med 2005;352:2211–2221. (An excellent basic review of the P450 system and drug–drug interactions.) Zhang D, Zhu M, Humphreys WG, eds. Drug metabolism in drug design and development: basic concepts and practice. Hoboken, NJ: John Wiley & Sons; 2007. (Drug metabolism as it pertains to development of new pharmaceuticals.) Zhou S, Gao Y, Jiang W, Huang M, Xu A, Paxton JW. Interactions of herbs with cytochrome P450. Drug Metab Rev 2003;35:35–98. (Review of P450 interactions with herbal medicines.)

5 Drug Toxicity Michael W. Conner, Catherine Dorian-Conner, Laura C. Green, Sarah R. Armstrong, Cullen Taniguchi, Armen H. Tashjian, Jr., and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . 56-57 MECHANISMS OF DRUG TOXICITY . . . . . . . . . . . . . . . . . . . . 57 On-Target Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Off-Target Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Idiosyncratic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 CONTEXTS OF DRUG TOXICITY . . . . . . . . . . . . . . . . . . . . . . . 61 Drug Overdose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Drug–Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Pharmacokinetic Drug–Drug Interactions . . . . . . . . . . . 61 Pharmacodynamic Drug–Drug Interactions . . . . . . . . . . 61 Drug–Herb Interactions . . . . . . . . . . . . . . . . . . . . . . . . . 61 Cellular Mechanisms of Toxicity: Apoptosis and Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Organ and Tissue Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . 62 Harmful Immune Responses and Immunotoxicity . . . . . . . 62 Drug-Induced Hepatotoxicity . . . . . . . . . . . . . . . . . . . . . 65 Drug-Induced Renal Toxicity . . . . . . . . . . . . . . . . . . . . . 66 Drug-Induced Neurotoxicity . . . . . . . . . . . . . . . . . . . . . 66 Drug-Induced Skeletal Muscle Toxicity . . . . . . . . . . . . . 66 Drug-Induced Cardiovascular Toxicity . . . . . . . . . . . . . . 67 Drug-Induced Pulmonary Toxicity . . . . . . . . . . . . . . . . . 67 Carcinogenesis Due to Drug Therapy . . . . . . . . . . . . . . 67 Teratogenesis Due to Drug Therapy . . . . . . . . . . . . . . . 67 PRINCIPLES FOR TREATING PATIENTS WITH DRUG-INDUCED TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 69 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

 INTRODUCTION

characteristics—such as polymorphisms of enzymes that detoxify harmful metabolites, comorbidities, or reduced functional reserves in key organs—render patients more or less capable of defending against toxicity. This is one reason why, all other things being equal, new medications should be initiated at the lowest doses likely to be therapeutic. Drug toxicity is critically important in drug development (see Chapter 49, Drug Discovery and Preclinical Development, and Chapter 50, Clinical Drug Evaluation and Regulatory Approval). Early in drug development, preclinical and clinical studies are used to evaluate compound potency, selectivity, pharmacokinetic and metabolic profiles, and toxicity. Prior to marketing, the regulatory agencies responsible for drug approval review the test data and decide whether the benefits of the drug outweigh its risks. Once a drug is marketed and many more patients are exposed, the appearance of unexpected types or frequencies of adverse effects may cause a re-evaluation of the drug, such that its use may be restricted to specific patient populations or withdrawn entirely (as in the cases, for example, of the nonsteroidal anti-inflammatory drug rofecoxib and the antidiabetic drug troglitazone). In this chapter, categories of drug toxicities that derive from inappropriate activation or inhibition of the intended drug target (on-target adverse effects) or unintended targets (off-target adverse effects) are discussed first. The phenotypic effects of these drug toxicities are then discussed at the physiologic, cellular, and molecular levels. General principles and specific examples are also illustrated in this

Like many medical interventions, the use of drugs for therapeutic benefit is subject to the law of unintended consequences. These consequences—termed side effects, adverse effects, or toxic effects—are a function of the mechanisms of drug action, the size of the drug dose, and the characteristics and health status of the patient. As such, the principles of pharmacology, presented in the preceding chapters, apply to drug toxicology as well. Many subsequent chapters contain Drug Summary Tables that list, among other properties, the specific adverse effects that can be caused by each drug. This chapter focuses on the mechanisms underlying these adverse effects. As a general matter, adverse effects range from those that are common and relatively benign to those that pose serious risk of organ damage or death. Even the former group of side effects, however, can cause considerable discomfort and lead patients to avoid or reduce their use of medication. Also, in general, the type and risk of adverse effects depend on the margin of safety between the dose required for efficacy and the dose that causes side effects. When the margin of safety is large, toxicity results primarily from overdoses; when this margin is small or nonexistent, side effects may be manifest at therapeutic doses. These principles apply to over-the-counter drugs as well, such as acetaminophen and aspirin. Note that safety margins are a function not only of the drug but also of the patient, in that genetic or other 56

58 Fundamental Principles of Pharmacology Intended tissue

Unintended tissue

D

D

Drug metabolism

Drug metabolism

D-X

D-X

Intended receptor "On-target" adverse effects Dose too high Chronic activation or inhibition effects

Unintended receptor

Intended receptor

"Off-target" adverse effects

"On-target" adverse effects

Incorrect receptor is activated or inhibited

Correct receptor, but incorrect tissue Dose too high Chronic activation or inhibition effects

Unintended receptor "Off-target" adverse effects Incorrect receptor is activated or inhibited

Toxic cellular effects

FIGURE 5-1. On-target and off-target adverse drug effects. Drug D is intended to modulate the function of a specific receptor (Intended receptor) in a particular tissue (Intended tissue). On-target adverse effects in the intended tissue could be caused by a supratherapeutic dose of the drug or by chronic activation or inhibition of the intended receptor by Drug D or its metabolite D–X. The same on-target effects could occur in a second tissue (Unintended tissue); in addition, the intended receptor could mediate an adverse effect because the drug is acting in a tissue for which it was not designed. Off-target effects occur when the drug and/or its metabolites modulate the function of a target (Unintended receptor) for which it was not intended.

or kidney disease or to interactions with other drugs), or by changes in the pharmacodynamics of the drug–receptor interaction that alter the pharmacologic response (e.g., changes in receptor number). All such changes can lead to an increase in the effective concentration of the drug and thus to an increased biological response. Because on-target effects are mediated via the desired mechanism of action of the drug, these effects are often shared by every member of the therapeutic class and are thus also known as “class effects.” An important set of on-target adverse effects may occur because the drug, or one of its metabolites, interacts with the appropriate receptor but in tissues other than those affected by the disease condition being treated. Many drug targets are expressed in more than one cell type or tissue. For example, the antihistamine diphenhydramine is an H1 receptor antagonist used to ameliorate the effects of histamine release in allergic conditions. Diphenhydramine also crosses the blood–brain barrier to antagonize H1 receptors in the central nervous system, leading to somnolence. This adverse effect led to the design of second-generation H1 receptor antagonists that do not cross the blood–brain barrier and thus do not induce drowsiness. Notably, the first of these second-generation H1 antagonists, terfenadine, produced an off-target effect (interaction with cardiac potassium channels) that led to a different and serious side effect—an increased risk of cardiac death. This example is discussed later in this chapter.

Local anesthetics such as lidocaine and bupivacaine provide a second example of an on-target adverse effect. These drugs are intended to prevent axonal impulse transmission by blocking sodium channels in neuronal membranes near the site of injection. Blockade of sodium channels in the central nervous system (CNS) following overdose or inappropriate administration (e.g., intravascular administration) can lead to tremors, seizures, and death. These on-target effects are discussed in greater detail in Chapter 11, Local Anesthetic Pharmacology. The antipsychotic agent haloperidol produces its beneficial effect through blockade of mesolimbic and mesocortical D2 receptors. One consequence of blocking D2 receptors in the pituitary gland is an increase in prolactin secretion, leading in some cases to amenorrhea, galactorrhea, sexual dysfunction, and osteoporosis. These on-target effects are discussed in Chapter 13, Pharmacology of Dopaminergic Neurotransmission. Sometimes on-target adverse effects unmask important functions of the biological target. A prominent example of this phenomenon occurs with administration of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (so-called statins), which are used clinically to decrease cholesterol levels. The intended target tissue of these drugs is the liver, where they inhibit HMG-CoA reductase, the rate-limiting enzyme of isoprenoid synthesis. A rare adverse effect of statin therapy is muscle toxicity, including

CHAPTER 5 / Drug Toxicity 59

A Mast cell

Hapten 1

2 Hapten-bound protein

Protein

Complement-mediated RBC lysis

B Cytotoxic T cell

Antigen 1

2

Antigen-bound RBC

RBC

3

RBC lysis

Antibodies bind RBC

RBC removal by reticuloendothelial system

C

Macrophage Antigen

1

Antibodies

2

Antigen–antibody complexes

3

Immune complex deposition in tissues

D Hapten 1

Protein

2

3

Hapten-bound protein Antigen phagocytosis

Antigen presented Activated T cell

FIGURE 5-2.

Mechanisms of hypersensitivity reactions. A. Type I hypersensitivity reactions occur when a hapten binds to a protein (1). The antigen crosslinks IgE antibodies on the surface of a mast cell, leading to mast cell degranulation (2). Mast cells release histamine and other inflammatory mediators. B. Type II hypersensitivity reactions occur when an antigen binds to the surface of a circulating blood cell, usually a red blood cell (RBC) (1). Antibodies to the antigen then bind the surface of the RBC (2), attracting cytotoxic T cells (3), which release mediators that lyse the RBC. Binding of antibody to RBCs can also directly stimulate complement-mediated RBC lysis and RBC removal by the reticuloendothelial system. C. Type III hypersensitivity reactions occur when antibodies bind to a soluble toxin, acting as an antigen (1). The antigen–antibody complexes are then deposited in the tissues (2), attracting macrophages (3) and starting a complement-mediated reaction sequence (not shown). D. Type IV hypersensitivity reactions occur when a hapten binds to a protein (1) and the hapten-bound protein is phagocytosed by a Langerhans cell (2). The Langerhans cell migrates to a regional lymph node, where it presents the antigen to a T cell, thereby activating the T cell (3).

rhabdomyolysis and myositis; the fact that this effect occurs highlights the physiologic role of HMG-CoA reductase in regulating the post-translational modification of several muscle proteins through a lipidation process called geranylgeranylation. Statins, as examples of drugs causing skeletal muscle injury, are also referenced later in this chapter.

Off-Target Effects Off-target adverse effects occur when a drug interacts with unintended targets. Indeed, few drugs are so selective that they interact with only one molecular target. A prominent example of an off-target effect is the interaction of numerous compounds with cardiac IKr potassium channels. (Because the human ether-à-go-go-related gene [hERG] codes for one subunit of the human IKr channel, these channels are also called hERG channels.) Inhibition of potassium

currents carried by IKr channels can lead to delayed repolarization of cardiac myocytes (see Chapter 23, Pharmacology of Cardiac Rhythm). In turn, delayed repolarization can lead to increases in the heart-rate corrected QT interval (QTc), cardiac arrhythmias including torsades de pointes, and sudden death. The antihistamine terfenadine was one of the earliest examples of compounds found to interfere with cardiac potassium channel currents, leading to potentially fatal arrhythmias. This drug was designed to avoid drowsiness, an adverse effect of the first-generation H1 antagonists (see earlier discussion). The observation of increased deaths due to cardiac arrhythmia in patients receiving terfenadine led to withdrawal of this compound from the market and vigorous efforts to understand how to prevent such events. Surveys have shown that, although many compounds inhibit the hERG channel, compounds with a half-maximal inhibitory concentration (IC50) more than 30-fold greater than the

62 Fundamental Principles of Pharmacology

products contain alkaloids that may deplete hepatic glutathione stores, increasing the risk of acetaminophen toxicity. In combination with selective serotonin reuptake inhibitors, St. John’s wort may cause a mild serotonergic syndrome.

Cellular Mechanisms of Toxicity: Apoptosis and Necrosis Cells are equipped with various mechanisms to avoid or repair damage: toxicity occurs if and when these defenses are overwhelmed. In some cases, toxicity can be minimized in the short term, but repeated insults (for example, those leading to fibrosis) can eventually compromise organ function. The primary cellular responses to a potentially toxic drug are illustrated in Fig. 5-3A and 5-3B, using the hepatocyte as an example. Depending on the severity of the toxic insult, a cell may undergo apoptosis (programmed cell death) or necrosis (uncontrolled cell death). Apoptosis allows the cell to undergo ordered self-destruction by the coordinated activation of a number of dedicated proteins. Apoptosis can be beneficial when it eliminates damaged cells without damage to surrounding tissue. Inhibition of apoptosis is common in many cancer cells. If the toxic insult is so severe that ordered cell death cannot be accomplished, the cell undergoes necrosis. Necrosis is characterized by enzymatic digestion of cellular contents, denaturation of cellular proteins, and disruption of cellular membranes. While apoptotic cells undergo cell death with minimal inflammation and disruption of adjacent tissue, necrotic cells attract inflammatory cells and can damage nearby healthy cells.

Organ and Tissue Toxicity Most chapters in this book contain tables that list the serious and common adverse effects of the drugs discussed in that chapter. Here, we consider common mechanisms of injury and repair pertaining to the toxic effects of drugs on the major organ systems. This chapter is not intended to catalogue every possible injury to each organ or organ system, since the range of drug-associated organ and tissue toxicity is so great that is not possible to discuss all the specific toxicities of all the individual drugs in a single chapter. Instead, a few specific examples of injury are provided to demonstrate the general features of drug toxicity. Harmful Immune Responses and Immunotoxicity Stimulation of the immune system plays a role in the toxicity of several drugs and drug classes. Drugs can be responsible for immune reactions (the classic type I through type IV reactions), syndromes that mimic some features of immune responses (red man syndrome), and skin rashes (eruptions)— including severe and life-threatening conditions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Drugs can also compromise the normal function of the immune system (immunotoxicity), leading to secondary effects such as increased risk of infection. Some drugs may be recognized by the immune system as foreign substances. Small-molecule drugs with a mass of less than 600 daltons are not direct immunogens, but can act as haptens, where the drug binds (often covalently) to a protein in the body and is then capable of triggering an immune response. If a drug is sufficiently large (e.g., a therapeutic peptide or protein), it may directly activate the immune system. The two principal immune mechanisms by which drugs

can produce damage are hypersensitivity responses (allergic responses) and autoimmune reactions. The hypersensitivity responses are classically divided into four types (Fig. 5-2). Table 5-1 provides information about the mediators and clinical manifestations of the four types of hypersensitivity reactions. Prior exposure to a substance is required for each of the four types of reactions. A type I hypersensitivity response (immediate hypersensitivity or anaphylaxis) results from the production of IgE after exposure to an antigen. The antigen may be a foreign protein, such as the bacterially derived thrombolytic drug streptokinase, or it may be an endogenous protein modified by a hapten to become immunogenic. Penicillin fragments—either in the administered drug formulation or formed in vivo—can act as haptens and activate the immune system. Subsequent exposure to the antigen causes mast cells to degranulate, releasing inflammatory mediators such as histamine and leukotrienes that promote bronchoconstriction, vasodilatation, and inflammation. Type I hypersensitivity responses manifest as a wheal-and-flare reaction in the skin. Symptoms of “hay fever” such as conjunctivitis and rhinitis may develop in the upper respiratory tract, while asthmatic bronchoconstriction may occur in the lower respiratory tract (see Chapter 47, Integrative Inflammation Pharmacology: Asthma). A type II hypersensitivity response (antibody-dependent cytotoxic hypersensitivity) occurs when a drug binds to cells, usually red blood cells, and is recognized by an antibody, usually IgG. The antibody triggers cell lysis by complement fixation, phagocytosis by macrophages, or cytolysis by cytotoxic T cells. Type II responses are rare adverse responses to several drugs, including penicillin and quinidine. Type III hypersensitivity responses (immune complexmediated hypersensitivity) occur when antibodies, usually IgG or IgM, are formed against soluble antigens. The antigen–antibody complexes are deposited in tissues such as kidneys, joints, and lung vascular endothelium. These complexes cause damage by initiating serum sickness, an inflammatory response in which leukocytes and complement are activated within the tissues. For example, type III hypersensitivity can be caused by the administration of antivenins, horse serum proteins obtained by inoculating a horse with the venom to be neutralized. Examples of other drugs that may pose a risk of serum sickness are bupropion and cefaclor. A type IV hypersensitivity response (delayed-type hypersensitivity) results from the activation of TH1 and cytotoxic T cells. It most commonly presents as contact dermatitis when a substance acts as a hapten and binds to host proteins. The first exposure does not normally produce a response, but subsequent dermal exposures can activate Langerhans cells, which migrate to local lymph nodes and activate T cells. The T cells then return to the skin and initiate an immune response. Well-known type IV hypersensitivity responses include reactions to poison ivy and the development of latex allergies. Repeated exposure to a drug recognized as foreign by the immune system can trigger a massive immune response. This “cytokine storm” can lead to fever, hypotension, and even organ failure. Thus, physicians should consider possible immune reactions to all administered drugs, even those that have appeared to be safe in broader populations. In the case presented at the beginning of the chapter, Ms. G’s fever and rash were likely caused by a T-cell mediated

CHAPTER 5 / Drug Toxicity 63

A

Drug

Quiescent Kupffer cell

Hepatocyte

Stressed hepatocyte

Cytotoxins (IL-1β, α-TNF)

Mitochondrial uncoupling Consumption of ATP Consumption of antioxidants Lipid and protein oxidation Disturbance of Ca2+ homeostasis

Activated Kupffer cell

ROS

IL-1 EC-GF

ROS

ROS TGF-β

RNI

ROS TGF-β

Apoptotic hepatocyte

Endothelial cells (activation and proliferation)

Ito cell Fibrosis

B Drug Circulating monocytes

Quiescent Kupffer cell

Hepatocyte

Chemotactic activating factors (LTB4, LPO)

Injured hepatocyte

Plasma membrane blebs Increased cellular volume Mitochondrial swelling Dilated endoplasmic reticulum

Cytotoxins (RNI, ROS)

ROS RNI

Necrotic hepatocyte

Activated Kupffer cell IL-1

ROS

Endothelial cells (activation)

FIGURE 5-3. Subtoxic and toxic damage to hepatocytes in response to moderate and high doses of drug. A. Subtoxic damage. Moderate doses of a potentially toxic drug activate Kupffer cells and are metabolized by hepatocytes. The resulting hepatocyte stress may be exacerbated by the effects of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) elaborated by activated endothelial cells. Hepatocyte apoptosis and Ito cell activation may result, leading to fibrosis. B. Toxic damage. High doses of a toxic drug are metabolized by hepatocytes to reactive metabolites that can induce cell injury. Chemotactic activating factors released by the injured hepatocytes activate Kupffer cells and endothelial cells, which elaborate toxic ROS and RNI. The end result of this toxic cascade is hepatocyte necrosis. EC-GF, endothelial cell growth factor; IL-1, interleukin-1; IL-1␤, interleukin-1␤; LPO, lipid peroxidation; LTB4, leukotriene B4; TGF-␤, transforming growth factor ␤; ␣TNF, tumor necrosis factor ␣.

CHAPTER 5 / Drug Toxicity 65

at concentrations of drug that are required for efficacy. For these agents, there is generally little safety margin for damage to normal tissues, and successful therapy depends on a greater sensitivity of the cancer cells compared to normal tissues. An increased risk of infection often accompanies therapy with agents that are cytotoxic to white blood cells. The margin between adverse effects and therapeutic effects may be increased by the use of agents that stimulate leukocyte production (filgrastim). Targeting of the immune system may be appropriate when the disease is exacerbated by a deleterious immune response (see Chapter 45). For example, inhaled corticosteroids may be used to control symptoms in patients with frequent, severe exacerbations of chronic obstructive pulmonary disease (see Chapter 47). By inhibiting immune responses to pathogenic micro-organisms, however, such treatment is also associated with an increased risk of pneumonia. Some immunotherapies target specific cell types in the immune system and are associated with an increased risk of serious infection. Rituximab is a monoclonal antibody (mAb) that targets CD20-positive B cells, which are involved in the pathogenesis of non-Hodgkin’s lymphoma (malignant CD20-positive B cells) and rheumatoid arthritis (antibody-producing CD20-positive B cells). Two potentially serious adverse effects that have been observed with the use of rituximab are progressive multifocal leukoencephalopathy (PML), an infection caused by a polyomavirus, the JC virus (JCV), and hepatitis B reactivation with the potential for fulminant hepatitis. These infectious agents are generally present in latent form in patients prior to treatment with rituximab, but the loss of immunocompetence as a result of treatment allows expression of these serious infections. Similarly, efalizumab is a monoclonal antibody that targets CD11a, the ␣ subunit of leukocyte function-associated antigen-1 (LFA-1), which is expressed on all leukocytes. By decreasing the cell surface expression of CD11a and inhibiting the binding of LFA-1 to the intercellular adhesion molecule-1 (ICAM-1), efalizumab inhibits leukocyte adhesion and acts as an effective immunotherapy for psoriasis. However, because CD11a is also expressed on the surface of B cells, monocytes, neutrophils, natural killer cells, and other leukocytes, efalizumab can affect the activation, adhesion, migration, and destruction of these cells as well. Like rituximab, efalizumab has been associated with PML; this serious adverse effect led to its withdrawal from the market in 2009. A similar increase in the frequency of PML has been found in patients receiving natalizumab for multiple sclerosis. Natalizumab binds to the ␣4 subunit of ␣4␤1 and ␣4␤7 integrins expressed on the surface of all leukocytes except neutrophils; by inhibiting the ␣4-mediated adhesion of leukocytes to their target cells, further leukocyte recruitment and activation are prevented. Drug-Induced Hepatotoxicity As described in Chapter 4, many drugs are metabolized in the liver, and some of these metabolites can cause liver damage. A clinically significant example is acetaminophen, a widely used analgesic and antipyretic. In its therapeutic dose range, acetaminophen is metabolized predominantly by glucuronidation and sulfation, resulting in readily excreted metabolites; a small fraction of the dose is also excreted unchanged. As shown in Figure 5-4, however, acetaminophen can also be oxidized to a reactive and potentially toxic species,

O

O NH O

NH NH

-

O

O OH

O

O

Glucuronyl transferase

Sulfotransferase -

O

O S

OH OH

O

OH

Acetaminophen

Acetaminophen glucuronide

O

Acetaminophen sulfate

P450 enzyme or PHS O O

NH N

Gly S

Glutathione

O

OH

NH Glu

O

N-acetyl-p-benzoquinoneimine (NAPQI)

Glutathione conjugate

Hepatotoxicity

Excretion

O H N OH O HS

N-acetylcysteine (NAC)

FIGURE 5-4. Mechanism of acetaminophen poisoning and treatment. Therapeutic doses of acetaminophen are nontoxic, but one metabolite formed at supratherapeutic doses can cause potentially lethal hepatotoxicity. Under ordinary circumstances, acetaminophen is metabolized primarily by glucuronidation (⬃55–60%) and sulfation (⬃30–35%); another 5% or less is excreted unchanged. The remaining 5–10% is oxidized to a reactive intermediate, N-acetyl-p-benzoquinoneimine (NAPQI). This oxidation is catalyzed by cytochrome P450 enzymes (CYP), primarily CYP2E1, as well as CYP3A4 and CYP1A2, and by prostaglandin H synthase (PHS). At therapeutic doses, NAPQI reacts rapidly with glutathione to form a nontoxic metabolite that is readily excreted. Under conditions of overdosing, however, NAPQI formation exceeds glutathione production, allowing free NAPQI to attack mitochondrial and cellular proteins. If this process is unchecked, hepatocyte necrosis and acute liver failure can result. Timely administration of the antidote N-acetylcysteine (NAC) can be lifesaving (within ⬃10 hours of acetaminophen overdosing), in that NAC both reacts directly with NAPQI and serves as a precursor to glutathione.

N-acetyl-p-benzoquinoneimine (NAPQI). Glutathione can conjugate with and thus detoxify NAPQI, but overdosing with acetaminophen depletes glutathione reserves (as can other conditions), leaving NAPQI free to attack cellular and mitochondrial proteins, resulting ultimately in the necrosis of hepatocytes. Timely (within about 10 hours of acetaminophen overdosing) administration of the antidote N-acetylcysteine (NAC) replenishes glutathione stores and can avert liver failure and death. This example underscores the importance of

66 Fundamental Principles of Pharmacology

dose: although acetaminophen is used safely by millions of individuals every day, the same drug, when taken in excess, is responsible for some 50% of the cases of acute liver failure in the United States. Unexpected hepatotoxicity is the most frequent reason for drug withdrawals in the United States. Many cases of fulminant hepatitis following drug therapy are idiosyncratic—that is, the mechanism by which the patient develops hepatic injury is not known—making it difficult to identify at-risk patients. In some cases, failure to determine the mechanism(s) responsible for hepatic injury is due to the inability to reproduce the injury in laboratory animals. A further challenge is that hepatotoxicity may not be anticipated based on preclinical studies, because compounds exhibiting significant hepatotoxicity in animal studies at doses near the anticipated therapeutic exposures in humans are generally eliminated from development. Further confounding the prevention of hepatotoxicity is that clinical trials of a drug typically include a few thousand patients, even though a risk of drug-induced hepatotoxicity in the range of 1 in 10,000 to 1 in 100,000 patients would be of sufficient concern to result in withdrawal. In other words, many clinical trials are too small, or have been designed with exclusion criteria not maintained once the drug is marketed, to detect unacceptable risks of hepatotoxicity. Withdrawal of the insulin-sensitizing agent troglitazone, for example, occurred only after it was noted that approximately 1 in 10,000 patients taking the drug died from acute liver failure. The serum activities of certain enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase [ALP]) and bilirubin are often used to monitor for potential hepatotoxicity in patients. The combination of hepatocellular injury (indicated by increased serum activity of ALT, AST, and ALP) and decreased hepatic function (indicated by elevated bilirubin) is the best predictor of outcome for drug-induced hepatotoxicity. Elevation of serum ALT to ⬎3 times the upper limit of normal, combined with elevation of serum bilirubin to ⬎2 times the upper limit of normal, is associated with a mortality rate of at least 10%. This predictor has become known as “Hy’s Rule,” named for the hepatologist Hyman Zimmerman. Drug-Induced Renal Toxicity The kidney is the major route of elimination of many drugs and their metabolites. Nephrotoxicity may manifest as alterations in renal hemodynamics, tubular damage and obstruction, glomerular nephropathy, and interstitial nephritis. Progressive renal failure, characterized by progressive increases in serum creatinine, may result from loss of function of a sufficient number of nephrons. Examples of drug classes that can cause renal failure include certain antibiotics, NSAIDs, antineoplastic agents, immunomodulators, and angiotensin converting enzyme (ACE) inhibitors. Here, we describe the mechanisms of nephrotoxicity caused by the aminoglycoside antibiotic gentamicin and the antifungal agent amphotericin B. Renal injury is a common adverse effect of treatment with both of these agents. Gentamicin causes renal injury in part through its inhibition of lysosomal hydrolases (sphingomyelinase, phospholipases) in proximal tubules of the kidney, leading to the lysosomal accumulation of electron-dense lamellar structures containing undegraded phospholipids. This process is called renal phospholipidosis. Lysosomal rupture leads to cell death in the form of acute tubular necrosis. Renal tubular injury by

gentamicin and other aminoglycoside antibiotics is reversible upon cessation of treatment, provided that the initial injury is not too severe. The polyene amphotericin B damages fungal cell membranes by interacting with ergosterol and forming membrane pores through which potassium leaks, leading to cell death. Amphotericin-induced renal injury appears to occur via a similar mechanism, with initial binding of drug to sterols in the membranes of renal tubular epithelial cells. Because the mechanism responsible for efficacy is shared by the mechanism responsible for toxicity, the margin between the exposures required for antifungal activity and those required for renal injury is small, leading to a high frequency of renal injury in patients receiving amphotericin B. Liposomal formulations of amphotericin B have been developed in an attempt to reduce this toxicity and to increase the plasma half-life of the drug. If the initial injury is not too severe, cessation of treatment with amphotericin often results in recovery of renal function. Contrast media is administered intra-arterially or intravenously to provide radiographic delineation of the vasculature in organs such as the heart and the brain. These agents appear to cause renal injury both by direct toxicity to renal tubular epithelial cells and by constriction of the vasa recta leading to reduced renal medullary blood flow. The nephrotoxicity of contrast media is dose-related, and patients with preexisting reductions in medullary blood flow—due, for example, to renal insufficiency, intravascular volume depletion, heart failure, diabetes, or diuretic or NSAID use—are at higher risk. Drug-Induced Neurotoxicity Drug-induced neurotoxicity is most often associated with the use of cancer chemotherapeutic agents. In most cases, neurotoxicity manifests in the peripheral nerves, but the central nervous system may be affected as well. Peripheral neuropathy has been associated with vinca alkaloids (e.g., vincristine, vinblastine), taxanes (e.g., paclitaxel), and platinum compounds (e.g., cisplatin). The neuropathy caused by vinca alkaloids and taxanes is directly related to their primary mechanism of action, microtubule disruption (see Chapter 38). In peripheral nerves, microtubule disruption is thought to result in altered axonal trafficking and both sensory and motor neuropathy. Platinum-containing compounds may have direct toxic effects on peripheral nerves. Drug-Induced Skeletal Muscle Toxicity Drug classes associated with skeletal muscle injury include HMG-CoA reductase inhibitors (statins), corticosteroids (dexamethasone, betamethasone, prednisolone, hydrocortisone), and zidovudine (AZT or ZDV). Statin-induced myopathy appears to relate to the inhibition of geranylgeranylation of several muscle proteins. Corticosteroid-induced muscle injury is complex, involving altered carbohydrate metabolism, decreased protein synthesis, and alterations in mitochondrial function that reduce oxidative capacity. Patients treated with corticosteroids can manifest weakness, atrophy, myalgia, and microscopic decreases in muscle fiber size. Corticosteroid-associated muscle injury is reversible, albeit slowly. Understanding the pathogenesis of zidovudine-induced myopathy is complicated by the ability of HIV, the viral infection for which zidovudine is administered, to induce myopathy in the absence of drug therapy. Nonetheless, the improvement in

CHAPTER 5 / Drug Toxicity 67

Drug-Induced Pulmonary Toxicity Adverse effects in the lungs range from acute, reversible exacerbations of asthmatic symptoms to chronic injury characterized by remodeling and/or fibrosis. Reversible airway obstruction can be associated with beta-agonist therapy, whereas chronic injury is observed in some patients receiving the chemotherapeutic agent bleomycin or the antiarrhythmic drug amiodarone. The response to injury after cellular damage is largely determined by the regenerative capacity of the target organ. Repeated insults to the lung, particularly to the epithelial cells lining conducting airways and alveoli, may be followed by regeneration. Repeated cycles of epithelial injury can lead to excessive deposition of collagen and extracellular matrix proteins in alveolar septa and the alveolar spaces, causing fibrosis. Pulmonary fibrosis is manifested as loss of function. Bleomycin and amiodarone are contraindicated in patients with existing disease of the lung parenchyma because both of these agents can cause pulmonary fibrosis.

types of DNA damage (these agents are termed initiators) or by facilitating proliferation of cells carrying precancerous mutations (these agents are promoters). Initiators act by damaging DNA, interfering with DNA replication, or interfering with DNA repair mechanisms. Most initiators are reactive species that covalently modify the structure of DNA, preventing accurate replication and, if unrepaired or misrepaired, leading to a mutation(s). If the mutation(s) affects a gene(s) that controls cell cycle regulation, neoplastic transformation may be initiated. Carcinogenesis is a complex process, involving multiple genetic and epigenetic changes, that usually takes place over years or decades. For most therapeutic areas, compounds that cause direct DNA damage are avoided. Yet DNA damage and/or interference with DNA repair is the desired therapeutic effect of many agents used to treat neoplasia. Damage to normal blood cell progenitors is an important on-target adverse effect of cytotoxic alkylating agents used in cancer chemotherapy (chlorambucil, cyclophosphamide, melphalan, nitrogen mustards, and nitrosoureas). These agents can cause myelodysplasia and/or acute myeloid leukemia (AML). Indeed, 10% to 20% of cases of AML in the United States arise secondary to treatment of other cancers with such anticancer drugs. Tamoxifen, a nongenotoxic estrogen receptor modulator, is an effective treatment in patients with estrogen-sensitive breast cancer. However, this agent also increases the risk of some tumors. Although tamoxifen is an antagonist of estrogen receptors in the breast, it acts as a partial agonist in other tissues that express the estrogen receptor, most notably the uterus. Therefore, an adverse effect of breast cancer treatment with tamoxifen can be the development of endometrial cancer. Newer estrogen receptor modulators, such as raloxifene, do not stimulate uterine estrogen receptors and may be used to treat or prevent breast cancer with a lower risk of endometrial cancer (see Chapter 29, Pharmacology of Reproduction). Product labels describe the preclinical assessment of each drug in the section entitled “Carcinogenesis, Mutagenesis, Impairment of Fertility.” In this section, it is not unusual to find descriptions of rodent studies that suggest carcinogenic potential for drugs. Since mutagens are not typically developed as drugs (with the exceptions noted above), the treatmentrelated tumors observed in these lifetime studies in rodents administered high doses of drug are generally attributed to nongenotoxic (epigenetic) mechanisms. To assess whether the rodent findings represent a risk to the intended patient population, it is important to understand the mechanism by which these tumors occur. The proton pump inhibitor omeprazole, for example, causes tumors of the gastric enterochromaffinlike (ECL) cells in rodents. The development of these tumors results from a dose-related and sustained increase in gastrin, which is secondary to the desired effect of the compound (decreased acid secretion). However, the exposures required for sustained gastrin elevation and tumor formation in rodents far exceed the exposures required for efficacy in patients. Further, the gastrin elevations noted in patients are of low magnitude and are not sustained. Thus, the carcinogenic finding in the rodent studies is not considered to signal a risk for tumor development in patients.

Carcinogenesis Due to Drug Therapy Drugs (and other agents) that can cause cancer are termed carcinogens. More broadly, a carcinogen is a chemical, physical, or biological insult that acts by causing specific

Teratogenesis Due to Drug Therapy Drugs given to pregnant patients may adversely affect the fetus. Teratogenesis is the induction of structural defects in the fetus, and a teratogen is a substance that can induce

muscle function upon withdrawal of zidovudine and the independent demonstration of zidovudine-induced myopathy in rodents suggest that the drug itself causes myopathy, at least in some patients. The mechanism of zidovudine-associated myopathy is not well understood, but accumulation of the drug in skeletal muscle, disruption of mitochondrial cristae, and diminished oxidative phosphorylation are thought to play a role. Drug-Induced Cardiovascular Toxicity Three major mechanisms of drug-induced cardiovascular toxicity have been recognized. First, as discussed earlier, many drugs interact with cardiac potassium channels to cause QTc prolongation, delayed repolarization, and cardiac arrhythmia. Second, some drugs are directly toxic to cardiac myocytes. The anthracycline antineoplastic agent doxorubicin avidly binds to iron; in the presence of oxygen, the iron can cycle between the iron(II) and iron(III) states, leading to the production of reactive oxygen species (ROS). These ROS promote cytotoxicity and death of cardiac myocytes, which possess low activity of antioxidant enzyme systems. Cardiotoxicity, leading to heart failure and arrhythmia, is often the dose-limiting toxicity in patients receiving this drug. Third, as noted earlier, some drugs are toxic to heart valves. The amphetamine analog fenfluramine exerts its desired anorectic effect by both increasing the release of serotonin and decreasing the uptake of serotonin. Fenfluramine and its metabolite norfenfluramine also bind with high affinity to 5-HT2B receptors. Drug binding to 5-HT2B receptors in heart valves activates mitogenic pathways, resulting in proliferation of valvular myofibroblasts that form myxoid plaques on the atrioventricular valves, leading to valvular insufficiency and death in some patients. The 5-HT activity of fenfluramine can also increase vascular resistance and remodel the pulmonary arterial system, leading to the development of pulmonary hypertension. Because of the potential severity of these cardiovascular toxicities, there is a concerted effort to avoid selection of compounds for drug development that exhibit significant prolongation of the QTc interval or binding affinity for 5-HT2B receptors.

CHAPTER 5 / Drug Toxicity 69

Another example of an on-target teratogenic effect is in utero exposure of the fetus to ACE inhibitors. Although ACE inhibitors were previously not contraindicated in the first trimester of pregnancy, recent data indicate that fetal exposure during this period significantly increases the risks of cardiovascular and central nervous system malformations. ACE inhibitors can cause a group of conditions including oligohydramnios, intrauterine growth retardation, renal dysplasia, anuria, and renal failure, reflecting the importance of the angiotensin pathway on renal development and function.

 PRINCIPLES FOR TREATING PATIENTS WITH DRUG-INDUCED TOXICITY Treatment of drug-induced toxicity may include: (1) reducing or eliminating exposure to the drug; (2) administering specific treatments based on antagonizing the mechanism of action of the drug or altering its metabolism; and/or (3) providing supportive measures. Reduction of exposure to a therapeutic agent in a patient who experiences adverse effects may seem intuitive, but it is not always the correct choice. The appearance of an adverse effect during therapy does not necessarily indicate that the effect is due to the drug, despite the temporal relationship between the initiation of therapy and the appearance of the adverse effect. Even if the adverse effect most likely occurred because of the drug, the risks of cessation must be weighed against the benefits of continuing that drug. Cessation of therapy is more obviously a correct choice when the adverse effects have been previously associated with the drug and are life-threatening, such as anaphylaxis due to a beta-lactam antibiotic. Needless to say, for such patients, future therapy with this class of antibiotics would also be contraindicated. Adverse effects that are irreversible and/or likely to increase in severity with continued treatment may also lead to the appropriate decision to terminate therapy. Many adverse effects, however, are considered tolerable and reversible. Depending on the severity of the disease condition being treated, it may be that the overall benefit to the patient is greater with drug treatment than without. An example of such circumstances is the leukopenia that often occurs in patients receiving chemotherapy with cytotoxic drugs. Thus, the decision to withdraw or reduce therapy can be complex and often requires evaluation of many factors affecting the patient’s immediate and long-term health. Therapies designed to counteract the adverse effects produced by a given drug are often based on antagonizing the pharmacologic (on-target) activity of the drug or interfering with effects related to metabolism of the drug. Antagonizing the pharmacologic activity of a drug is a useful approach in overdoses of opioids, benzodiazepines, and acetylcholinesterase (AChE) inhibitors. Interference with the toxic effects of drug metabolites is a useful approach in the treatment of acetaminophen toxicity. These examples are briefly discussed below. Conceptually, the simplest treatment for drug overdose is the administration of an antagonist that blocks the action of a drug that, directly or indirectly, results in supraphysiologic activation of a receptor. For example, an opioid overdose can be treated with naloxone, a pharmacologic antagonist of the opioid receptor. By competitively binding to opioid receptors, naloxone prevents or reverses the toxic effects of natural or synthetic opioids, including

respiratory depression, sedation, and hypotension. Naloxone has a rapid onset of action and is highly potent; indeed, if no clinical improvement is observed within 10 minutes after naloxone doses of up to 10 mg, a different diagnosis or multiple toxic entities should be considered. Naloxone has a relatively short half-life, so it must be given every 1 to 4 hours to provide adequate receptor antagonism while the opioid is being cleared. Flumazenil, a pharmacologic antagonist at the GABAA (benzodiazepine) receptor, is used to treat benzodiazepine overdose. Flumazenil acts by competitive inhibition at benzodiazepine receptors in the central nervous system to completely or partially reverse the sedative effects of benzodiazepines. Like naloxone, it has a rapid onset of action and is highly potent; its effects should be seen within 5 minutes at a dose of not more than 3 mg. Flumazenil also has a short half-life (approximately 1 hour) and must be given frequently to provide adequate receptor antagonism while the benzodiazepine is being cleared. Pharmacologic antagonism can also be used when the toxic agent is not a direct agonist but rather indirectly increases the concentration of the natural ligand for a receptor. AChE inhibitors produce a supraphysiologic concentration of acetylcholine in the synaptic cleft and a characteristic toxidrome of cholinergic excess—bradycardia, miosis, hypersalivation, sweating, diarrhea, vomiting, bronchoconstriction, weakness, respiratory paralysis, and convulsions. Although it is sometimes possible to restore AChE activity, the treatment of AChE inhibition generally depends on administering an anticholinergic agent such as atropine. By antagonizing the muscarinic acetylcholine receptor, atropine restores cholinergic balance and prevents bronchoconstriction, the most common cause of death in patients exposed to AChE inhibitors. As noted earlier, a consequence of acetaminophen overdose is depletion of intracellular glutathione by the drug’s metabolite N-acetyl-p-benzoquinoneimine (NAPQI). Glutathione stores can be replenished by administering Nacetylcysteine (NAC), a metabolic precursor of glutathione (see Fig. 5-4 for details). In addition to supportive therapy (gastric lavage and/or charcoal), NAC is given orally or intravenously within 8 to 10 hours following ingestion of a potentially hepatotoxic dose of acetaminophen to prevent or lessen hepatic injury. Finally, supportive therapy can be provided in the face of drug-induced toxicity. One example is the administration of intravenous fluids to patients with renal injury in order to maintain adequate renal blood flow. In cases of severe renal injury, dialysis may be required until renal function is regained. Another example is the treatment of bone marrow suppression resulting from the administration of cytotoxic agents in cancer chemotherapy. Filgrastim, a recombinant human granulocyte colony-stimulating factor (G-CSF), can be used to stimulate leukocyte production and provide supportive therapy until endogenous production of leukocytes resumes in the bone marrow upon completion of the cytotoxic therapy.

 CONCLUSION AND FUTURE DIRECTIONS This chapter has presented a mechanism-based approach to understanding drug toxicity and provided examples to

6 Pharmacogenomics Liewei Wang and Richard M. Weinshilboum

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 71-72 PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Genomic Variation and Pharmacogenomics . . . . . . . . . . . . 71 PHARMACOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Variation in Enzymes of Drug Metabolism: Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Variation in Drug Targets: Pharmacodynamics . . . . . . . . . . 75

Pathway-Based PharmacogeneticsPharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Idiosyncratic Drug Reactions . . . . . . . . . . . . . . . . . . . . . . . 77 Modern Pharmacogenetics-Pharmacogenomics. . . . . . . . . 77 Pharmacogenomics and Regulatory Science . . . . . . . . . . . 78 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 79 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

 INTRODUCTION

approximately 25,000 genes that, through alternative splicing and post-translational modification, may encode 100,000 or more proteins. Any two people differ on average at about one nucleotide in every 1,000 in their genome, totaling an average interindividual difference of 3 million base pairs throughout the genome. The majority of these differences are so-called single nucleotide polymorphisms or SNPs (pronounced “snips”), in which one nucleotide is exchanged for another at a given position. SNPs and other differences in DNA sequence can occur anywhere in the genome, in both coding regions and noncoding regions. If a SNP changes the encoded amino acid, it is called a nonsynonymous coding SNP (cSNP). The remaining differences in DNA sequence involve insertions, deletions, duplications, and reshufflings, sometimes of just one or a few nucleotides but occasionally of whole genes or larger DNA segments that include many genes. Functionally significant DNA sequence differences that we currently understand tend to fall within genes, either within their coding sequences or in the promoters, enhancers, splice sites, or other sequences that control gene transcription or mRNA stability. Taken together, these differences constitute each person’s genetic individuality. Some of that individuality affects the way in which each person will respond to drug treatment.

Although modern pharmacologic agents can be used to treat or control diseases that range from hypertension to human immunodeficiency virus (HIV) infection, there are large individual variations in response to drug therapy. These variations can range from potentially life-threatening adverse drug reactions to equally serious lack of therapeutic efficacy. Many factors can influence the drug response phenotype, including age, gender, and underlying disease, but genetic variation also plays an important role. Interindividual differences in the genes that encode drug targets, drug transporters, or enzymes that catalyze drug metabolism can profoundly affect the success or failure of pharmacotherapy. Pharmacogenetics is the study of the role of inheritance in variation in drug response. The convergence of recent advances in genomic science and equally striking advances in molecular pharmacology has resulted in the evolution of pharmacogenetics into pharmacogenomics. The promise of pharmacogenetics-pharmacogenomics is the possibility that knowledge of a patient’s DNA sequence could be used to enhance pharmacotherapy, maximize drug efficacy, and reduce the incidence of adverse drug reactions. Therefore, pharmacogenetics and pharmacogenomics represent an important aspect of the aspiration to “personalize” or “individualize” medicine—in this case, drug therapy. This chapter describes the principles of pharmacogenetics and pharmacogenomics as well as recent developments in this discipline. Several key examples are cited in which knowledge of pharmacogeneticspharmacogenomics may help to individualize drug therapy.

 PHYSIOLOGY Genomic Variation and Pharmacogenomics The human genome contains approximately three billion nucleotides. According to current estimates, the genome contains

 PHARMACOLOGY The concept that inheritance might be an important determinant of individual variation in drug response emerged half a century ago. It originally grew out of clinical observations of striking differences among patients in their response to “standard” doses of a drug. Those observations, in addition to twin and family studies that showed inherited variations in plasma drug concentrations and other pharmacokinetic parameters, led to the birth of pharmacogenetics. Many of those original examples of pharmacogenetic variation, and many of the most striking examples even today, involve pharmacokinetics—factors 71

74 Fundamental Principles of Pharmacology A

CYP2D6 pharmacogenetics

Ultrarapid metabolizers

Extensive metabolizers

Poor metabolizers

Number of subjects

120

80

40

0 0.01

0.10

1

10

100

Debrisoquine: 4-Hydroxydebrisoquine metabolic ratio

B

AmpliChip CYP450 array

FIGURE 6-1. CYP2D6 pharmacogenetics. A. Frequency distribution of the metabolic ratio for the cytochrome P450 2D6 (CYP2D6)-catalyzed metabolism of debrisoquine to form its 4-hydroxy metabolite. Data for 1,011 Swedish subjects are plotted as the ratio of metabolites in the urine. Most subjects metabolize debrisoquine extensively, while some subjects metabolize the compound ultrarapidly and others metabolize the compound poorly. B. The AmpliChip CYP450 array can be used to determine variant genotypes for cytochrome P450 genes that influence drug metabolism.

the neuroleptic haloperidol, the opioids codeine and dextromethorphan, and the antidepressants fluoxetine, imipramine, and desipramine, among many others (Table 6-1). Therefore, poor metabolizers for CYP2D6 can potentially experience an adverse drug effect when treated with standard doses of agents such as metoprolol that are inactivated by CYP2D6, whereas codeine is relatively ineffective in poor metabolizers because it requires CYP2D6-catalyzed metabolism to form the more potent opioid morphine. Conversely, ultrarapid metabolizers may require unusually high doses of drugs that are inactivated by CYP2D6, but those same subjects can be “overdosed” with codeine, suffering respiratory depression or even respiratory arrest in response to “standard” doses. In one tragic case, a nursing infant whose mother was an ultrarapid CYP2D6 metabolizer died when the mother was prescribed a standard dose of codeine and the baby was overdosed on morphine present in the breast milk.

CYP2D6 genetic polymorphisms are also important for the efficacy of the breast cancer drug tamoxifen. Tamoxifen is used to block the estrogen receptor (ER) in approximately 60% of breast cancer patients with ER-positive tumors. However, tamoxifen is a prodrug that requires metabolic activation to form 4-hydroxytamoxifen and 4-hydroxy-Ndesmethyltamoxifen (endoxifen) (Fig. 6-2A). These metabolites are approximately 100 times more potent as antagonists of the ER than the parent drug. As a result, patients who are CYP2D6 poor metabolizers (Fig. 6-1) are relatively unable to form the active 4-hydroxy metabolites of tamoxifen. Poor-metabolizer patients have worse outcomes with respect to breast cancer recurrence than do CYP2D6 extensive metabolizers (EMs) (Fig. 6-2B). Furthermore, if EM patients are co-administered other drugs such as antidepressants that are good CYP2D6 substrates, they receive less benefit from tamoxifen therapy than do CYP2D6 EMs who are not co-administered drugs that compete with tamoxifen for CYP2D6-catalyzed metabolism. In the past, an individual’s genotype for CYP2D6 and many other genes encoding drug-metabolizing enzymes was inferred from phenotype (e.g., the urinary metabolic ratio that can be measured by assaying the urinary excretion of a specific metabolite after the administration of a probe drug) (Fig. 6-1A). As discussed below, genotype assignment is now increasingly dependent on DNA-based tests performed with devices such as the “chip” shown in Figure 6-1B. TPMT represents another example of an important and clinically relevant genetic polymorphism for drug metabolism. TPMT catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine and azathioprine (see Chapter 38, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance). Among other indications, these cytotoxic and immunosuppressive agents are used to treat acute lymphoblastic leukemia of childhood and inflammatory bowel disease. Although thiopurines are useful drugs, they have a narrow therapeutic index (i.e., the difference between the toxic and therapeutic dose is small), with occasional patients suffering from life-threatening thiopurine-induced myelosuppression. In Caucasians, the most common variant allele for TPMT is TPMT*3A; the frequency of this allele is approximately 5%, so 1 in 300 subjects carries two copies of the TPMT*3A allele. TPMT*3A is predominantly responsible for the trimodal frequency distribution of the level of red blood cell TPMT activity shown in Figure 6-3. TPMT*3A has two nonsynonymous cSNPs, one in exon 7 and another in exon 10 (Fig. 6-3). The presence of TPMT*3A results in a striking decrease in tissue levels of TPMT protein. Mechanisms responsible for the observed decrease in TPMT*3A protein level include both accelerated TPMT*3A degradation and intracellular TPMT*3A aggregation, probably as a result of protein misfolding. As a result, drugs such as 6mercaptopurine are poorly metabolized and may reach toxic levels. Subjects homozygous for TPMT*3A are at greatly increased risk for life-threatening myelosuppression when treated with standard doses of thiopurine drugs. These patients have to be treated with approximately one-tenth to one-fifteenth the standard dose. There are striking ethnic differences in the frequency of variant alleles for TPMT. For example, TPMT*3A is rarely observed in East Asian populations, whereas TPMT*3C, which has only the exon 10 SNP, is the most common variant allele in those populations.

CHAPTER 6 / Pharmacogenomics 75

A N

% Subjects per 0.5 units of activity

N

O

O

CYP2D6

SULT1A1

(CYP2B6, CYP2C9, CYP2C19, CYP3A)

Tamoxifen (TAM)

OH

4-hydroxyTAM

CYP3A4/5 (CYP2C9 + other CYP isoforms)

CYP3A4/5

N O

N H

O

TPMTH/TPMTH 10

TPMTL/TPMTH 5

TPMTL/TPMTL

0 5

0

H

10

15

20

TPMT activity (units/ml RBC) CYP2D6

SULT1A1 OH

N-desmethylTAM

1

TPMT*1 (wild type)

Endoxifen

2

3

4

5

6

7

8

9

10

VNTR

TPMT*3A B

VNTR

Relapse-free survival (%)

100

FIGURE 6-3. TPMT pharmacogenetics. Frequency distribution of red blood cell (RBC) thiopurine S-methyltransferase (TPMT) activity for 298 unrelated Caucasian subjects. TPMT L indicates an allele or alleles for the trait of low activity, while TPMT H refers to the “wild type” (TMPT*1) allele for high activity. The observed trimodal frequency distribution for RBC TPMT activity is due mainly to the effect of TPMT*3A, the most common variant allele for low activity in a Caucasian population. TMPT*1 and TPMT*3A differ by two nonsynonymous single nucleotide polymorphisms (SNPs), one in exon 7 and one in exon 10. VNTR, variable number tandem repeat.

80 EM

60

G460A A719G Ala154Thr Tyr240Cys

IM

40 PM

20 P=0.013

0 0

2

4

6

8

10

12

Years after randomization

Disease-free survival (%)

100 80 EM

60

IM

40 PM Second pain

20 P=0.009

0 0

2

4

6

8

10

12

Years after randomization

FIGURE 6-2.

Tamoxifen pharmacogenetics. A. Tamoxifen is metabolized by two cytochrome P450 pathways to form the active metabolites 4-hydroxytamoxifen (4-hydroxyTAM) and endoxifen, which are further metabolized by sulfotransferase (SULT) 1A1 (not shown). Genetic variations in CYP2D6 can influence the extent of tamoxifen metabolism. B. Kaplan-Meier curves showing the influence of CYP2D6 “metabolizer” status on the survival of women with estrogen receptor-positive [ER(⫹)] breast cancer who were treated with tamoxifen. Patients who were extensive metabolizers (EM) of tamoxifen had improved relapse-free survival and diseasefree survival relative to intermediate metabolizers (IM) and poor metabolizers (PM).

Because of its clinical significance, TPMT was the first example selected by the FDA for public hearings on the inclusion of pharmacogenetic information in drug labeling. For the same reason, clinical testing for TPMT genetic polymorphisms is widely available. The phenomenon of marked changes in the level of a protein as a result of the alteration of only one or two amino acids in the protein has been observed repeatedly for many other genes of pharmacogenetic significance and is a common explanation for the functional effects of nonsynonymous cSNPs. The BChE, NAT2, CYP2D6, and TPMT genetic polymorphisms all behave as monogenic (single-gene) Mendelian traits, as do many other early examples from pharmacogenetics. However, pharmacogenetics-pharmacogenomics has now moved beyond monogenic pharmacokinetic traits, and the focus increasingly involves functionally and clinically significant variation in drug targets as well as drug-metabolizing enzymes. Variation can also involve multiple genes that influence both pharmacokinetics and pharmacodynamics.

Variation in Drug Targets: Pharmacodynamics Drugs generally exert their effects by interacting with specific target proteins. Therefore, genetic variations in these target proteins, or in signaling pathways downstream from the target proteins, can influence the outcome of pharmacotherapy (Table 6-2). Furthermore, variation in drug targets

Precursors of clotting factors

O2

Active clotting factors CO2

Vitamin K-dependent

γ-glutamyl carboxylase

Vitamin K reduced

Vitamin K epoxide

Vitamin K epoxide reductase

6-Hydroxywarfarin 7-Hydroxywarfarin CYP2C9

S-Warfarin

78 Fundamental Principles of Pharmacology

rs4363657 P=4x10-9

8 7

-Log10 P Value

EM

6 IM

5 PM

4 3

P=0.013

2 1 0 1

2

3

4

5

6

7

8

9

10

Chromosome

11 12 13 14 15 16 17 19 21 X 18 20 22

FIGURE 6-5.

Genome-wide association study of statin-induced myopathy. In the first genome-wide association study of drug response, patients who developed myopathy while taking the statin medication simvastatin were compared to controls who did not develop myopathy. The statistical association of myopathy with each single nucleotide polymorphism (SNP) was plotted against the chromosomal location of the SNP. Genome-wide association revealed a single SNP that was highly associated (p value ⫽ 4 ⫻ 10⫺9) with the development of myopathy. The arrow points to the SNP in the SLCO1B1 gene, which encodes an organic anion transporter that mediates hepatic uptake of statins. Patients with this variant may have higher plasma levels of statins at any given dose of drug.

Attaining the goal of truly personalized drug therapy and translating genomic knowledge into clinical practice rapidly will require the application of high-throughput genotyping technologies. An excellent example involves the use of GWAS to identify a genomic biomarker for statin-induced myopathy. Cholesterol-lowering HMG-CoA reductase inhibitors, such as simvastatin and atorvastatin (see Chapter 19, Pharmacology of Cholesterol and Lipoprotein Metabolism), are among the most widely prescribed drugs worldwide. Although these drugs are generally very safe, statins can rarely cause serious myopathy with rhabdomyolysis and renal failure. In an attempt to predict and prevent this serious adverse drug reaction, the SEARCH collaborative group performed a GWAS in which approximately 300,000 SNPs across the genome were genotyped using DNA from 85 patients who had developed severe statin-induced myopathy and 90 control subjects who had not developed this adverse drug reaction. The results, depicted in Figure 6-5, showed that rs4363657, a single SNP located within the SLCO1B1 gene on chromosome 12, had a strong association with myopathy ( p value of 4 ⫻ 10⫺9). The odds ratio for myopathy risk in subjects homozygous for the variant nucleotide at the SNP was 16.9, and it was estimated that more than 60% of the cases of myopathy in this 12,064patient trial were associated with this one SNP. The SLCO1B1 gene encodes an organic anion transporter that mediates statin uptake by the liver; patients homozygous for the variant SNP may have higher plasma levels of statins and therefore be more likely to develop rhabdomyolysis at any given dose of drug. This example is undoubtedly only the first of many applications of genome-wide techniques to pharmacogenomics.

Pharmacogenomics and Regulatory Science To achieve individualized drug therapy, we need not only to understand the science underlying pharmacogenetics and

pharmacogenomics and to develop state-of-the-art technologies to detect and assay DNA sequence data, but also to translate that knowledge into the clinic. That translation process will require the active involvement of the FDA and the pharmaceutical industry, which develops virtually all new drugs. In 2003, the FDA issued a Draft Guidance with regard to pharmacogenomic data, and that draft was approved in 2005. The FDA also initiated a series of public hearings with regard to the incorporation of pharmacogenomic data into drug labeling. Those hearings began with thiopurine drugs and TPMT and were followed by hearings on a genetic polymorphism in UGT1A1, a gene encoding a phase II enzyme involved in biotransformation of the antineoplastic agent irinotecan. Public hearings have also been held on CYP2C9, VKORC1, and warfarin—resulting in relabeling—and on tamoxifen and CYP2D6. The attention given to pharmacogenetics-pharmacogenomics by the FDA is having an impact on the pharmaceutical industry, especially within the context of the unfortunate series of events that resulted in the withdrawal of the COX-2 inhibitor rofecoxib (Vioxx) from the market for reasons of safety. It is unclear whether pharmacogenetics played a role in the Vioxx-induced cardiovascular disease that led to the withdrawal of that drug. However, pharmacogenetics almost certainly could contribute to postmarketing surveillance, not only to help avoid adverse reactions, but also to “rescue” drugs that might be of benefit to groups of patients selected on the basis of genetic variation in drug response. The latter situation was highlighted by reports that a polymorphism in the ␤1-adrenoceptor influences response to the ␤1-adrenergic antagonist bucindolol—both in vitro and in patients with heart failure. This ␤-antagonist had initially failed in a clinical trial that did not include genotyping, perhaps because only patients with the wild-type ␤1-adrenoceptor genotype displayed the desired clinical response.

CHAPTER 6 / Pharmacogenomics 79

 CONCLUSION AND FUTURE DIRECTIONS Pharmacogenetics and pharmacogenomics involve the study of ways in which DNA sequence variation affects the response of individual patients to medications. The goal of pharmacogenetics and pharmacogenomics is to maximize efficacy and minimize toxicity, based on knowledge of an individual’s genetic composition. Although many factors other than inheritance influence differences among patients in their response to drugs, the past half-century has demonstrated that genetics is an important factor responsible for variation in the occurrence of adverse drug reactions or the failure of individual patients to achieve the desired therapeutic response. Pharmacogenetics has evolved during that half-century from classical examples, such as CYP2D6 and TPMT, to include more complex situations such as that represented by the pharmacogenetics of warfarin, a drug that displays both pharmacokinetic and pharmacodynamic pharmacogenetic variation. This area of genomic medical science also presents unique challenges in its translation into the clinic. However, there can no longer be any doubt that pharmacogenetics and pharmacogenomics will be applied to clinical medicine with increasing breadth and depth and that, ultimately, they will enhance our ability to individualize drug therapy.

Suggested Reading Broder S, Venter JC. Sequencing the entire genomes of free-living organisms: the foundation of pharmacology in the new millennium.

Ann Rev Pharmacol Toxicol 2000;40:97–132. (Overview of genome sequencing and a primer on the possible implications of genetic diversity for pharmacology.) Drazen JM, Yandava CN, Dube L, et al. Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment. Nat Med 1999;22:168–171. (Original study that showed different pharmacologic responses in people with different polymorphisms of the ALOX5 gene.) Evans WE, McLeod HL. Pharmacogenomics—drug disposition, drug targets, and side effects. N Engl J Med 2003;348:538–549. (Review describing the integration of genomics with pharmacogenetics.) Mallal S, Phillips E, Carosi G, et al. HLA-B*5701 screening for hypersensitivity to abacavir. N Engl J Med 2008;358:568–579. (A double-blind randomized study of a genetic biomarker for an idiosyncratic adverse drug response.) Rieder MJ, Reiner AP, Gage BF, et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med 2005;352: 2285–2293. (Description of VKORC1 haplotypes, CYP2C9 genotypes, and their relationship to warfarin dose.) The SEARCH Collaborative Group. SLCO1B1 variants and statin-induced myopathy—a genomewide study. N Engl J Med 2008;359:789–799. (The first genome-wide association study of a drug response.) Wang L, Weinshilboum RM. Pharmacogenomics: candidate gene identification, functional validation and mechanisms. Hum Mol Genet 2008;17:R174–R179. (Overview of the evolution of pharmacogenetics into pharmacogenomics with the incorporation of genome-wide techniques.) Weinshilboum RM, Wang L. Pharmacogenetics and pharmacogenomics: development, science and translation. Annu Rev Genomics Hum Genet 2006;7:223–245. (Review of pharmacokinetic and pharmacodynamic pharmacogenomic variation, as well as challenges to the translation of this science into the clinic.)

II Principles of Neuropharmacology

IIA Fundamental Principles of Neuropharmacology

7 Principles of Cellular Excitability and Electrochemical Transmission Lauren K. Buhl, John Dekker, and Gary R. Strichartz

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . . 82-83 CELLULAR EXCITABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Ohm’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Channel Selectivity, the Nernst Equation, and the Resting Potential . . . . . . . . . . . . . . . . . . . . . . . . . . 84 The Goldman Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 The Action Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

PHARMACOLOGY OF ION CHANNELS . . . . . . . . . . . . . . . . . . 89 ELECTROCHEMICAL TRANSMISSION . . . . . . . . . . . . . . . . . . 89 Synaptic Vesicle Regulation . . . . . . . . . . . . . . . . . . . . . . . . 91 Postsynaptic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Transmitter Metabolism and Reuptake . . . . . . . . . . . . . . . . 92 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . 92 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

 INTRODUCTION

Many drugs modify chemical or electrical signaling to increase or decrease cellular excitability and electrochemical transmission. To appreciate how such drugs act, the present chapter explains the electrochemical foundations that underlie these phenomena. These general principles are applicable to many areas of pharmacology, including those discussed in Chapters 9 to 11 (Section IIB, Principles of Autonomic and Peripheral Nervous System Pharmacology), Chapters 12 to 18 (Section IIC, Principles of Central Nervous System Pharmacology), and Chapter 23, Pharmacology of Cardiac Rhythm.

Cellular communication is essential for the effective functioning of any complex multicellular organism. The major mode of intercellular communication is the transmission of chemical signals, such as neurotransmitters and hormones. In excitable tissues, such as nerves and muscles, rapid intracellular communication relies on the propagation of electrical signals—action potentials—along the plasma membrane of the cell. Both chemical and electrical transmission commonly involve the movement of ions across the plasma membrane that separates the cell from its environment or across the membranes of internal organelles such as the endoplasmic reticulum or mitochondria. Ionic movements can directly change the cytoplasmic concentration of ions, such as Ca2⫹, that are key regulators of biochemical and physiologic processes such as phosphorylation, secretion, and contraction. Ionic movements also change the electrical potential across the membrane through which the ions flow, thus regulating various voltage-dependent functions such as the opening of other ion channels. Some of these events are brief, with durations and actions of several milliseconds (0.001 sec). Others can take many seconds, with biochemical consequences—such as phosphorylation of proteins—that can persist for minutes or hours. Even gene expression can be regulated by changes in ion concentrations, resulting in long-term changes in cellular physiology, growth, differentiation, and death. 82

 CELLULAR EXCITABILITY Excitability refers to the ability of a cell to generate and propagate electrical action potentials. Neuronal, cardiac, smooth muscle, skeletal muscle, and many endocrine cells have an excitable character. Action potentials may propagate over large distances, as in peripheral nerve axons that conduct over several meters, or they may stimulate activity in cells of much smaller size such as the 30- to 50-␮m-diameter interneurons that are contained within a single autonomic ganglion. The function of action potentials differs depending on the cell in which they occur. Propagating waves of action potentials rapidly conduct encoded information with fidelity over long distances along axons. Within a small cell, action potentials excite the whole cell at once, causing an increase in intracellular ions (e.g., Ca2⫹) followed by a rapid release

I  V/R

Equation 7-1a Outward current I I=gV

Negative potential

I  gV

V

Equation 7-1b

Inward current

Positive potential

84 Fundamental Principles of Neuropharmacology

Ion Channels How does current actually flow across a cell membrane? Biological membranes are composed of a lipid bilayer within which some proteins are embedded and to which other proteins are attached (Fig. 7-2). Pure lipid membranes are virtually impermeable to most polar or charged substances, thus having a very high intrinsic resistance. From an electrical perspective, the lipid bilayer also acts as a capacitor by separating the extracellular and intracellular ions. To allow the passage of ions that carry electrical current, ion channels span the membrane. Most ion channels discriminate among the various types of ions and remain closed until specific signals dictate their opening, showing the respective properties of ion selectivity and of gating. From an electrical perspective, a set of ion channels is a variable conductor: it provides many individual conductances for ion flow between the extracellular and intracellular environments. The magnitude of the overall conductance depends on the fraction of channels in the open state and the conductance of the individual open channels.

Channel Selectivity, the Nernst Equation, and the Resting Potential By itself, the hypothetical I-V relation in Figure 7-1 does not explain the electrical behavior of most real cells. If a cell behaved according to Equation 7-1, then the potential difference across the membrane would be zero in the absence of an externally applied current. Instead, most cells maintain a negative potential difference across their plasma membrane. This voltage difference is most pronounced in neuronal and cardiac ventricular cells, where a resting potential (the voltage difference

Capacitor (plasma membrane)

Resistor (ion channel)

Ii

Ic

IT = Ii + Ic

FIGURE 7-2.

Electric circuit model of the cell membrane. The cell membrane can be modeled as a simple electric circuit containing a resistor and a capacitor. Ion-selective channels function as resistors (identical to conductors), through which ions can flow down their electrochemical gradient. The lipid bilayer acts as a capacitor by maintaining a separation of charges between the extracellular and intracellular spaces. This circuit (referred to as an RC, or resistor-capacitor, circuit) changes the timing between the flow of charges across the membrane (current) and changes in transmembrane potential (voltage), because the lipid bilayer, acting as a capacitor, stores some of the charge that passes across the membrane. Time is required to store this charge; therefore, the initial change in voltage associated with a step of current is slow. As the capacitor (lipid bilayer) fills with charges and the voltage change grows, more of the charge passes through the resistor, until a new steady state is reached and the current–voltage relationship becomes more linear. (Ic, capacitor current; Ii, ionic current; IT, total current.)

across the membrane in the absence of external stimuli) of ⫺60 to ⫺80 mV can be recorded. The resting potential results from three factors: (1) an unequal distribution of positive and negative charges on each side of the plasma membrane; (2) a difference in selective permeabilities of the membrane to the various cations and anions; and (3) the current-generating action of active (energy-requiring) and passive pumps that help to maintain the ion gradients. The effects of these interrelated factors can be explained best with an example. Consider the case when there are only potassium ions (K⫹) and protein-bound anions (A⫺) inside the cell and no other ions outside the cell (Fig. 7-3). If this cell’s membrane is permeable to potassium alone, then K⫹ will flow outward, while A⫺ will remain inside. The K⫹ ions flow outward because of a chemical gradient; that is, K⫹ efflux is favored because the K⫹ concentration inside the cell is greater than that outside the cell. A potential efflux of the anion, A⫺, is also favored by its chemical gradient, but the absence of transmembrane channels permeable to A⫺ prevents this anion from flowing across the membrane. Because of this selective permeability for K⫹, every K⫹ ion that exits the cell leaves one net negative charge (an A⫺ ion) on the inside of the cell and adds one net positive charge (a K⫹ ion) on the outside of the cell. This separation of charges across the membrane creates a negative membrane potential. If a negative membrane potential were not established as K⫹ leaves the cell, then K⫹ ions would continue to exit until the extracellular concentration of K⫹ was equal to the intracellular concentration of K⫹. However, the establishment of a voltage difference creates an electrostatic force that eventually prevents net K⫹ efflux (Fig. 7-3B). Thus, the electrical gradient (Vm) and the chemical gradient “pull” the K⫹ ions in opposite directions: the electrical gradient favors an inward flow of K⫹ ions, while the chemical gradient favors an outward flow of K⫹ ions. These forces combine to create an electrochemical gradient, which is equal to the sum of the electrical gradient and the chemical gradient. The transmembrane electrochemical gradient is the net driving force for ion movement across biological membranes. As a result of the electrochemical gradient, the extracellular concentration of K⫹ does not equilibrate with the intracellular concentration. Instead, an equilibrium is established in which the electrostatic force “pulling” K⫹ back into the cell is balanced exactly by the chemical gradient favoring K⫹ efflux. The electrical potential at which this equilibrium occurs, for any permeant ion X, is a function of the charge of the ion (z), the temperature (T), and the intracellular and extracellular concentrations of the ion. This relationship is expressed as the Nernst equation: Vx  Vin  Vout 

[X]out RT ln [X]in zF

Equation 7-2

where Vx is the transmembrane potential that a membrane selectively permeable to ion X would reach at equilibrium (i.e., the Nernst potential for that ion), Vin ⴚ Vout is the transmembrane voltage difference, RT/zF is a constant for a given temperature and charge (this number simplifies to 26.7 mV for a charge of ⫹1 at a temperature of 37°C), and [X]out and [X]in are the extracellular and intracellular concentrations, respectively, of ion X. The electrochemical driving force on ion X is equal to the difference between the actual membrane potential and the Nernst potential for that ion, Vm ⴚ Vx.

A

K+ selective channel

B

+

K

K

+

C

K

+

K

+

+

K +

K +

A

K

-

A

A

-

A K

+

-

A

+

K

K

-

-

A +

K

K -

A

A

K

+

+

-

-

+

A K

K

+

K

-

+

A

A

+

-

K A

-

-

A

K

A

A

+

K

+

A +

-

A

-

-

-

K -

-

A

Electrochemical gradient = chemical force + electrical force

+

K

Chemical force Electrical force

A

A

A

ZERO ZERO

-

+ -

86 Fundamental Principles of Neuropharmacology

different channels that are selective for different ions, all of which contribute to the overall resting membrane potential. When the resting potential is determined by two or more species of ions, the influence of each species is governed by its concentrations inside and outside the cell and by the relative permeability of the membrane to that ion. Quantitatively, this relationship is expressed as the Goldman-Hodgkin–Katz Equation:

I(nA)

IK

INet = IK + INa

VK

VNa VR

V(mV)

P [K ]  PNa[Na ]o  PCl[Cl ]i RT ln K  o Equation 7-3 F PK [K ]i  PNa[Na ]i  PCl [Cl ]o

gK : gNa = 5:1

INa -92

Vm 

-70

+40 IK VK gK INa VNa gNa VR INet

K+ current K+ Nernst potential K+ conductance Na+ current Na+ Nernst potential Na+ conductance Resting membrane potential Net current

Relative contribution of K⫹ and Na⫹ to the resting membrane potential. The relative membrane permeabilities of K⫹, Na⫹, and other ions, and the Nernst (electrochemical equilibrium) potentials of these ions, together determine the resting membrane potential. In the example shown, the conductance of K⫹ is five times the conductance of Na⫹ (shown by the slopes of the I versus V lines for IK and INa, respectively). That is, the membrane is five times_more permeable to K⫹ than to Na⫹. The K⫹ current is described by IK [IK ⫽ gK(V ⫺ VK)], while Na⫹ current is described by INa _ _ the _ [INa ⫽ gNa(V ⫺ VNa)]. (In this example, gK and gNa are constant conductances over all voltages.) INet, the net membrane current, is the sum of these two currents (INet ⫽ IK ⫹ INa). The “resting” membrane potential (VR) is the value of V at which INet equals zero. In this example, note that VR is close to, but greater than, VK. This is because, although K⫹ is the primary determinant of the resting potential, the minor Na⫹ current depolarizes VR to a value more positive than VK.

where Px is the membrane permeability of ion x. (Px is expressed as a fraction, with a value of 1 indicating maximum permeability.) Essentially, this expression states that the higher the concentration gradient of a particular ion and the greater its membrane permeability, the greater its role is in determining the membrane potential. In the extreme case, when the permeability of one ion is exclusively dominant, the Goldman equation reverts to the Nernst equation for that ion. For example, if PK ⬎⬎ PCl, PNa, the equation becomes Vm 

FIGURE 7-4.

[K ] RT ln  o F [K ]i

Alternatively, if PNa greatly exceeds PK, PCl, then Vm ⬃ VNa, and the membrane is strongly depolarized. This important concept links changes in ion channel permeability to changes in membrane potential. Whenever an ion-selective channel opens, the membrane potential shifts toward the Nernst potential for that ion. The relative contribution of a given channel to the overall membrane potential depends on the extent of ion flow through that channel. Time-dependent changes in the membrane permeabilities of Na⫹ and K⫹ (and, in cardiac cells, Ca2⫹) account for the major distinguishing feature of electrically excitable tissues—the action potential.

The Action Potential ⫹

potential for K (about ⫺90 mV). In reality, the additional, weak permeabilities of other ionic species raise the resting membrane potential above that for K⫹. Thus, although K⫹ is the most permeant ion, the permeability of the other ions and the action of the “electrogenic” pumps also contribute to the overall resting potential. At the steady state that describes the true resting membrane potential (Fig. 7-4), Vm does not equal the Nernst potential for any of the individual ions, and each ionic species experiences a net electrochemical force. In other words, (Vm ⴚ Vion) is nonzero, and small ion fluxes occur. The algebraic sum of these inward and outward currents is small and is balanced by currents from active, electrogenic pumps, so there is no net current across the resting membrane. It has been estimated that up to 25% of all cellular energy in excitable tissues is expended in maintaining ion gradients across cellular membranes.

The Goldman Equation The example shown in Figure 7-3 addresses a situation where only one ionic species flows across the plasma membrane. In reality, many cells possess a number of

According to Ohm’s law, passage of a small amount of current across a cell membrane causes the voltage across the membrane to change, reaching a new steady-state value that is determined by the membrane’s resistance (see above). The time course of this voltage change is determined by the product of the resistance rm and the capacitance cm of the membrane, with a rate constant equal to [rm ⴛ cm]⫺1. (The membrane’s capacitance results from having an insulator, the hydrocarbon core of the phospholipids in the membrane, between two conductors, the ionic solutions on either side of the membrane [see Fig. 7-2]. Capacitors store charge at both surfaces and require time to change the magnitude of this charge.) If the stimulated potential change is less than the threshold value, then the membrane voltage changes smoothly and returns to its resting value when the stimulating current is turned off (Fig. 7-5A). On the other hand, if the membrane voltage changes positively by more than the threshold value, then a dramatic event occurs: the membrane voltage rises much more rapidly, to a value of approximately ⫹50 mV, and then drops to its resting value of approximately ⫺80 mV (Fig. 7-5B). This “suprathreshold” event is known as the action potential (AP). Importantly, hyperpolarizing stimuli cannot trigger an AP (Fig. 7-5C).

CHAPTER 7 / Principles of Cellular Excitability and Electrochemical Transmission 87

Voltage (mV)

A 0 Threshold voltage

-50 -90

Small depolarizing stimulus

Voltage (mV)

B

0 Threshold voltage

-50 -90

Large depolarizing stimulus

INa  gNa (Vm  VNa )

C

Voltage (mV)

0

dependence of Po, shown in Figure 7-6A. Rapid membrane depolarizations to ⫺50 mV or above cause Na⫹ channels to open, with a probability that increases to a maximum value of 1.0 at about 0 mV. The open channel probability represents the fraction of all Na⫹ channels that open in response to a single voltage step. For example, at very negative potentials (e.g., ⫺85 mV), essentially no Na⫹ channels are open; as the membrane is depolarized through 0 mV, most or all Na⫹ channels open; and fast depolarizations to ⫺25 mV open about half of the Na⫹ channels. These are the relations that occur when a constant depolarization is imposed on the membrane (in a process called voltage-clamping); when the brief depolarization of an AP stimulates the membrane, fewer Na⫹ channels have time to reach the open state, and a large reserve of unopened channels provides a margin of safety for impulse transmission. Recall that ionic current is the product of the ionic conductance (g) and a potential difference. For ions, the potential difference is the same as the electrochemical driving force, Vm ⴚ Vx, where Vx is the Nernst potential for the specified ion. For example, for Na⫹ current:

Large hyperpolarizing stimulus Threshold voltage

-50

or INa  gNa Po (Vm  VNa ) _

-90

Time

FIGURE 7-5.

The action potential. A. In the example shown, a resting cell has a membrane potential of approximately ⫺80 mV. If a small depolarizing stimulus is applied to the cell (for example, a stimulus that opens a few voltage-gated Ca2⫹ channels), the membrane slowly depolarizes in response to the influx of Ca2⫹ ions. Once the stimulus ends and the Ca2⫹ channels close, the membrane returns to its resting potential. The time course of the voltage change is determined by the membrane capacitance (see Fig. 7-2). B. If a larger depolarizing stimulus is applied to the cell, such that the membrane potential exceeds its “threshold” voltage, the membrane rapidly depolarizes to about ⫹50 mV and then returns to its resting potential. This event is known as an action potential; its magnitude, time course, and shape are determined by voltage-gated Na⫹ and K⫹ channels that open in response to membrane depolarization. C. In comparison, application of a hyperpolarizing stimulus to a cell does not generate an action potential, regardless of the magnitude of hyperpolarization.

In most neurons, the balance between voltage-gated Na⫹ and K⫹ channels regulates the AP. (In cardiac cells, voltagegated Ca2⫹ channels are also involved in AP regulation; see Chapter 23.) Voltage-gated Na⫹ channels conduct an inward current that depolarizes the cell at the beginning of the AP. Voltage-gated K⫹ channels conduct an outward current that repolarizes the cell at the end of the AP, in preparation for the next excitatory event. Figure 7-6 shows the current–voltage (I-V) relationships for the voltage-gated Na⫹ channel and the “resting” K⫹ channel. The total Na⫹ conductance of the membrane is the product of the conductance of a single open Na⫹ channel, the total number of Na⫹ channels, and the probability that an individual Na⫹ channel is open, Po. Key to the excitability of the membrane is the voltage

Equation 7-4

Here, gNa is the Na⫹ conductance of the membrane when all Na⫹ channels are open, and Po is, as above, the probability that any individual Na⫹ channel is open. The graphic illustration of this equation is shown in Figure 7-6B, where the Na⫹ current for a “fully activated” membrane is described by the straight line that passes with positive slope through VNa. If there were no voltage dependence_ to the Na⫹ conductance (i.e., if gNa were always equal to gNa), this line would extend throughout the negative voltage range, as shown by its dashed-line extrapolation. However, the voltage dependence of Po (Fig. 7-6A) causes the actual Na⫹ conductance gNa to be voltage-dependent, resulting in deviation of the real INa from this theoretical “fully activated” condition. Thus, increasing depolarizations from rest (caused, for example, by an applied stimulus) result in inward Na⫹ currents that first become larger as more channels open and then become smaller as Vm approaches VNa (Fig. 7-6B). Potassium channels conduct outward currents that oppose the depolarizing actions of inward Na⫹ currents. Although there are many types of K⫹ channels with diverse “gating” properties, only two types need to be considered in order to appreciate the role of K⫹ channels in excitability. These two K⫹ channel types include the voltage-independent “leak” channels and the voltage-gated “delayed rectifier” channels. Leak channels are the K⫹ channels that contribute to the resting membrane potential by remaining open throughout the negative range of membrane potentials. The K⫹ current that flows through these channels is shown by the dashed line in Figure 7-6B; for these channels, K⫹ current is outward for all Vm ⬎ VK. The summation of INa and IK(leak) is represented by the dashed blue line in Figure 7-6C. Three important points on this line define three critical aspects of the AP. The net ionic current (INet) is zero at all three of these points. First, at rest, Vm ⬇ VK. Under this condition, small, transient membrane depolarizations caused by “external” stimuli result in net

88 Fundamental Principles of Neuropharmacology A

FIGURE 7-6.

1

P0 0 -50

0

B Outward Current

50

V (mV)

INa, IK INa Na+ channels begin to open

-90

IK

-50

50

V (mV)

VNa

Inward Current

All Na+ channels open

C Outward Current

INa, IK, INet IK INa VK -90

VT

VP

-50

50

V (mV)

Inward Current

INet

outward currents from ion conductances that repolarize the membrane back to rest when the external stimulus ends. Second, at Vm ⴝ VT, the outward potassium currents are matched by inward sodium currents, and the net current is also zero. Under this condition, however, even a small further depolarization results in a net inward current that further depolarizes the membrane, which leads to a larger inward current and further membrane depolarization. This positive feedback loop constitutes the rising phase of the AP. Thus, the AP occurs in response to any rapid depolarization beyond VT, which is defined as the threshold potential. Third, Vp is the potential at the peak of the AP. Once Vm reaches this maximum depolarization, the net current switches sign from inward to outward, and consequently, the membrane begins to be repolarized. Voltage-gated (delayed rectifier) K⫹ channels contribute to the rapid repolarization phase of the AP. Although membrane depolarization opens these channels, they open and close more slowly than do Na⫹ channels in response to depolarization. Therefore, inward Na⫹ current dominates the early (depolarization) phase of the AP, and outward K⫹ current dominates the later (repolarization) phase (Fig. 7-7). This is why the AP is characterized by an initial rapid depolarization (caused by fast inward Na⫹ current) followed by a prolonged repolarization (caused by slower and more sustained outward K⫹ current). The final feature determining membrane excitability is the limited duration of Na⫹ channel opening in response to membrane depolarization. After opening in response to rapid

Voltage dependence of channel activity. A. Po, the probability that an individual voltage-gated Na⫹ channel will open, is a function of the membrane voltage (V ). At voltages more negative than ⫺50 mV, there is a very low probability that a voltage-gated sodium channel will open. At voltages more positive than ⫺50 mV, this probability begins to increase and approaches 1.0 (i.e., a 100% chance of opening) at 0 mV. These probabilities are also generalizable to a population of voltage-gated Na⫹ channels, so that virtually 100% of voltage-gated Na⫹ channels in the membrane will open at 0 mV. B. The Na⫹ current across a membrane (INa) is a function of the voltage dependence of the Na⫹ channels that carry the current. At voltages more negative than ⫺50 mV, the Na⫹ current is zero. As the voltage increases above ⫺50 mV, Na⫹ channels begin to open, and there is an increasingly inward (negative) Na⫹ current. The maximum inward Na⫹ flux is reached at 0 mV, when all the channels are open. As the voltage continues to increase above 0 mV, the Na⫹ current is still inward, but decreasing, because inward flow of the positively charged Na⫹ ions is opposed by the increasingly positive intracellular potential. The Na⫹ current is zero at VNa (the Nernst potential for Na⫹) because, at this voltage, the electrical and chemical gradients for Na⫹ ion flow are balanced. At voltages more positive than VNa, the Na⫹ current is outward (positive). The dashed line indicates the relationship that would exist between Na⫹ current and voltage if the open probability of the Na⫹ channels were not voltage-dependent. The potassium current that flows through voltage-independent K⫹ “leak channels” is shown by the dashed line (IK). C. The summation of plasma membrane Na⫹ currents (INa ) and K⫹ currents (IK ) demonstrates three key transition points in the I-V graph (denoted by blue circles) at which the net current is zero. The first of these points occurs at a membrane potential of ⫺90 mV, where V ⫽ VK. At this voltage, a small increase in potential (i.e., a small depolarization) results in an outward (positive) K⫹ current that brings the membrane potential back toward VK. The second point occurs at VThreshold, the threshold voltage (VT ). At this voltage, INa ⫽ ⫺IK; further depolarization results in the opening of more voltage-dependent Na⫹ channels and a net negative (inward) current, which initiates the action potential. The third point occurs at VPeak, the peak voltage (VP). At this voltage, the transition occurs from a net negative current to a net positive (outward) current. As the Na⫹ channels inactivate, the net positive current is dominated by IK, and the membrane potential returns toward VK (i.e., the membrane is repolarized).

membrane depolarization, most Na⫹ channels enter a closed state in which they are inactivated (i.e., prevented from subsequent opening). Recovery from inactivation occurs only when the membrane is repolarized, whereupon the Na⫹ channels return to the closed, resting state from which they can open in response to a stimulus. This inactivation of Na⫹ conductance, combined with the slowly decaying voltage-gated K⫹ conductance, produces dynamic changes in membrane excitability. Following _just one AP, fewer Na⫹ channels are available to open (i.e., gNa is temporarily smaller), more K⫹ channels are open (i.e., gK is larger), the corresponding ionic currents are changed, and VT is more positive than it was before the AP. An excitable membrane is in its so-called refractory state during this period, which lasts from just after the AP until the conditions of fast gNa inactivation and slow gK activation have returned to their resting values. Very slow depolarizing stimuli will fail to induce an AP, even when the membrane reaches the threshold potential defined by a rapid depolarizing stimulus, because of the accumulation of inactivated Na⫹ channels during the slow depolarizing stimulus. The inactivation property of Na⫹ channels is important in the concept of use-dependent block, as discussed in Chapter 11, Local Anesthetic Pharmacology, and Chapter 23, Pharmacology of Cardiac Rhythm. Also, under pathologic conditions, cells may express Na⫹ channels that inactivate incompletely and therefore continue to carry an inward current after termination of the AP. Such currents may be adequate to raise the

VNa

Voltage

Vm

VT Vr VK 0

1

2

3

4

2

3

4

Conductance

gNa gK

0

0

1

Time (ms) Depolarizating stimulus

90 Fundamental Principles of Neuropharmacology

Presynaptic neuron Action potential Neurotransmitter transporter

2 1 Precursor

6b Na

Na+

+

Neurotransmitter

Ca2+

3 4

Synaptic cleft 5a Ca2+

K+

5b

6a

Adenylyl cyclase

Na+

α β GDP

γ

E

α GTP

ATP

cAMP

Postsynaptic cell

7

Phosphodiesterase AMP

FIGURE 7-8. Steps in synaptic transmission. Synaptic transmission can be divided into a series of steps that couple electrical depolarization of the presynaptic neuron to chemical signaling between the presynaptic and postsynaptic cells. 1. Neuron synthesizes neurotransmitter from precursors and stores the transmitter in vesicles. 2. An action potential traveling down the neuron depolarizes the presynaptic nerve terminal. 3. Membrane depolarization activates voltage-dependent Ca2⫹ channels, allowing Ca2⫹ entry into the presynaptic nerve terminal. 4. The increased cytosolic Ca2⫹ enables vesicle fusion with the plasma membrane of the presynaptic neuron, with subsequent release of neurotransmitter into the synaptic cleft. 5. Neurotransmitter diffuses across the synaptic cleft and binds to one of two types of postsynaptic receptors. 5a. Neurotransmitter binding to ionotropic receptors causes channel opening and changes the permeability of the postsynaptic membrane to ions. This may also result in a change in the postsynaptic membrane potential. 5b. Neurotransmitter binding to metabotropic receptors on the postsynaptic cell activates intracellular signaling cascades; the example shows G protein activation leading to the formation of cAMP by adenylyl cyclase. In turn, such a signaling cascade can activate other ion-selective channels (not shown). 6. Signal termination is accomplished by removal of transmitter from the synaptic cleft. 6a. Transmitter can be degraded by enzymes (E ) in the synaptic cleft. 6b. Alternatively, transmitter can be recycled into the presynaptic cell by reuptake transporters. 7. Signal termination can also be accomplished by enzymes (such as phosphodiesterase) that degrade postsynaptic intracellular signaling molecules (such as cAMP).

3. Depolarization of the nerve terminal membrane causes opening of voltage-dependent Ca2⫹ channels and influx of Ca2⫹ through these open channels into the presynaptic nerve terminal. In many neurons, this Ca2⫹ influx is regulated by P/Q-type (Cav 2.1) or N-type (Cav 2.2) Ca2⫹ channels. 4. In the presynaptic terminal, the rapid rise in cytosolic free Ca2⫹ concentration is sensed by specialized protein machinery, causing neurotransmitter-filled vesicles to fuse with the presynaptic plasma membrane (see the next section, Synaptic Vesicle Regulation). After vesicle fusion, neurotransmitter is released into the synaptic cleft. 5. Released neurotransmitter diffuses across the synaptic cleft, where it can bind to two classes of receptors in the postsynaptic membrane: a. Binding of neurotransmitter to ligand-gated ionotropic receptors opens channels that mediate ion flux across the postsynaptic membrane. Within milliseconds, this ion flux leads to excitatory or inhibitory postsynaptic potentials. b. Binding of neurotransmitter to metabotropic receptors (e.g., G protein-coupled receptors) causes activation of intracellular second messenger signaling cascades. These signaling events can then modulate ion channel function, leading to changes in the postsynaptic potential, although the time course of these changes is slower (generally seconds to minutes). Some neurotransmitters may also bind to a third class of receptors on the presynaptic membrane. These receptors are called autoreceptors because they regulate neurotransmitter release. 6. Excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) propagate passively (i.e., without generating an AP) along the membrane of the postsynaptic cell. A large number of EPSPs can summate to cause the postsynaptic membrane potential to exceed threshold voltage (VT). If this occurs, an AP can be generated in the postsynaptic cell. (This process is not shown in Fig. 7-8.) 7. Stimulation of the postsynaptic cell is terminated by removal of the neurotransmitter, desensitization of the postsynaptic receptor, or a combination of both. Neurotransmitter removal occurs by two mechanisms: a. Degradation of the neurotransmitter by enzymes in the synaptic cleft. b. Uptake of the neurotransmitter by specific transporters into the presynaptic terminal (or the surrounding glial cells), which terminates synaptic action and allows the neurotransmitter to be recycled into synaptic vesicles in preparation for a new release event. 8. For G protein-coupled metabotropic receptors in the postsynaptic cell, termination of the response to a transmitter stimulus is also dependent on intracellular enzymes that inactivate second messengers (e.g., phosphodiesterases that convert cAMP to its inactive metabolite AMP). The prototypic chemical synapse is that of the neuromuscular junction (see Fig. 9-4 for more detail). At this junction, terminal branches of the motor axon lie in a synaptic trough on the surface of the muscle cells. When the neuron fires, acetylcholine (ACh) is released from the motor neuron

CHAPTER 7 / Principles of Cellular Excitability and Electrochemical Transmission 91

terminals. The released ACh diffuses across the synaptic cleft to bind to ligand-gated ionotropic receptors located on the postsynaptic muscle membrane. This binding of ACh to its receptors causes a transient increase in the probability of opening of receptor-associated ion channels. The channel pore is equally permeable to Na⫹ and K⫹, and these channels have a reversal potential (i.e., a potential at which there is no net current flowing through the channel) of approximately 0 mV (the average of the individual Na⫹ and K⫹ Nernst potentials). The net inward current passing through these open channels depolarizes the muscle cell membrane. Although this particular end-plate potential is sufficiently large to stimulate an AP in the muscle, its magnitude is unusual, because most neuronal excitatory postsynaptic potentials are of insufficient magnitude to stimulate an AP. More commonly, several neuronal excitatory postsynaptic potentials must occur together, within a short time (⬃10 ms) and at closely spaced synapses, in order for the postsynaptic depolarization to reach the threshold value for firing of an AP. The following discussion highlights steps in the basic processes of neurotransmission that can be modified by pharmacologic agents.

Synaptic Vesicle Regulation Nerve terminals contain two types of secretory vesicles: small, clear-core synaptic vesicles and large, dense-core synaptic vesicles. The clear-core vesicles store and secrete small organic neurotransmitters such as acetylcholine, GABA, glycine, and glutamate. Dense-core vesicles are more likely to contain peptide or amine neurotransmitters. The larger dense-core vesicles are similar to the secretory granules of endocrine cells because their release is not limited to “active zones” on the presynaptic cell. Dense-core vesicle release is also more likely to follow a train of impulses (continuous or rhythmic stimulation) than a single AP. Hence, the smaller clear-core vesicles are involved in rapid chemical transmission, while the larger dense-core vesicles are implicated in slow, modulatory, or distant signaling. Over the past several years, many of the proteins that control synaptic vesicle trafficking have been identified. Synapsin is a protein with dynamic affinity for synaptic vesicles that also binds to actin. This binding allows it to link vesicles to the cytoplasmic actin cytoskeleton at nerve terminals. Because synapsin is a major substrate for a variety of protein kinases, including those regulated by cAMP and Ca2⫹/ calmodulin, it is thought that these second messengers act

in neurotransmitter release by controlling the availability of synaptic vesicles for Ca2⫹-dependent exocytosis. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) present in both the vesicle membrane (synaptobrevin) and presynaptic plasma membrane (syntaxin and SNAP-25) provide the driving force for both Ca2⫹-regulated and Ca2⫹-independent vesicle exocytosis (Fig. 7-9). Certain neurotoxins, such as tetanus toxin and botulinum toxin (see Chapter 9), appear to act by selectively cleaving SNAREs Neurotransmitter

A Synaptic vesicle membrane Cytoplasm SNARE complex Presynaptic plasma membrane Voltage-gated calcium channel (closed)

B

Synaptotagmin-1 (Ca2+ sensor)

Ca2+

Ca2+

C

FIGURE 7-9.

Current model of neurotransmitter release. A. Synaptic vesicles are tethered close to the plasma membrane of the presynaptic neuron by several protein–protein interactions. The most important of these interactions involve SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins present in both the vesicle membrane and the plasma membrane. The SNARE proteins include synaptobrevin (red), syntaxin-1 (yellow), and SNAP-25 (green). Voltage-gated Ca2⫹ channels are located in the plasma membrane in close proximity to these SNARE complexes; this facilitates the sensing of Ca2⫹ entry by Ca2⫹-binding proteins (synaptotagmin-1, in blue) localized to the presynaptic plasma membrane and/or the synaptic vesicle membrane. B–D. Voltage-gated calcium channels open in response to an action potential, allowing entry of extracellular Ca2⫹ into the cell. The increase in intracellular Ca2⫹ triggers binding of synaptotagmin-1 to the SNARE complex and fusion of the vesicle membrane with the plasma membrane, releasing neurotransmitter molecules into the synaptic cleft. Several additional proteins (Munc18-1, Munc13-1, complexin-1, and others) may also be involved in the regulation of synaptic vesicle fusion (not shown).

Action potential

D

92 Fundamental Principles of Neuropharmacology

and thereby inhibiting synaptic vesicle exocytosis. SNAREassociated proteins such as synaptotagmin and complexin are critical for the Ca2⫹ sensitivity of vesicle release; together with synapsin, the SNAREs, and other recently discovered proteins involved in neurotransmitter release, these SNAREassociated proteins may provide future targets for pharmacologic control of synaptic transmission.

Postsynaptic Receptors A large number of neuropharmacologic drugs act on postsynaptic receptors. These integral membrane proteins fall into two classes: ionotropic and metabotropic. Ionotropic receptors, such as nicotinic acetylcholine receptors and “A” type GABA receptors, are almost always composed of four to five subunits that oligomerize in the membrane to form a ligand-gated channel. Binding of one (or sometimes two) ligand molecule to the receptor leads to an allosteric conformational change that opens the channel pore. The subunits composing the same functional receptor often differ among different tissues and, as a consequence, the detailed molecular pharmacology of the receptors is tissue-dependent. For example, although acetylcholine is the endogenous transmitter for all nicotinic cholinergic receptors, a number of synthetic agonists (or antagonists) selectively activate (or inhibit) these receptors in skeletal muscle, autonomic ganglia, or the central nervous system (see Chapter 9). Metabotropic receptors are similarly diverse. Although most are G protein-coupled receptors, the extracellular and cytoplasmic domains of these receptors differ significantly. These differences enable the development of agonists (or antagonists) that activate (or inhibit) specific subtypes of metabotropic receptors.

Transmitter Metabolism and Reuptake Altering the metabolism of the neurotransmitter provides an important mechanism for pharmacologic intervention at the synapse. The two major types of intervention involve inhibition of neurotransmitter degradation and antagonism of neurotransmitter reuptake. Acetylcholinesterase, the enzyme responsible for degrading acetylcholine, is an example of the first type of drug target. Acetylcholinesterase inhibitors are the mainstays of treatment for myasthenia gravis (see Chapter 9). The transporters that facilitate neurotransmitter reuptake from the synaptic cleft into the presynaptic cell are of even greater importance. Because these reuptake transporters are

crucial for the termination of synaptic transmission, their inhibition has profound effects. For example, the psychotropic effects of cocaine derive from this drug’s ability to inhibit dopamine and norepinephrine reuptake in the brain, and the therapeutic benefit of antidepressants such as fluoxetine likely results from inhibition of serotonin-selective reuptake (see Chapter 14, Pharmacology of Serotonergic and Central Adrenergic Neurotransmission). Because reuptake transporters tend to be substrate-specific, it is anticipated that new drugs can be designed to selectively target other specific transporter subtypes as well.

 CONCLUSION AND FUTURE DIRECTIONS Cellular excitability is a crucial component of intercellular communication. The fundamental basis for cellular excitability lies in the electrochemical gradients that are established by ion pumps across the lipid bilayer of the plasma membrane. Ion-selective channels enable cellular membranes to regulate the permeability of the membrane selectively for different ionic species, allowing a change in membrane voltage to be coupled to a chemical stimulus or response. The action potential, a special type of stereotyped response found in excitable cells, is made possible by the voltage-dependent properties of Na⫹ and K⫹ channels. The basic processes of electrochemical transmission provide the substrate for pharmacologic modulation of cellular excitation and communication, topics that are addressed in more detail throughout this book.

Acknowledgment We thank Michael Ty for his valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Nestler EJ, Hyman SE, Malenka RC. Molecular neuropharmacology: a foundation for clinical neuroscience. 2nd ed. New York: McGraw-Hill Professional; 2008. (An overview of neuropharmacology.) Rizo J, Rosenmund C. Synaptic vesicle fusion. Nat Struct Mol Biol 2008;15:665–674. (Review of mechanisms that regulate synaptic vesicle fusion.) Rizzoli SO, Betz WJ. Synaptic vesicle pools. Nat Rev Neurosci 2005;6:57–69. (Advances in synaptic vesicle biology.) Sutton MA, Schuman EM. Partitioning the synaptic landscape: distinct microdomains for spontaneous and spike-triggered neurotransmission. Sci Signal 2009;2:pe19. (Recent research on regulation of synaptic transmission.)

8 Principles of Nervous System Physiology and Pharmacology Joshua M. Galanter, Susannah B. Cornes, and Daniel H. Lowenstein

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . . 93-94 NEUROANATOMY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Anatomy of the Peripheral Nervous System . . . . . . . . . . . . 93 Autonomic Nervous System . . . . . . . . . . . . . . . . . . . . . 94 Peripheral Motor and Sensory Systems . . . . . . . . . . . . . 96 Anatomy of the Central Nervous System . . . . . . . . . . . . . . 96 Cerebrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Diencephalon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Cerebellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Brainstem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Cellular Organization of the Nervous System . . . . . . . . . . . 99 Long Tract Neuronal Organization . . . . . . . . . . . . . . . . . 99

Local Circuit Neuronal Organization . . . . . . . . . . . . . . 100 Single-Source Divergent Neuronal Organization . . . . . 100 NEUROPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Amino Acid Neurotransmitters. . . . . . . . . . . . . . . . . . . 102 Biogenic Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Other Small Molecule Neurotransmitters . . . . . . . . . . . 106 Neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 The Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . 106 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 107 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

 INTRODUCTION

 NEUROANATOMY

The nervous system contains more than 10 billion neurons. Most neurons form thousands of synaptic connections, giving the nervous system complexity unlike that seen in any other organ system. Interactions among neuronal circuits mediate functions ranging from primitive reflexes to language, mood, and memory. To perform these functions, the individual neurons that comprise the nervous system must be organized into functional networks, which, in turn, are organized into larger anatomical units. The previous chapter reviewed the physiology of individual neurons by describing electrical transmission within a neuron and chemical transmission from one neuron to another. This chapter discusses neuronal systems by examining two levels of organization. First, the gross anatomical organization of the nervous system is presented, to place in context the sites of action of pharmacologic agents that act on this system. Second, the major patterns of neuronal connectivity (so-called neuronal tracts) are presented, because knowledge of the ways in which neuronal cells are organized to transmit, process, and modulate signals facilitates a deeper understanding of the actions of drugs on these tracts. This chapter also discusses the major types of neurotransmitters and the blood–brain barrier; these functional and metabolic concepts have important pharmacologic consequences for drugs that act on the nervous system.

The nervous system can be divided structurally and functionally into peripheral and central components. The peripheral nervous system includes all nerves traveling between the central nervous system and somatic and visceral sites. It is divided functionally into the autonomic (involuntary) nervous system and the sensory and somatic (voluntary) nervous system. The central nervous system (CNS) includes the cerebrum, diencephalon, cerebellum, brainstem, and spinal cord. The CNS relays and processes signals received from the peripheral nervous system; the processing results in responses that are formulated and relayed back to the periphery. The CNS is responsible for important functions such as perception—including sensory, auditory, and visual processing—wakefulness, language, and consciousness.

Anatomy of the Peripheral Nervous System The autonomic nervous system regulates involuntary responses of smooth muscle and glandular tissue. For example, it controls vascular tone, heart rate and contractility, pupillary constriction, sweating, salivation, piloerection (“goose bumps”), uterine contraction, gastrointestinal (GI) motility, and bladder function. The autonomic nervous system is divided into the sympathetic nervous system, responsible for 93

Dorsal root ganglion Dorsal root Gray matter White matter

Sympathetic nerve trunk Spinal cord Skin

Ventral root Prevertebral ganglion Smooth muscle Sensory neuron Paravertebral chain ganglion

Somatic motor neuron Preganglionic neuron Postganglionic neuron

Adrenal medulla

Skeletal muscle

CHAPTER 8 / Principles of Nervous System Physiology and Pharmacology 95

Sympathetic nervous system

Parasympathetic nervous system Eyes Oculomotor nerve (CN III)

Salivary glands Facial nerve (CN VII)

Respiratory tract

Glossopharyngeal nerve (CN IX)

Cranial Vagus nerve (CN X)

C1 C2 C3 C4 C5 C6 C7 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 L1 L2

Heart Cervical

Skin

Liver

Stomach

Thoracic Celiac ganglion Adrenal gland

Pancreas Intestines

Kidney

Lumbar

L3

Bladder

L4

Superior mesenteric ganglion

L5 S1 S2 S3 S4 S5

Peripheral blood vessel

Sacral

Sympathetic trunk Inferior mesenteric ganglion

Paravertebral ganglia

Prevertebral ganglia

External genitalia Sympathetic preganglionic fibers Sympathetic postganglionic fibers Parasympathetic preganglionic fibers Parasympathetic postganglionic fibers

FIGURE 8-2. Patterns of sympathetic and parasympathetic innervation. Sympathetic preganglionic neurons arise in the thoracic and lumbar segments of the spinal cord. Sympathetic preganglionic neurons project onto postganglionic neurons in ganglia that lie close to the spinal cord, most notably the paravertebral ganglia, and in the prevertebral ganglia located near the aorta. Parasympathetic ganglia generally lie close to the organs they innervate. Thus, parasympathetic preganglionic neurons, which arise in nuclei in the brainstem and the sacral segments of the spinal cord, are generally long and project onto short postganglionic neurons. organ. As discussed below, the anatomical location of these connections differs for neurons of the sympathetic and parasympathetic divisions of the autonomic nervous system. Anatomy of the Sympathetic Nervous System

The sympathetic nervous system is also known as the thoracolumbar system, because its preganglionic fibers arise

from the first thoracic segment to the second or third lumbar segment of the spinal cord (Fig. 8-2). Specifically, the preganglionic nerve cell bodies arise from the intermediolateral columns in the spinal cord. Preganglionic nerves exit the spinal cord at the ventral roots of each vertebral level and make synaptic connections with postganglionic neurons in sympathetic ganglia. Most sympathetic ganglia are located in

96 Fundamental Principles of Neuropharmacology

the sympathetic chain, which consists of 25 pairs of interconnected ganglia that lie on either side of the vertebral column. The first three ganglia, termed the superior cervical ganglion, middle cervical ganglion, and inferior cervical ganglion, send their postganglionic fibers via the cranial and cervical spinal nerves. The superior cervical ganglion innervates the pupil, salivary glands, and lacrimal glands, as well as blood vessels and sweat glands in the head and face (Fig. 8-2). Postganglionic neurons arising in the middle and inferior cervical ganglia, as well as the thoracic ganglia, innervate the heart and lungs. Fibers arising from the remaining paravertebral ganglia innervate sweat glands, pilomotor muscles, and blood vessels of skeletal muscle and skin throughout the body. Postganglionic neurons that innervate the GI tract down to the sigmoid colon, including the liver and pancreas, arise from ganglia that are located anterior to the aorta, at the origins of the celiac, superior mesenteric, and inferior mesenteric blood vessels (Fig. 8-2). Hence, these ganglia, collectively known as prevertebral ganglia, are named the celiac ganglion, superior mesenteric ganglion, and inferior mesenteric ganglion, respectively. In contrast to the paravertebral ganglia, the prevertebral ganglia have long preganglionic fibers and short postganglionic fibers. The adrenal medulla is contained within the adrenal glands that lie on the superior surface of the kidneys. The adrenal medulla contains postsynaptic neuroendocrine cells (Fig. 8-2). Unlike sympathetic postganglionic neurons, which synthesize and release norepinephrine, neuroendocrine cells of the adrenal medulla synthesize primarily epinephrine (85%) and release this neurotransmitter into the bloodstream rather than at synapses on a specific target organ (see Chapter 10, Adrenergic Pharmacology). Many pharmacologic agents modulate sympathetic nervous system activity. As discussed in Chapter 10, the sympathetic nervous system has an organ-specific distribution of adrenergic receptor types. This organ-specific receptor expression allows drugs to modulate sympathetic activity selectively. For example, certain sympathetic agonists, such as albuterol, can dilate bronchioles selectively, while certain sympathetic antagonists, such as metoprolol, can selectively decrease heart rate and contractility. Anatomy of the Parasympathetic Nervous System

Nearly all of the parasympathetic ganglia lie in or near the organs they innervate. The preganglionic fibers of the parasympathetic nervous system arise in the brainstem or in sacral segments of the spinal cord; thus, the parasympathetic system is also called the craniosacral system (Fig. 8-2). In some cases, parasympathetic preganglionic neurons can travel almost one meter before synapsing with their postganglionic targets. Preganglionic nerve fibers of cranial nerve (CN) III, the oculomotor nerve, arise from a region of the midbrain termed the Edinger-Westphal nucleus and innervate the pupil, stimulating it to constrict. The medulla of the brain contains nuclei for parasympathetic nerve fibers in CNs VII, IX, and X. Parasympathetic fibers in the facial nerve (CN VII) stimulate salivary secretion by the submaxillary and sublingual glands as well as tear production by the lacrimal gland. Parasympathetic fibers in the ninth cranial nerve, the glossopharyngeal nerve, stimulate the parotid gland. The 10th cranial nerve, termed the vagus nerve, provides parasympathetic innervation to the major organs in the chest and abdomen, including the heart, tracheobronchial

tree, kidneys, and GI system down to the proximal colon. Parasympathetic nerves originating in the sacral region of the spinal cord innervate the remainder of the colon, urinary bladder, and genitalia. Many pharmacologic agents modulate parasympathetic nervous system activity. For example, bethanechol is a parasympathomimetic that promotes GI and urinary tract motility. Antagonists of parasympathetic activity include atropine, a drug used locally to dilate the pupils or systematically to increase heart rate, and ipratropium, a drug used to dilate bronchioles. These agents and others are discussed in Chapter 9, Cholinergic Pharmacology. Peripheral Motor and Sensory Systems Fibers of the somatic nervous system innervate their target striated muscles directly (Fig. 8-1). The first-order neurons from the motor cortex send projections that cross in the lower medulla and descend through the spinal cord in the lateral corticospinal tract before synapsing on the second-order neurons in the ventral horns of the spinal cord. Projections from the second-order neurons exit through the ventral roots and join the dorsal roots, carrying sensory nerve fibers, to form the spinal nerves. Spinal nerves exit the vertebral column through the intervertebral foramina, after which they separate into peripheral nerves. Somatic components of the peripheral nerves innervate muscles directly. Muscles are innervated in a myotomal distribution. That is, neurons originating from a particular ventral root level of the spinal cord (e.g., C6) innervate specific muscles (e.g., flexor muscles of the forearm). Sensory neurons have cell bodies in the dorsal root ganglia. The endings of sensory nerves lie in the skin and joints and enter the spinal cord through the dorsal roots. Neurons for vibration and position sense (proprioception) ascend through the ipsilateral dorsal columns in the spinal cord and synapse with secondary neurons in the contralateral lower medulla. Sensory neurons that carry sensations of pain and temperature synapse with secondary neurons in the posterior horn of the spinal cord and then cross within the spinal cord to ascend in the contralateral spinothalamic tract. Both the spinothalamic tract and the dorsal column tracts connect with third-order neurons in the thalamus, part of the diencephalon (see below), before ultimately reaching the somatosensory cortex. Sensory information is encoded in a dermatomal distribution. That is, neurons originating from a particular dorsal root level of the spinal cord (e.g., C6) carry sensory information corresponding to a particular area of the skin (e.g., the lateral aspects of the forearm and hand). A number of pharmacologic agents modulate the activity of the somatic nervous system. For example, antagonists of neuromuscular junction activity, such as pancuronium, are used to induce paralysis during surgery. In contrast, drugs that increase neuromuscular junction activity, such as edrophonium and neostigmine, are used in the diagnosis and treatment of myasthenia gravis, an autoimmune disease characterized by decreased skeletal muscle stimulation at the neuromuscular junction. These agents and others are discussed in Chapter 9.

Anatomy of the Central Nervous System The CNS is divided anatomically into seven major divisions, namely, the cerebral hemispheres, diencephalon, cerebellum, midbrain, pons, medulla, and spinal cord (Fig. 8-3).

CHAPTER 8 / Principles of Nervous System Physiology and Pharmacology 97

Cerebral hemisphere: Cerebral cortex Basal ganglia

Brainstem

Midbrain Pons Medulla

Diencephalon

Cerebellum

perception, planning and ordering motor functions, cognitive functions, such as abstract reasoning, and language. The cortex is divided anatomically and functionally into the frontal, temporal, parietal, and occipital lobes (Fig. 8-4A). Subregions of the cortex have specific functions. For example, stimulation of part of the precentral gyrus, which lies in the frontal cortex, induces peripheral motor function (movement), and ablation of this structure inhibits movement. From a pharmacologic perspective, the cerebral cortex is a site of action of many drugs, sometimes as

Cervical A Frontal lobe

Parietal lobe

Temporal lobe

Spinal cord Thoracic

Occipital lobe

B Cingulate gyrus

Corpus callosum

Lumbar

Sacral

FIGURE 8-3. Anatomic organization of the central nervous system. The central nervous system is divided into seven major regions: the cerebral hemispheres, diencephalon (thalamus), cerebellum, midbrain, pons, medulla, and spinal cord. The cerebral hemispheres include the cerebral cortex, underlying white matter (not shown), and basal ganglia. The midbrain, pons, and medulla together make up the brainstem. The spinal cord is further divided into cervical, thoracic, lumbar, and sacral segments.

The midbrain, pons, and medulla are collectively known as the brainstem and together connect the spinal cord with the cerebrum, diencephalon, and cerebellum. Cerebrum The cerebral hemispheres constitute the largest division of the human brain. These structures contain several subdivisions, including the cerebral cortex, its underlying white matter, and the basal ganglia (Fig. 8-4). The cerebral hemispheres are divided into left and right sides that are connected by the corpus callosum. The cerebral cortex is responsible for high-level functions, including sensory

C

Inte

r nal capsule

Caudate Thalamus Putamen

FIGURE 8-4.

Anatomy of the cerebral hemispheres. A. In this lateral view, the cerebral hemispheres are divided into four lobes—frontal, parietal, occipital, and temporal—which are structurally and functionally distinct from each other. B. A sagittal view of the cerebral hemispheres shows the corpus callosum and cingulate gyrus. The corpus callosum connects the left and right hemispheres and coordinates their actions. The cingulate gyrus is part of the limbic system; it lies immediately superior to the corpus callosum. C. The basal ganglia include the caudate and putamen, which are together known as the striatum, and the globus pallidus (medial to the putamen, not shown). The thalamus lies medial to the basal ganglia. Arrows indicate the trajectory of neurons in the internal capsule, a bundle of white matter that carries motor commands from the cortex to the spinal cord.

Flocculonodular lobe

Cerebellar vermis Cerebellar hemispheres

CHAPTER 8 / Principles of Nervous System Physiology and Pharmacology 99

Brainstem The midbrain, pons, and medulla are collectively known as the brainstem. The brainstem connects the spinal cord to the thalamus and cerebral cortex. It is arranged with the midbrain superior, the medulla inferior, and the pons bridging the midbrain and medulla (Fig. 8-3). White matter pathways interconnecting the spinal cord, cerebellum, thalamus, basal ganglia, and cerebral cortex course through this small region of the brain. In addition, the brainstem gives rise to most of the cranial nerves. Some of these nerves are conduits for sensation from the head and face, including hearing, balance, and taste. The cranial nerves also control the motor output to the skeletal muscles of mastication, facial expression, swallowing, and eye movement. The brainstem also regulates parasympathetic output to the salivary glands and the iris. The medulla contains several control centers that are essential for life, including centers that direct the output of the autonomic nuclei, pacemakers that regulate heart rate and breathing, and centers that control reflex actions such as coughing and vomiting. Several relay structures in the pons also play a role (in conjunction with the midbrain) in regulating vital functions such as respiration. The base of the pons contains white matter tracts connecting the cerebral cortex and the cerebellum. Neurons in the periaqueductal gray, especially in the midbrain, send descending projections to the spinal cord that modulate pain perception (see Chapter 17, Pharmacology of Analgesia). Clusters of diffusely projecting neurons lie throughout the brainstem, hypothalamus, and the surrounding base of the brain. These nuclei, which include the locus ceruleus, raphe nucleus, and several others, comprise the reticular activating system, which is responsible for consciousness and sleep regulation. The nuclei each use a different neurotransmitter system (see below), and thus a variety of classes of medications can have effects on this system. For example, it is via these nuclei that antihistamines cause sedation and stimulants such as cocaine cause heightened alertness. Spinal Cord The spinal cord is the most caudal division of the central nervous system. It runs from the base of the brainstem (medulla) at the level of the first cervical vertebra down to the first lumbar vertebra. Like the cerebrum, the spinal cord is organized into white matter tracts and regions of gray matter. The white matter tracts connect the periphery and spinal cord to more rostral divisions of the CNS, while the gray matter forms the nuclear columns that lie in an “H-shaped” pattern in the center of the spinal cord (Fig. 8-6). Neurons in the spinal cord can be defined by their spatial location relative to the gray matter “H.” These neurons include sensory neurons located in the dorsal horns of the “H,” motor neurons located in the ventral horns of the “H,” and spinal interneurons. The sensory neurons relay information from the periphery to more rostral divisions of the CNS via the dorsal columns or spinothalamic tracts (see above). The motor neurons relay commands arising in the central motor areas and descend in the corticospinal tract to peripheral muscles. Interneurons connect sensory and motor neurons and are responsible for mediating reflexes, such as the deep tendon reflexes, by coordinating the action of opposing muscle groups. Because the spinal cord carries sensory signals— including sensations of pain—to the central nervous system, it is an important target for analgesic drugs such as opioids (see Chapter 17).

Gray matter

Ventral horn

White matter Dorsal horn

Dorsal root

Dorsal root ganglion

Dura

FIGURE 8-6. Anatomy of the spinal cord. The spinal cord has an H-shaped wedge of gray matter that includes the dorsal and ventral horns. The dorsal horn is responsible for sensory relays to the brain, and the ventral horn is responsible for motor relays to skeletal muscle. The white matter carries signals to and from more rostral divisions of the CNS.

Cellular Organization of the Nervous System Cellular organization in the autonomic and peripheral nervous system involves a limited number of neurons that make few connections. For example, somatic and sensory information is carried directly between the spinal cord and the periphery. Autonomic nerves are slightly more complex, in that the signal must undergo synaptic transmission between a preganglionic and a postganglionic neuron. In both cases, however, few ancillary neuronal connections are made, and little or no modification of information occurs. In contrast, cellular organization in the central nervous system is far more complex. Information is not simply relayed from one area to another; instead, central neurons receive signals from numerous sources and distribute their own axons widely. Some neurons synapse with hundreds of thousands of other neurons. Moreover, not every synaptic connection is excitatory (i.e., designed to depolarize the postsynaptic neuron). Some connections are inhibitory (i.e., designed to hyperpolarize the postsynaptic neuron). Other neurons projecting onto a target neuron can modulate the relative excitability of the cell, affecting the response of the postsynaptic neuron to other signals. The complexity generated by this variability is needed to carry out the many intricate processes performed by the brain. Although the CNS possesses immense complexity at the level of neuronal connectivity, three major motifs are used to organize neurons into functional units in the nervous system: the long tract neuronal systems, local circuits, and singlesource divergent systems (Fig. 8-7). The peripheral nervous system is organized exclusively as a long tract system, while the central nervous system uses all three motifs. Long Tract Neuronal Organization Long tract neuronal organization involves neural pathways that connect distant areas of the nervous system to one another (Fig. 8-7A). It is the organization used by the peripheral

100 Fundamental Principles of Neuropharmacology

A Long-tract

B Local circuit

C Single-source divergent

Convergent signaling

Divergent signaling

FIGURE 8-7. Cellular organization of the central nervous system. The CNS has three main organizational motifs. A. Long-tract neurons act as relays between the periphery and higher sites in the CNS. Long-tract neurons receive signals from many different neurons (convergent signaling) and synapse with many downstream neurons (divergent signaling). B. Local circuit neurons show a complicated structural motif, arranged in layers, which includes both excitatory and inhibitory neurons. These circuits are used to process information. C. Single-source divergent neurons typically originate in a nucleus in the brainstem and have axonal terminals that innervate thousands of neurons, usually in the cerebral cortex.

nervous system, and it is important for the transmission of signals from one region to another within the central nervous system. In the peripheral nervous system, signals are transmitted with little modification. Sensory neurons respond to stimuli such as touch, temperature, pressure, vibration, and noxious chemicals and, if the initial membrane depolarization is strong enough, transmit an action potential directly to the spinal cord. There, sensory neurons synapse directly with somatic motor neurons, forming reflex arcs, and with ascending spinal neurons that transmit the information to higher levels. Motor neurons carry information directly from the spinal cord out through the ventral roots and project directly on the motor end plates of the muscles they innervate. The long axon tracts of the peripheral sensory and motor neurons are bundled together and travel as peripheral nerves. As described above, preganglionic neurons of the autonomic nervous system form synaptic connections with postganglionic neurons at ganglia that are located prevertebrally, paravertebrally, or near the innervated visceral organs. One preganglionic neuron typically makes synaptic connections with up to several thousand postganglionic neurons, an organization that is termed divergent signaling. Although divergent signaling does result in some processing and modification of information, the autonomic nervous system does not generally modify neural signals appreciably. In contrast to neurons in the peripheral pathways, neurons in long tract systems of the central nervous system not only relay but also integrate and modify signals. CNS long tract neurons display divergent signaling like autonomic neurons, but also receive synaptic connections from many upstream neurons (convergent signaling). The CNS uses both excitatory and inhibitory neurotransmitters to localize a signal, a strategy that is known as center-surround signaling. For example, sensory perception in the CNS can precisely localize a signal by activating cortical neurons that map to one area of the body and inhibiting neurons that map to surrounding areas of the body.

Local Circuit Neuronal Organization Local circuit neurons maintain connectivity primarily within the immediate area. These neurons are generally responsible for modulating signal transmission (Fig. 8-7B). For example, neurons in the cerebral cortex are organized in layers, usually six in number. While information flows into one layer and out of a different layer through long tract connections, links between the layers process the signals and interpret the inputs. Local synaptic connections can be both excitatory and inhibitory, ensuring that only certain patterns of inputs are passed along. For example, information originating in the lateral geniculate neurons enters the primary visual cortex through a long tract connection called the optic tract. In an area of the cortex designed to perceive lines, the outgoing neurons will be excited only if the incoming neurons fire in a particular pattern, in this case designating a line in a particular orientation. The outgoing signal might then serve as the input to another area of the brain that recognizes shapes. If this area receives an appropriate pattern of lines from the appropriate sources, it might recognize a particular object, such as the grid on a tic-tac-toe board. Single-Source Divergent Neuronal Organization Nuclei in the brainstem, hypothalamus, and basal forebrain follow single-source divergent circuit organization (Fig. 8-7C), in which neurons originating in one nucleus innervate many target cells. Because single-source divergent neuronal organization involves the action of signals on a wide variety of neurons, it is also commonly referred to as a diffuse system of organization. Instead of stimulating their targets directly, divergent neurons typically exert a modulatory influence by using neurotransmitters—generally, biogenic amines (see below)— that act on G protein-coupled receptors. These receptors alter the resting potential and ion channel conductance of the neuronal membranes in which they are embedded, thereby altering the ease of depolarization of these neurons. Neurons constituting single-source divergent circuits do not generally have myelin sheaths, because their modulatory influences vary over the

A Dopaminergic and cholinergic pathways Medial septal nuclei

Striatum

Nucleus basalis Ventral tegmental area Substantia nigra Dopaminergic neurons

Pedunculopontine nucleus Cholinergic neurons

B Noradrenergic and serotonergic pathways

Locus ceruleus Raphe nuclei Spinal cord Noradrenergic neurons

Serotonergic neurons

102 Fundamental Principles of Neuropharmacology

projects to the cortex and regulates alertness, while the latter nucleus controls sleep–wake cycles and arousal. Cells in the basal forebrain that receive inputs from the pedunculopontine nucleus degenerate in several diseases, including Alzheimer’s disease. The tuberomamillary nucleus uses the neurotransmitter histamine (see below) and may help maintain arousal through its actions on the forebrain. The somnolence induced by first-generation antihistamines—histamine H1 receptor antagonists used to treat allergies (see Chapter 43, Histamine Pharmacology)—may be caused by inhibition of transmission involving tuberomamillary nucleus neurons.

 NEUROPHYSIOLOGY Neurotransmitters The peripheral nervous system uses only two neurotransmitters, acetylcholine and norepinephrine (Fig. 8-9). In contrast, the CNS uses not only a wide variety of small molecule neurotransmitters, including acetylcholine and norepinephrine (Table 8-2), but also many neuroactive peptides. These peptides may be transmitted concurrently with the small

A Sympathetic

Preganglionic neuron

molecule neurotransmitters, and they generally have a neuromodulatory role. The small molecule neurotransmitters can be organized into several broad categories, based on both their structure and function (Fig. 8-10). The first category, the amino acid neurotransmitters, includes glutamate, aspartate, GABA, and glycine. The biogenic amine neurotransmitters, which are derived from decarboxylated amino acids, include dopamine, norepinephrine, epinephrine, serotonin, and histamine. Acetylcholine, which is neither an amino acid nor a biogenic amine, is used as a neurotransmitter in both the CNS and the peripheral nervous system. The purines adenosine and adenosine triphosphate (ATP) are also used in central neurotransmission, although their roles have not been studied in as much detail as those of other neurotransmitters. The lipid-soluble gas nitric oxide (NO), which has many effects in peripheral tissues, has recently been shown to act as a diffusible neurotransmitter in the CNS. Amino Acid Neurotransmitters The amino acid neurotransmitters are the primary excitatory and inhibitory neurotransmitters in the CNS. Two types of

B Parasympathetic

C Somatic

Acetylcholine Nicotinic receptors

Acetylcholine Nicotinic receptors Postganglionic neuron

Norepinephrine or Acetylcholine Tissue receptor

Adrenergic Muscarinic (sweat glands)

Acetylcholine

Muscarinic

Acetylcholine

Nicotinic

FIGURE 8-9. Neurotransmitters in the peripheral nervous system (A-C). Only two neurotransmitters are required to mediate neurotransmission in the peripheral nervous system. Acetylcholine is released by sympathetic and parasympathetic preganglionic neurons, parasympathetic postganglionic neurons, somatic motor neurons, and sympathetic postganglionic neurons that innervate sweat glands. All other sympathetic postganglionic neurons release norepinephrine. Acetylcholine stimulates nicotinic acetylcholine receptors on sympathetic and parasympathetic postganglionic neurons and at the neuromuscular junction. Acetylcholine stimulates muscarinic acetylcholine receptors on sweat glands and on tissues innervated by parasympathetic postganglionic neurons. Norepinephrine stimulates ␣- and ␤-adrenergic receptors on tissues (except for sweat glands) innervated by sympathetic postganglionic neurons.

Amino Acid Neurotransmitters O

O

H2N

H2N

OH

OH

HO O

HO

Aspartic acid

O Glutamic acid

O

O H2N

H2N

OH

OH

γ-Aminobutyric acid (GABA)

Glycine

Biogenic Amine Neurotransmitters OH HO

NH2

HO

H N

HO

HO Dopamine

Epinephrine

OH HO

NH2

HO Norepinephrine

NH2

HN

NH2

N

OH

HN Histamine

Serotonin

Other Neurotransmitters O

N H2N

N+

N

N

N

O Acetylcholine

O OH

HO Adenosine

OH

NO Nitric oxide

CHAPTER 8 / Principles of Nervous System Physiology and Pharmacology 105

O OH NH2

HO Tyrosine Tetrahydrobiopterin O2, Fe2+

Tyrosine hydroxylase (TH)

O HO OH NH2

HO L-DOPA

Aromatic L-amino acid decarboxylase

Pyridoxal phosphate

HO

Clonidine is a partial agonist that acts on presynaptic ␣2receptors. Some antidepressants increase the synaptic concentration of norepinephrine by blocking its reuptake (tricyclic antidepressants [TCAs]), while others increase the intracellular pool of norepinephrine available for synaptic release by inhibiting its chemical degradation (monoamine oxidase inhibitors [MAOIs]). 5-Hydroxytryptamine (5-HT, also known as serotonin) is formed from the amino acid tryptophan by enzymatic oxidation at the 5 position followed by enzymatic decarboxylation. This sequence of reactions is similar to that used in the synthesis of dopamine, although the enzymes that carry out the reactions are different (Fig. 8-12). Several classes of drugs target serotonergic neurotransmission. Tricyclic antidepressants, which block norepinephrine reuptake, also block serotonin reuptake. Selective serotonin reuptake inhibitors (SSRIs), which act more selectively on serotonin reuptake transporters, are also used to treat depression. The role of serotonergic neurons in depression and the various therapies for depression that target serotonergic neurotransmission are discussed in more detail in Chapter 14.

NH2

O HN

HO

OH

Dopamine Ascorbic acid O2, Cu2+

NH2

Dopamine ß-hydroxylase Tryptophan

OH HO

Tryptophan hydroxylase (TPH)

NH2

O

HO HN

Norepinephrine

OH NH2

Phenylethanolamine N-methyltransferase

S-adenosylmethionine

OH

OH

5-Hydroxytryptophan

H N

HO

Aromatic L-amino acid decarboxylase

HO Epinephrine

FIGURE 8-11.

Synthesis of catecholamines. Catecholamines are all synthesized from tyrosine. Sequential enzymatic reactions result in hydroxylation of tyrosine to form L-DOPA, decarboxylation of L-DOPA to form dopamine, hydroxylation of dopamine to form norepinephrine, and methylation of norepinephrine to form epinephrine. Depending on the enzymes (shown in blue lettering) expressed in a particular type of presynaptic neuron, the reaction sequence may stop at any of the last three steps, so that dopamine, norepinephrine, or epinephrine can be the final product that is synthesized and used as a neurotransmitter.

NH2

HN

OH 5-Hydroxytryptamine (Serotonin)

FIGURE 8-12.

Synthesis of 5-hydroxytryptamine (serotonin). Tryptophan is first oxidized by tryptophan hydroxylase (TPH) and then decarboxylated by aromatic L-amino acid decarboxylase to yield serotonin.

106 Fundamental Principles of Neuropharmacology

Histamine is formed by decarboxylation of the amino acid histidine. Histamine functions as a diffuse neurotransmitter in the CNS; it also has a particular role in the maintenance of arousal via the tuberomamillary nucleus of the hypothalamus and in the sensation of nausea via the area postrema in the floor of the fourth ventricle. Few therapeutics intentionally target central histaminergic neurotransmission. Instead, most drugs in this class act on peripheral histamine H1 receptors, at which histamine mediates the inflammatory response to allergic stimuli, or on H2 receptors in the treatment of peptic ulcer disease (see Chapters 43 and 46). Peripherally acting antihistamines are sometimes used to effect sedation or as antiemetics, acting via the central neuroanatomic substrates noted above. Other Small Molecule Neurotransmitters Acetylcholine plays a major role in peripheral neurotransmission. At the neuromuscular junction, this molecule is used by somatic motor neurons to depolarize striated muscle. In the autonomic nervous system, acetylcholine is the neurotransmitter used by all preganglionic neurons and by parasympathetic postganglionic neurons. The multiple functions of acetylcholine in the peripheral nervous system have spurred the development of a wide range of drugs that target peripheral cholinergic neurotransmission. These include muscle paralytics, which interfere with neurotransmission at the motor end plate, acetylcholinesterase inhibitors, which increase local acetylcholine concentration by interfering with the metabolic breakdown of the neurotransmitter, and receptor-specific agonists and antagonists. In the CNS, acetylcholine acts as a diffuse system neurotransmitter. Like the biogenic amines, it is thought to regulate sleep and wakefulness. Donepezil, a reversible acetylcholinesterase inhibitor that acts at central cholinergic synapses, helps to “brighten” patients with dementia (see Chapter 9). Peripheral anticholinergic agents may cause central cholinergic blockade and thereby result in major adverse effects. For example, the antimuscarinic drug scopolamine can cause drowsiness, amnesia, fatigue, and dreamless sleep. In contrast, cholinergic agonists such as pilocarpine can induce adverse effects of cortical arousal and alertness. The purinergic neurotransmitters adenosine and adenosine triphosphate have a role in central neurotransmission. This role is most evident in the effects of caffeine, which is a competitive antagonist at adenosine receptors and causes a mild stimulant effect. In this case, the adenosine receptors, which are located on presynaptic noradrenergic neurons, act to inhibit the release of norepinephrine. Antagonism of these adenosine receptors by caffeine causes the release of norepinephrine to be disinhibited, which causes the characteristic stimulatory effects of the drug. Nitric oxide (NO), which has generated significant interest as a peripheral vasodilator, acts in the brain as a neurotransmitter. Unlike the other small molecule neurotransmitters, NO diffuses through the neuronal membrane and binds to its receptors within the target cell. Receptors for NO are thought to reside in presynaptic neurons, allowing NO to act as a retrograde messenger. While many therapeutics target the peripheral vasodilatory effects of NO, none as of yet target its actions as a central neurotransmitter. Neuropeptides The neuroactive peptides are the last major class of neurotransmitters. Many neuropeptides also have endocrine,

autocrine, and paracrine actions. Major examples of neuroactive peptide families are the opioids, tachykinins, secretins, insulins, and gastrins. Neuropeptides also include the pituitary hormone release and inhibiting factors, including corticotropin-releasing hormone (CRH), gonadotropinreleasing hormone (GnRH), thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone (GRH), and somatostatin. The opioid peptide family includes the enkephalins, dynorphins, and endorphins. Opioid receptors, which are distributed widely in areas of the spinal cord and brain that are involved in pain sensation, are the principal pharmacologic targets of opioid analgesics such as morphine (see Chapter 17) and of some drugs of abuse such as heroin (see Chapter 18).

The Blood–Brain Barrier In the case of Ms. P, L-DOPA, the immediate precursor of dopamine, is administered rather than the neurotransmitter itself. Unlike L-DOPA, which is able to cross from the blood to the brain tissue where it acts to treat Ms. P’s Parkinson’s disease, dopamine is unable to cross that boundary. The reason for this exclusion is the existence of a selective filter, termed the blood–brain barrier, which regulates the transport of many molecules from the blood into the brain (Fig. 8-13). The blood–brain barrier protects the brain tissue both from toxic substances that circulate in the blood and from neurotransmitters such as epinephrine, norepinephrine, glutamate, and dopamine that have systemic effects in body tissues but that would bind receptors in the CNS and cause undesirable effects if access were permitted. The structural basis for the blood–brain barrier resides in the unique design of the cerebral microcirculation. In most tissues, there are small gaps, called fenestrae, between the endothelial cells that line the microvasculature. These gaps allow water and small molecules to diffuse across the lining without resistance but filter out large proteins and cells. In the CNS, the endothelial cells form tight junctions that prevent diffusion of small molecules across the vessel wall. Also, unlike peripheral endothelial cells, CNS endothelial cells do not generally have pinocytotic vesicles that transport fluid from the blood vessel lumen to the extracellular space. In addition, blood vessels in the CNS are covered by cellular processes derived from astroglia. These processes play an important role in selectively transporting certain nutrients from the blood to central neurons. In the absence of a selective transport mechanism, the blood–brain barrier generally excludes water-soluble substances. In contrast, lipophilic substances, including important lipid-soluble gases such as oxygen and carbon dioxide, can usually diffuse across the endothelial membranes. The oil/water partition coefficient is a good indicator of the ease with which a small molecule can enter the CNS. Lipophilic substances with high oil/water partition coefficients can generally diffuse across the blood–brain barrier, whereas hydrophilic substances with low oil-water partition coefficients are typically excluded (Fig. 8-14). Many important hydrophilic nutrients, such as glucose and a number of amino acids, would not be able to cross the blood–brain barrier without the existence of specific transporters. Glucose, for example, is transported across the barrier by a hexose transporter that allows this nutrient to move down its concentration gradient in a process called

Relative penetration into brain

1.00

Peripheral capillary

Fenestra

Diazepam Ethanol

Heroin Glucose

Chloramphenicol L-DOPA

0.10

Phenytoin

Phenobarbital

Dopamine Methotrexate Mannitol

0.01

Pinocytotic vesicles

Nicotine

Morphine Penicillin

Sodium

Endothelial cell 0.001

0.01

0.1

1.0

10

Oil/Water partition coefficient

Brain capillary

Pericyte

Astroglial process Basement membrane Mitochondria Tight junction

100

108 Fundamental Principles of Neuropharmacology

drugs as examples, the focus is on the general principles of anatomy and neurotransmission that are important for understanding the action of all pharmacologic agents affecting the nervous system. The remaining chapters in this section discuss specific neurotransmitter systems and specific agents that act on the peripheral and central nervous systems. Thus, Chapters 9 and 10 describe peripheral cholinergic and adrenergic systems, and Chapter 11 discusses the production of local anesthesia by inhibition of electrical transmission through peripheral and spinal neurons. Chapter 12 describes central excitatory and inhibitory neurotransmission. Although few therapeutics currently take advantage of glutamatergic neurotransmission, two major classes of drugs, the benzodiazepines and the barbiturates, affect GABAergic neurotransmission by potentiating the effect of GABA at the GABAA receptor. Chapter 13 discusses dopaminergic systems, describing in more detail the concept, introduced in the present chapter, that some of the symptoms of Parkinson’s disease can be alleviated by drugs that increase dopaminergic transmission. Chapter 13 also explains how inhibiting dopaminergic transmission can alleviate some of the symptoms of schizophrenia, implying that dopamine may play a role in this disease. Chapter 14 discusses drugs that modify affect, the outward

manifestations of mood. These agents include antidepressants, which block reuptake or inhibit metabolism of the biogenic amines norepinephrine and serotonin, as well as the “mood stabilizer” lithium, which is thought to affect a signal transduction pathway. Chapter 15 explores the pharmacology of abnormal electrical neurotransmission, including the action of channel blockers, such as phenytoin, which block the propagation of action potentials and thereby inhibit many types of seizures. Chapter 16 describes the pharmacology of general anesthetics, agents whose mechanism of action remains an area of active investigation. Chapter 17 discusses the pharmacology of analgesia, including opioid receptor agonists and nonopioid analgesics. To conclude, Chapter 18 focuses on the pharmacology of drugs of abuse.

Suggested Reading Blumenfeld H. Neuroanatomy through clinical cases. 2nd ed. Sunderland, MA: Sinauer Associates, Inc.; 2010. (Thorough review of human neuroanatomy with an emphasis on clinical correlation; includes many exemplary clinical cases.) Squire LR, Berg D, Bloom F, du Lac S, Ghosh A. Fundamental neuroscience. 3rd ed. Academic Press; 2008. (Comprehensive textbook containing detailed information on human neuroanatomy and neurophysiology.)

IIB Principles of Autonomic and Peripheral Nervous System Pharmacology

9 Cholinergic Pharmacology Alireza Atri, Michael S. Chang, and Gary R. Strichartz

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 110-111 BIOCHEMISTRY AND PHYSIOLOGY OF CHOLINERGIC NEUROTRANSMISSION . . . . . . . . . . . . . . . . . 110 Synthesis of Acetylcholine . . . . . . . . . . . . . . . . . . . . . . . . 110 Storage and Release of Acetylcholine . . . . . . . . . . . . . . . . 111 Cholinergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . 113 Nicotinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Degradation of Acetylcholine . . . . . . . . . . . . . . . . . . . . . . 114 Physiologic Effects of Cholinergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Neuromuscular Junction . . . . . . . . . . . . . . . . . . . . . . . 115 Autonomic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 CNS Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 119 Inhibitors of Acetylcholine Synthesis, Storage, and Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Acetylcholinesterase Inhibitors . . . . . . . . . . . . . . . . . . . . . 120 Structural Classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Clinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Muscarinic Receptor Agonists . . . . . . . . . . . . . . . . . . . 122 Nicotinic Receptor Agonists. . . . . . . . . . . . . . . . . . . . . 123 Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Muscarinic Receptor Antagonists . . . . . . . . . . . . . . . . 124 Nicotinic Receptor Antagonists . . . . . . . . . . . . . . . . . . 126 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 126 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

 INTRODUCTION

of ACh at a particular cholinergic synapse are largely determined by the ACh receptor type at that synapse. Cholinergic receptors are divided into two broad classes. Muscarinic cholinergic receptors (mAChR) are G protein-linked and expressed at the terminal synapses of all parasympathetic postganglionic fibers and a few sympathetic postganglionic fibers, at autonomic ganglia, and in the CNS. Nicotinic cholinergic receptors (nAChR) are ligand-gated ion channels that are concentrated postsynaptically at many excitatory autonomic synapses and presynaptically in the CNS. Acetylcholinesterase (AChE), the enzyme responsible for acetylcholine degradation, is also an important pharmacologic target. In this section, a description of the biochemistry of each of these pharmacologic targets is followed by a discussion of the physiologic effects of acetylcholine at the neuromuscular junction, in the autonomic nervous system, and in the CNS.

Cholinergic pharmacology is centered on the properties of the neurotransmitter acetylcholine (ACh). The functions of cholinergic pathways are complex, but generally involve the neuromuscular junction (NMJ), the autonomic nervous system, and the central nervous system. Despite the many important physiologic actions of ACh, the current therapeutic uses for cholinergic and anticholinergic drugs are limited by the ubiquitous and complicated nature of cholinergic pathways, and thus, by the inherent difficulty in effecting a specific pharmacologic intervention without inducing adverse effects. Nonetheless, medications with somewhat targeted cholinomimetic and anticholinergic actions are in widespread clinical use for their effects on the brain (especially cognition and behavior), neuromuscular junction, heart, eyes, lungs, and genitourinary and gastrointestinal tracts. Other relevant chapters discussing applications of cholinergic pharmacology are Chapter 17, Pharmacology of Analgesia; Chapter 46, Integrative Inflammation Pharmacology: Peptic Ulcer Disease; and Chapter 47, Integrative Inflammation Pharmacology: Asthma.

 BIOCHEMISTRY AND PHYSIOLOGY OF CHOLINERGIC NEUROTRANSMISSION Acetylcholine synthesis, storage, and release follow a similar set of steps in all cholinergic neurons. The specific effects 110

Synthesis of Acetylcholine Acetylcholine is synthesized in a single step from choline and acetyl coenzyme A (acetyl CoA) by the enzyme choline acetyltransferase (ChAT): Acetyl Coenzyme A ⴙ Choline

ChAT Æ ææææ

Acetylcholine ⴙ Coenzyme A ⴙ H2O

Equation 9-1

In the CNS, choline used for the synthesis of acetylcholine arises from one of three sources. Approximately 35%

112 Principles of Autonomic and Peripheral Nervous System Pharmacology

Na+ Choline

Cholinergic neuron AcCoA + Choline

Hemicholinium

Choline acetyltransferase

Vesamicol

ACh

Calcium channel

Calcium channel

Ca2+

Ca2+ ACh H

LEMS (autoantibody)

+

Botulinum toxin ACh

Muscarinic ACh receptor (M2, M4)

Nicotinic ACh receptor ACh

Choline + acetate

Synaptic cleft AChE

AChE Inhibitors

Acetylcholinesterase

Nicotinic ACh receptor Postsynaptic Muscarinic ACh receptor cell M1, M3, M5 M2, M4

Opens Na+/K+ channel

Gq

Gi

PLC

AC, K+ channel

Excitatory

Inhibitory

Excitatory

FIGURE 9-1. Acetylcholine synthesis, storage, release, and degradation pathways, and pharmacologic agents that act on these pathways. Choline is transported into the presynaptic cholinergic nerve terminal by a high-affinity Na⫹-choline co-transporter. This transporter is inhibited by hemicholinium. The cytosolic enzyme choline acetyltransferase catalyzes the formation of acetylcholine (ACh) from acetyl coenzyme A (AcCoA) and choline. Newly synthesized ACh is packaged (together with ATP and proteoglycans) into vesicles for storage. Transport of ACh into the vesicle is mediated by a H⫹-ACh antiporter, which is inhibited by vesamicol. The ACh-containing vesicles fuse with the plasma membrane when intracellular calcium levels rise in response to a presynaptic action potential, releasing the neurotransmitter into the synaptic cleft. Lambert–Eaton myasthenic syndrome (LEMS) results from an autoantibody that blocks the presynaptic Ca2⫹ channel. Botulinum toxin prevents the exocytosis of presynaptic vesicles, thereby blocking ACh release. Acetylcholine diffuses in the synaptic cleft and binds to postsynaptic and presynaptic receptors. Acetylcholine receptors are divided into nicotinic and muscarinic receptors. Nicotinic receptors are ligand-gated ion channels that are permeable to cations, while muscarinic receptors are G protein-coupled receptors that alter cell signaling pathways, including activation of phospholipase C (PLC), inhibition of adenylyl cyclase (AC), and opening of K⫹ channels. Postsynaptic nicotinic receptors and M1, M3, and M5 muscarinic receptors are excitatory; postsynaptic M2 and M4 muscarinic receptors are inhibitory. Presynaptic nicotinic receptors enhance Ca2⫹ entry into the presynaptic neuron, thereby increasing vesicle fusion and release of ACh; presynaptic M2 and M4 muscarinic receptors inhibit Ca2⫹ entry into the presynaptic neuron, thereby decreasing vesicle fusion and release of ACh. Acetylcholine in the synaptic cleft is degraded by membrane-bound acetylcholinesterase (AChE) into choline and acetate. Numerous inhibitors of AChE exist; most clinically relevant anticholinesterases are competitive inhibitors of the enzyme.

Transmission, for additional details on electrochemical transmission.) Two stores of ACh play distinct roles during the process of ACh release. One store, known as the “depot” pool, consists of vesicles positioned near the plasma membrane of the axon terminal. Axonal depolarization causes these vesicles to release ACh rapidly. The

“reserve” pool serves to refill the depot pool as it is being used. An adequate rate of reserve pool mobilization is required to sustain ACh release for an extended period of time. Of the two stores, the depot pool is replenished first by vesicles loaded with newly synthesized ACh; this process displaces some of the older depot pool vesicles into the reserve pool.

CHAPTER 9 / Cholinergic Pharmacology 113

Cholinergic Receptors After ACh has been released into the synaptic cleft, it binds to one of two classes of receptors, usually on the membrane surface of the postsynaptic cell. Muscarinic receptors (mAChR) are seven-transmembrane-domain G protein-coupled receptors (GPCRs), and nicotinic receptors (nAChR) are ligand-gated ion channels. Although muscarinic receptors are sensitive to the same neurotransmitter as nicotinic receptors, these two classes of cholinergic receptors share little structural similarity. Muscarinic Receptors Muscarinic cholinergic transmission occurs mainly at autonomic ganglia, at end organs innervated by the parasympathetic division of the autonomic nervous system, and in the CNS. Muscarinic receptors belong to the same family as a number of other cell surface receptors (such as the adrenergic receptors) that transduce signals across the cell membrane and interact with GTP-binding proteins. Because all the effects of muscarinic receptor activation occur through the actions of these G proteins, there is a latency of at least 100–250 ms associated with muscarinic responses to receptor activation. (In contrast, nicotinic channels have latencies on the order of 5 ms.) G protein activation by agonist binding to muscarinic receptors may have several different effects on cells. These include inhibition of adenylyl cyclase (via Gi) and stimulation of phospholipase C, both mediated by an ␣ subunit of the G protein. (See Chapter 1, Drug–Receptor Interactions, for a discussion of these signaling mechanisms.) Muscarinic activation also influences ion channels via second messenger molecules. The predominant effect of such mAChR stimulation is to increase the opening of specific potassium channels (G protein-modified inwardly rectifying K⫹ channels, or GIRKs), thereby hyperpolarizing the cell. This effect is mediated through the ␤␥ subunit of a G protein (Go), which binds to the channel and enhances its probability of being open. Five distinct cDNAs for human muscarinic receptors, denoted M1–M5, have been isolated and detected in cells. These receptor types form two functionally distinct groups. M1, M3, and M5 are coupled to G proteins responsible for the stimulation of phospholipase C. M2 and M4, on the other hand, are coupled to G proteins responsible for adenylyl cyclase inhibition and K⫹ channel activation. The receptors of each functional group can be distinguished based on their responses to pharmacologic antagonists (Table 9-1). Generally, M1 is expressed in cortical neurons and autonomic ganglia, M2 in cardiac muscle, and M3 in smooth muscle and glandular tissue. Because stimulation of M1, M3, and M5 receptors facilitates excitation of the cell, while stimulation of M2 and M4 receptors suppresses cellular excitability, there is a predictable correlation between the receptor subtype and the effect of ACh on the cell. The various muscarinic receptor subtypes account for much of the diversity in cellular responses to mAChR agonists. Nicotinic Receptors Nicotinic cholinergic transmission results from the binding of ACh to the nAChR (Fig. 9-2). This phenomenon is known as direct ligand-gated conductance. The binding of two ACh molecules to one nAChR elicits a conformational change in the receptor that creates a monovalent cation-selective pore

through the cell membrane. Open channels of the activated nAChR are equally permeable to K⫹ and Na⫹ ions. (Since the resting membrane potential is close to the Nernst potential for K⫹ and far below the Nernst potential for Na⫹, the predominant ion passing through the open nACR is Na⫹.) A relatively small permeability to Ca2⫹ ions nevertheless results in important elevations of intracellular [Ca2⫹]. Therefore, when open, these channels produce a net inward Na⫹ current that depolarizes the cell. Stimulation of multiple nAChRs may depolarize the cell sufficiently to generate action potentials and to open voltage-dependent calcium channels. This latter action, and the direct entry of Ca2⫹ through the nAChR pore, can lead to activation of several intracellular signaling pathways. Because ACh dissociates rapidly from active-state receptor molecules and acetylcholinesterase rapidly degrades free (unbound) ACh in the synaptic cleft (see below), the depolarization mediated by nAChRs is brief (⬍10 ms). Although the simultaneous binding of two ACh molecules is required for channel opening, it is not necessary for both molecules to dissociate for the channel to open again; binding of a second ACh molecule to a receptor that still has one ACh bound may, once again, result in channel opening. The kinetics of nAChR binding and channel opening are detailed in Figure 9-3. Structurally, the nicotinic acetylcholine receptor comprises five subunits, each of which has a mass of approximately 40 kilodaltons (Fig. 9-2A). Several types of subunits have been identified in the nAChR, designated ␣, ␤, ␥, ␦, and ␧. All of these subunits share 35–50% homology with one another. Each receptor at the NMJ is composed of two ␣ subunits, one ␤ and one ␦ subunit, and either one ␥ or one ␧ subunit. (The ␣2␤␧␦ form dominates at the neuromuscular junction in mature skeletal muscle, while the ␣2␤␥␦ form is expressed in embryonic muscle.) Agonist molecules bind at a hydrophobic pocket that is formed between each ␣ subunit and the adjacent, complementary subunit—this is the structural basis for the binding of two ACh molecules to each receptor. The conformational change in the ␣ subunits induced by the binding of ACh initiates the overall changes in the pore that permit ion flow through the receptor (i.e., that open the channel). Besides simply opening and closing in response to ACh binding, nicotinic receptors also modulate their responses to various concentration profiles of ACh. The receptors react differently to discrete, brief pulses of ACh than to neurotransmitter that is present continuously. As noted above, under normal conditions, a closed, resting-state channel responds to dual ACh binding by opening transiently, and the low affinity of the receptor for ACh causes rapid dissociation of ACh from the receptor and return of the receptor to its resting conformation. In comparison, continuous exposure of the receptor to ACh causes it to undergo a change to a “desensitized” conformation in which the channel is locked closed. The desensitized state is also characterized by a greatly increased affinity of the receptor for ACh, so that ACh remains bound to the receptor for a relatively long period of time. This prolonged binding of ACh to the desensitized conformation of the receptor delays the conversion of the receptor to its unstimulated, resting state. Nicotinic cholinergic receptors at autonomic ganglia and in the central nervous system (termed N2 or NN) are similar to receptors at the NMJ (N1 or NM), with the exception that

CHAPTER 9 / Cholinergic Pharmacology 115

A Overall Structure

α

δ

β

α

N

M4

ε

C

M1

M3

Physiologic Effects of Cholinergic Transmission

M2

B Acetylcholine Binding Site

δ

β

β α

α

ε

Amino acids

α

Y W W

ACh binding sites

Y N Y

Acetylcholine binding site

C C

Y

ε

C Ion Channel M2 M2

M2

M2

M2

M2

FIGURE 9-2.

M2

Amino acids

a minor role in early neural development as a co-regulator of ACh (it can hydrolyze ACh, but at rates much slower than AChE) and may be involved in the pathogenesis of Alzheimer’s disease. Because of its central importance to cholinergic transmission, an entire class of drugs known as acetylcholinesterase inhibitors has been designed to target AChE.

Leucine ring

Structural biology of the nicotinic acetylcholine receptor. A. Overall structure of the nicotinic acetylcholine receptor (NM type) and its five subunits (␣2␤␧␦). Each subunit is composed of a transmembrane protein that has four membrane-spanning (hydrophobic) alpha-helical regions (M1, M2, M3, M4). The large hydrophilic N-terminal domains of the two ␣ subunits contain the binding sites for acetylcholine. B. Acetylcholine binding site viewed from above (inset: lower magnification). The labeled amino acids of the ␣ subunit hydrophilic domain are particularly important in binding acetylcholine. The conformational change that results from the binding of two acetylcholine molecules opens the channel. C. The M2 domains of the five subunits all face the interior of the protein and together form the transmembrane channel (inset). Three negatively charged rings of five amino acids (one from each M2 subunit) draw positively charged ions through the channel. At the center, an uncharged leucine ring (purple ) participates in closing the ion channel when the receptor becomes desensitized to acetylcholine.

Neuromuscular Junction Acetylcholine is the principal neurotransmitter at the neuromuscular junction (Fig. 9-4). The binding of ACh released by ␣ motor neurons to nicotinic receptors in the muscle cell membrane results in motor end-plate depolarization. The extent of depolarization depends on the quantity of ACh released into the synaptic cleft. Release of ACh is quantal in nature; that is, ACh is released in discrete quantities by the presynaptic motor neuron. Each quantum of ACh corresponds to the contents of a single synaptic vesicle and elicits a small depolarization in the motor end-plate termed a miniature end-plate potential (MEPP). Under resting conditions, sporadic MEPPs are detected at the motor endplate, corresponding to a low baseline level of unstimulated ACh release that arises from spontaneous vesicle fusion with the motor axon’s presynaptic membrane. In contrast, the arrival of an action potential at the motor axon terminal causes many more vesicles (up to thousands) to fuse with the neuronal membrane and release their ACh. At the motor endplate, the result is a relatively large depolarization termed the end-plate potential (EPP) (Fig. 9-5). The magnitude of the EPP is more than sufficient to trigger a propagating action potential throughout the muscle fiber and, hence, to produce a single contraction or “twitch.” Acetylcholine not only triggers muscle contraction as its primary effect at the NMJ, but also modulates its own action at this site. Presynaptic cholinergic receptors, located on the axon terminal of the motor neuron, respond to ACh binding by facilitating the mobilization of synaptic vesicles from the reserve pool to the depot pool. This positive feedback loop, in which the release of ACh stimulates additional release, is necessary to ensure sufficient ACh release under high-frequency stimulation of the nerve (⬃100 Hz). Despite this mechanism, the ACh output per nerve impulse wanes rapidly during persistent high-frequency stimulation. Fortunately, because an excess of ACh is released and an excess of ACh receptors is present, there is a large safety margin. Only when 50% or more of the postsynaptic receptors are desensitized is a decline in muscle tension observed during tetanic stimulation (a phenomenon known as tetanic fade). Importantly, selective blockade of the modulatory presynaptic cholinergic receptors by antagonists such as hexamethonium prevents facilitation and causes rapid tetanic fade to occur under otherwise normal conditions (Fig. 9-6). Autonomic Effects Neurotransmission through autonomic ganglia is complicated because several distinct receptor types contribute to the complex changes observed in postganglionic neurons. The generalized postsynaptic response to presynaptic impulses can be separated into four distinct components (Fig. 9-7). The primary event in the postsynaptic ganglionic response is a rapid depolarization mediated by nicotinic ACh

116 Principles of Autonomic and Peripheral Nervous System Pharmacology ACh ACh

ACh binding sites

ACh

ACh

Receptor gate (closed)

ACh

kon 2A + R

ACh

A + AR

A 2R

k'off

ACh

β

k'on

koff

ACh

α

A2R

*

Receptor gate (open)

FIGURE 9-3. Kinetics of nicotinic acetylcholine receptor binding and channel opening. Each transition between states of receptor binding and channel opening is completely reversible, and it is not necessary to go through all of the possible conformations before returning to a given state. For example, a receptor with two associated ligands may lose one and then gain another to return to its initial state, without the need for both ligands to dissociate. A, ligand (ACh); R, nicotinic ACh receptor (closed); R*, nicotinic ACh receptor (open); kon, rate constant for association (binding) of the first ACh molecule to the receptor; k⬘on, rate constant for association of the second ACh molecule to the receptor; koff, rate constant for dissociation of the first ACh molecule from the receptor; k⬘off, rate constant for dissociation of the second ACh molecule from the receptor; ␤, rate constant of channel opening after both ACh molecules have bound; ␣, rate constant of channel closure. Note that channel opening and closing are much slower events than binding of ACh to the receptor.

receptors on the postganglionic neuron. The mechanism is similar to that in the NMJ, in that an inward current elicits a near-immediate excitatory postsynaptic potential (EPSP) of 10–50 ms in duration. Typically, the amplitude of such an EPSP is only a few millivolts, and many such events must sum for the postsynaptic cell membrane to reach the threshold for firing an action potential (Fig. 9-7A). The three remaining events of ganglionic transmission modulate this primary signal and are known as the slow EPSP, the IPSP (inhibitory postsynaptic potential), and the late, slow EPSP. The slow EPSP occurs after a latency of 1 second and is mediated by M1 muscarinic ACh receptors. The duration of this effect is 10–30 seconds (Fig. 9-7C). The IPSP is largely a product of catecholamine (i.e., dopamine and norepinephrine) stimulation of dopaminergic and ␣-adrenergic receptors (see Chapter 10, Adrenergic Pharmacology), although some IPSPs in a few ganglia are mediated by M2 muscarinic receptors. The latency and duration of the IPSPs generally vary between those of the fast and slow EPSPs. The late, slow EPSP is mediated by a decrease in potassium conductance induced by stimulation of receptors for peptide transmitters (i.e., angiotensin, substance P, and luteinizing hormone-releasing hormone). Lasting for several minutes, the late, slow EPSP is thought to play a role in the long-term regulation of postsynaptic neuron sensitivity to repetitive depolarization. One pharmacologic consequence of such a complex pattern of depolarization in autonomic ganglia is that drugs selective for the IPSP, slow EPSP, and late, slow EPSP are generally not capable of eliminating ganglionic transmission. Instead, such agents alter only the efficiency of transmission. For example, methacholine, a muscarinic receptor agonist, has modulatory effects on autonomic ganglia that resemble the stimulation of slow EPSPs (see below). Blockade of excitatory transmission through autonomic ganglia relies on inhibition of the nAChRs that mediate fast EPSPs. The overall effect of ganglionic blockade is complex and depends on the relative predominance of sympathetic and parasympathetic tone at the various end organs (Table 9-2).

For example, the heart is influenced at rest primarily by the parasympathetic system, whose tonic effect is a slowing of the heart rate. Thus, blockade of autonomic ganglia that innervate the heart by moderate to high doses of the antimuscarinic agent atropine results in blockade of vagal slowing of the sinoatrial node, and hence in relative tachycardia. It should be noted that in low doses, the central parasympathetic stimulating effects of atropine predominate, initially resulting in bradycardia prior to its peripheral vagolytic action. Blood vessels, in contrast, are innervated only by the sympathetic system. Because the normal effect of sympathetic stimulation is to cause vasoconstriction, ganglionic blockade results in vasodilation. It is important to realize, however, that the responses described above ignore the presence of muscarinic ACh receptors at many of the end organs. When stimulated directly by cholinergic agents, such receptors often mediate a response that overrides the response produced by ganglionic blockade. In general, the expected net cardiovascular effects of muscarinic blockade produced by clinical doses of atropine in a healthy adult with a normal hemodynamic state are mild tachycardia, with or without flushing of the skin, and no profound effect on blood pressure. The muscarinic receptor subtypes expressed in visceral smooth muscle, cardiac muscle, secretory glands, and endothelial cells mediate highly diverse responses to cholinergic stimulation. These effects are detailed in Table 9-3. In general, these end-organ effects tend to predominate over ganglionic influences. That is, for systemically administered cholinergic agents, the overall response is generally similar to that caused by direct stimulation of these postganglionic effector sites and often different from that caused by ganglionic stimulation alone. CNS Effects CNS functions of ACh include modulation of sleep, wakefulness, learning, and memory; suppression of pain at the spinal cord level; and essential roles in neural plasticity, early neural development, immunosuppression, and epilepsy. While the

CHAPTER 9 / Cholinergic Pharmacology 117

Neuron

Neuromuscular junction Muscle fiber

Myelin Axon Schwann cell sheath

Presynaptic boutons

End-plate region

Synaptic clefts

Mitochondria Synaptic vesicle (ACh) Dense bar (active zone) Presynaptic membrane Synaptic cleft Postsynaptic membrane Junctional fold

ACh receptors

Acetylcholinesterases

FIGURE 9-4. The neuromuscular junction (NMJ). At the neuromuscular junction, motor neurons innervate a group of muscle fibers. The area of muscle fibers innervated by an individual motor neuron is referred to as the end-plate region. Multiple presynaptic terminals extend from the axon of the motor neuron. When the motor neuron is depolarized, its synaptic vesicles fuse with the presynaptic membrane, releasing ACh into the synaptic cleft. ACh receptors of the neuromuscular junction are exclusively nicotinic, and stimulation of these receptors results in depolarization of the muscle cell membrane and generation of an end-plate potential.

past two decades have improved understanding of the diversity of subunits and molecular properties of neuronal nicotinic receptors, important questions remain about the anatomical distributions and functional roles of different neuronal receptor subtypes in the CNS, and of their changes in disease states and during nicotine abuse, as occurs with smoking. As part of the ascending reticular activating system, cholinergic neurons play an important role in arousal and attention (see Fig. 8-8). Levels of ACh throughout the brain increase during wakefulness and REM sleep and decrease during inattentive states and non-REM/slow-wave sleep (SWS). During an awake or aroused state, cholinergic projections from the pedunculopontine nucleus, the lateral tegmental nucleus, and the nucleus basalis of Meynert (NBM) are all active. Because the NBM projects diffusely throughout the cortex and hippocampus (see Fig. 8-8), activation of the NBM causes a global increase in ACh levels. Acetylcholine markedly potentiates the excitatory effects of other inputs to its cortical target cells without affecting the baseline activity of these neurons, an effect that likely derives from its modulation of excitatory neurotransmitter release. This primed state is thought to improve the ability of such neurons to process incoming inputs. For the brain as a whole, the result is a heightened state of responsiveness. The cholinergic link to memory processes is supported by evidence from diverse experimental models. Whereas elevated ACh levels during wakefulness appear to benefit memory encoding processes, consolidation of hippocampusmediated, episodic, explicit memories benefit from SWS, when ACh levels are at their minimum. By artificially keeping ACh levels elevated during SWS (e.g., by administration of an AChE inhibitor), consolidation of newly acquired explicit learning and episodic memories can be disrupted. Current understanding of the interplay among ACh, sleep, and memory is as follows. During awake states, ACh prevents interference in the hippocampus during initial learning by suppressing retrieval of previously stored memories (to prevent them from interfering with new encoding), but release of this suppression is necessary to allow consolidation of new memories. During sleep (in particular, during SWS), lower ACh levels are required for proper consolidation of newly acquired memories because stronger excitatory feedback transmission is needed to reactivate memories for consolidation within neocortical brain areas. Therefore, it may be useful to remember to sleep, as sleep is needed to remember, or at least to remember better. The clinical importance of ACh for cognitive function is illustrated by the pathophysiology and treatment of Alzheimer’s disease (AD) and other neurodegenerative dementias, including diffuse Lewy body dementia (DLB) and Parkinson’s disease with dementia (PDD). Neurodegenerative dementias and brain injury produce central cholinergic dysfunction. Patients with these conditions manifest cognitive, functional, and behavioral deficits that are at least partially related to cholinergic deficits and amenable to symptomatic treatment with procholinergic medications. An example is the symptomatic treatment of AD with acetylcholinesterase inhibitors. Acetylcholine also plays a role in pain modulation through inhibition of spinal nociceptive transmission. Cholinergic neurons located in the rostral ventromedial medulla extend processes to the superficial lamina of the dorsal horn at all levels of the spinal cord, where secondary neurons in afferent sensory pathways are located. ACh released by the cholinergic

Membrane potential (mV)

Threshold potential of postsynaptic cell

Action potential

Action potential

0 Resting potential

-55 -70 Q

A

Q

Q

Subthreshold, no summation

B

Q

2Q

Temporal summation

C

Spatial summation

FIGURE 9-5. Quantal release of acetylcholine and muscle contraction. Muscle contraction relies on the accumulation of a sufficient concentration of acetylcholine at the motor end-plate to depolarize the muscle beyond the threshold potential (typically, about ⫺55 mV). After local depolarization occurs, a self-propagating action potential is generated that can spread along the muscle fiber and result in muscle contraction. A. As a single cholinergic vesicle releases its contents into the NMJ, a small depolarization (Q), otherwise known as a miniature end-plate potential (MEPP), occurs in the local region of the muscle. This MEPP is insufficient to generate an action potential. When a sufficient number of individual cholinergic vesicles empty their contents into the NMJ, either in quick succession (B) or simultaneously (C), sufficient depolarization occurs (termed the end-plate potential, or EPP) that the motor end-plate threshold for action potential generation is exceeded, and muscle contraction occurs. An isolated action potential produces a twitch, while a train of action potentials may produce sustained contraction of the muscle. Note that although this example uses two MEPPs for simplicity, many more than two MEPPs are actually required to achieve threshold-level depolarization. In this figure, the x-axis is time.

A

A Fast EPSP

2 Hz

50 Hz

0.1 Hz

Voltage (mV)

0.1 Hz

B Slow IPSP

C Slow EPSP

B D Late, slow EPSP

0.1 Hz

FIGURE 9-6.

2 Hz

50 Hz

0.1 Hz

Tetanic fade and the effects of hexamethonium. A. Control stimulation. Rapid stimulation of muscle contraction relies on presynaptic acetylcholine autoreceptors that provide positive feedback and thereby increase the amount of acetylcholine released with each depolarization. The diagram shows control muscle responses to single-shock stimulation (0.1 Hz), a train of four stimulations (2 Hz), or tetanic stimulation (50 Hz). Positive feedback increases the amount of ACh released with each depolarization during tetanic stimulation, providing enhanced muscle contraction that gradually fades back to baseline during subsequent single-shock stimulation. B. Stimulation after the administration of hexamethonium. Note that although the response to isolated (0.1 Hz) stimuli is unchanged in the presence of hexamethonium, the drug prevents the increased effect that normally occurs with higher-frequency (50 Hz) stimulation. This is a result of hexamethonium’s antagonism of the acetylcholine autoreceptor on the presynaptic terminal that is normally responsible for positive feedback of ACh release. 118

Stimulus applied

15 sec

30 sec

5 min

Time

FIGURE 9-7. Four types of synaptic signals in an autonomic ganglion. The response of autonomic ganglia to neurotransmission is a complex event mediated by a number of different neurotransmitters and receptor types and occurring on several distinct time scales. A. The primary mode of neurotransmission is the action potential, which is produced by a sufficiently strong (suprathreshold) excitatory postsynaptic potential (EPSP). The fast EPSP is mediated by acetylcholine acting on postsynaptic nicotinic ACh receptors. B. The slow inhibitory postsynaptic potential (IPSP) is a membrane hyperpolarization response. This response is thought to be mediated by several different postsynaptic receptor types, including modulatory dopamine receptors and ␣-adrenergic receptors as well as M2 muscarinic ACh receptors. C. The slow EPSP is mediated by M1 muscarinic receptors, has a latency of about 1 second after an initial depolarization, and lasts for 10–30 seconds. D. The late, slow EPSP occurs on the order of minutes after a depolarization event. This excitatory response may be mediated by peptides that are co-released with acetylcholine.

120 Principles of Autonomic and Peripheral Nervous System Pharmacology

(12) bladder spasms and urinary incontinence, (13) cosmetic effects on skin lines and wrinkles, and (14) treatment of Alzheimer’s disease, cognitive dysfunction, and dementias. Slight variations in the pharmacologic properties of individual cholinergic and anticholinergic agents are responsible for their large differences in therapeutic utility. The relative selectivity of action of the most useful agents depends on both pharmacodynamic and pharmacokinetic factors, including inherent differences in receptor binding affinity, bioavailability, tissue localization, and resistance to degradation. These variations, in turn, derive from the molecular structure and charge of the drug. The structure of pirenzepine, for example, allows the drug to bind M1 muscarinic receptors (located in autonomic ganglia) with higher affinity than M2 and M3 receptors (located at parasympathetic end organs). As a result, the drug’s predominant effect at clinically used doses is ganglionic blockade (see Table 9-1). Similarly, the addition of a methyl group to acetylcholine yields methacholine, which is more resistant to degradation by AChE and, hence, possesses a longer duration of action. Charged agents such as muscarine generally do not cross membrane barriers. The absorption of such drugs through both the gastrointestinal (GI) mucosa and the blood–brain barrier is significantly impaired, unless specific carriers are available to transport the drug; therefore, such drugs typically have little or no effect on the CNS. In contrast, lipophilic agents have excellent CNS penetration. As one example, the high CNS penetration of physostigmine makes this drug the agent of choice for treating the CNS effects of anticholinergic overdose. The following discussion is ordered mechanistically. For each class of drugs, the selectivity of individual agents within the class is used as a basis to explain the therapeutic uses of each agent.

Inhibitors of Acetylcholine Synthesis, Storage, and Release Drugs that inhibit the synthesis, storage, or release of ACh have only recently begun to have clinical use (Fig. 9-1). Hemicholinium-3 blocks the high-affinity transporter for choline and thus prevents the uptake of choline required for ACh synthesis. Vesamicol blocks the ACh-H⫹ antiporter that is used to transport ACh into vesicles, thereby preventing the storage of ACh. Both of these compounds are utilized only in research settings, however. Botulinum toxin A, produced by Clostridium botulinum, degrades SNAP-25 and thus prevents synaptic vesicle fusion with the axon terminal (presynaptic) membrane. This paralysis-inducing property is currently used in the treatment of several diseases associated with increased muscle tone, such as torticollis, achalasia, strabismus, blepharospasm, and other focal dystonias. Botulinum toxin is also approved for cosmetic treatment of facial lines or wrinkles and is used to treat various headache and pain syndromes (e.g., by intrathecal delivery into the spinal fluid). Because it degrades a protein common to the synaptic-vesicle fusion machinery in multiple types of nerve terminals, botulinum toxin has a general effect on the release of many different neurotransmitters, not just ACh.

Acetylcholinesterase Inhibitors Agents in this class bind to and inhibit AChE, thereby elevating the concentration of endogenously released ACh in

the synaptic cleft. The accumulated ACh subsequently activates nearby cholinergic receptors. Agents in this class are also referred to as indirectly acting ACh receptor agonists because they generally do not activate receptors directly. It is important to note that a few AChE inhibitors have a direct action as well. For example, neostigmine, a quaternary carbamate, not only blocks AChE but also binds to and activates nAChRs at the neuromuscular junction. Structural Classes All indirectly acting cholinergic agonists interfere with the function of AChE by binding to the active site of the enzyme. There are three chemical classes of such agents, including (1) simple alcohols with a quaternary ammonium group, (2) carbamic acid esters of alcohols bearing either quaternary or tertiary ammonium groups, and (3) organic derivatives of phosphoric acid (Fig. 9-8). The most important functional difference among these classes is pharmacokinetic. Edrophonium is a simple alcohol that inhibits AChE by reversibly associating with the active site of the enzyme. Because of the noncovalent nature of the interaction between the alcohol and AChE, the enzyme–inhibitor complex lasts for only 2–10 minutes, resulting in a relatively rapid but completely reversible block. The carbamic acid esters neostigmine and physostigmine are hydrolyzed by AChE, so a labile covalent bond is formed between the drug and the enzyme. However, the rate at which this reaction occurs is many orders of magnitude slower than for ACh. The resulting enzyme–inhibitor complex has a halflife of approximately 15–30 minutes, corresponding to an effective inhibition lasting 3–8 hours. Organophosphates such as diisopropyl fluorophosphate have a molecular structure that resembles the transition state formed in carboxyl ester hydrolysis. These compounds are hydrolyzed by AChE, but the resulting phosphorylated enzyme complex is extremely stable and dissociates with a half-life of hundreds of hours. Furthermore, the enzyme– organophosphate complex is subject to a process known as aging, in which oxygen–phosphorus bonds within the inhibitor are broken spontaneously in favor of stronger bonds between the enzyme and the inhibitor. Once aging occurs, the duration of AChE inhibition is increased even further. Thus, organophosphate inhibition is essentially irreversible, and the body must synthesize new AChE molecules to restore AChE activity. However, if strong nucleophiles (such as pralidoxime) are administered before aging has occurred, it is possible to recover enzymatic function from the inhibited AChE. Clinical Applications Acetylcholinesterase inhibitors have a number of clinical applications, including (1) increasing transmission at the neuromuscular junction, (2) increasing parasympathetic tone, and (3) increasing central cholinergic activity (e.g., to treat symptoms of AD). Because of their ability to increase the activity of endogenous ACh, AChEs are especially useful in diseases of the neuromuscular junction, where the primary defect is an insufficient quantity of either ACh or AChR. In myasthenia gravis, autoantibodies are generated against NM receptors. These antibodies both induce NM receptor internalization and block the ability of ACh to activate the receptors. As a result, patients with myasthenia gravis present with significant

A

Simple Alcohols

HO

Carbamic Acid Esters

B

N+

N

C

Organophosphates O

N+

O

P O

O Edrophonium

Neostigmine

H N

Isoflurophate

O N O

O F

N Physostigmine

Choline Esters

A

B

Alkaloids

HO

O N+

N+

O O

Acetylcholine

Muscarine

O N+ O

N

O

Methacholine

O O

Pilocarpine N+

H2N

O

Carbachol

O N+ H2N

O

Bethanechol

N

CHAPTER 9 / Cholinergic Pharmacology 127

responses that are opposite to those produced by normal autonomic tone. Muscarinic receptors are G protein-coupled receptors that bind acetylcholine and initiate signaling through several intracellular pathways. These receptors are expressed in the autonomic ganglia and effector organs, where they mediate a parasympathetic response. The primary uses of muscarinic receptor agonists and antagonists are to modulate autonomic responses of effector organs. Both nicotinic and muscarinic receptors are ubiquitous in the CNS, where the effects of acetylcholine include analgesia, arousal, and attention. The relative roles of mAChRs and nAChRs in the brain and spinal cord are not fully understood, and the most effective currently available CNS drugs increase endogenous cholinergic transmission by inhibiting the action of acetylcholinesterase, the enzyme that hydrolyzes ACh. Although cholinergic pharmacology is a relatively mature field with a number of receptor-selective agents, the specificity of action of the various agents continues to be refined. The discovery of muscarinic-receptor subtype diversity may lead to the development of agents specific for subtypes that are expressed in a tissue-specific pattern. Similarly, elucidation of the role of nicotinic-receptor subunit diversity in the CNS may spur the development of more selective agents that modulate the activity of these receptor subtypes. Acetylcholinesterase inhibitors are now widely used in clinical practice and are standard-of-care in the treatment of AD and other dementias. Currently available AChE inhibitors provide modest symptomatic benefits, and several nicotinic and muscarinic agonists are in clinical development for treatment of cognitive impairment

and AD. Nicotinic receptors may also provide targets for future treatment approaches in epilepsy.

Suggested Reading Andersson KE. Antimuscarinics for treatment of overactive bladder. Lancet Neurol 2004;3:46–53. (Review of overactive bladder pathophysiology and pharmacology.) Atri A, Shaughnessy LW, Locascio JJ, Growdon JH. Long-term course and effectiveness of combination therapy in Alzheimer disease. Alzheimer Dis Assoc Disord 2008;22:209–221. (Reviews clinical efficacy data for anti-AD medications, including AChE inhibitors, and assesses their long-term impact on the course of AD.) Atri A, Sherman S, Norman KA, et al. Blockade of central cholinergic receptors impairs new learning and increases proactive interference in a word paired-associate memory task. Behav Neurosci 2004;118:223–236. (Reviews the theoretical and experimental basis of cholinergic influences on learning and memory and the effects of central blockade on cognitive processes.) Bertrand D, Elmslie F, Hughes E, et al. The CHRNB2 mutation I312M is associated with epilepsy and distinct memory deficits. Neurobiol Dis 2005;20:799–804. (Reviews the role of alterations in nicotinic ACh receptors in genetic epilepsy.) Caccamo A, Fisher A, LaFerla FM. M1 agonists as a potential disease-modifying therapy for Alzheimer’s disease. Curr Alzheimer Res 2009;6:112– 117. (Discusses the role and future research directions for M1 agonists in the treatment of AD.) Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Ann Rev Pharmacol Toxicol 2007;47:699–729. (A thorough yet readable review with many citations.) Fick DM, Cooper JW, Wade WE, Waller JL, Maclean JR, Beers MH. Updating the Beers criteria for potentially inappropriate medication use in older adults: results of a US consensus panel of experts. Arch Intern Med 2003;163:2716– 2724. (Recommendations regarding medications to avoid in older adults.) Jann MW, Shirly KL, Small GW. Clinical pharmacokinetics and pharmacodynamics of cholinesterase inhibitors. Clin Pharmacokinet 2002;41:719– 739. (Review of clinical pharmacology of oral cholinesterase inhibitors.)

10 Adrenergic Pharmacology Brian B. Hoffman and Freddie M. Williams

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 132-133 BIOCHEMISTRY AND PHYSIOLOGY OF ADRENERGIC FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Catecholamine Synthesis, Storage, and Release . . . . . . . 132 Reuptake and Metabolism of Catecholamines . . . . . . . . . 133 Catecholamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . 135 ␣1- and ␣2-Adrenoceptors . . . . . . . . . . . . . . . . . . . . . 135 ␤-Adrenoceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Regulation of Receptor Response. . . . . . . . . . . . . . . . . . . 137 Physiologic and Pharmacologic Effects of Endogenous Catecholamines . . . . . . . . . . . . . . . . . . . . 137 Epinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Norepinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 137 Inhibitors of Catecholamine Synthesis . . . . . . . . . . . . . . . 137 Inhibitors of Catecholamine Storage. . . . . . . . . . . . . . . . . 138 Inhibitors of Catecholamine Reuptake . . . . . . . . . . . . . . . 139 Inhibitors of Catecholamine Metabolism. . . . . . . . . . . . . . 139 Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 ␣-Adrenergic Agonists . . . . . . . . . . . . . . . . . . . . . . . . 139 ␤-Adrenergic Agonists . . . . . . . . . . . . . . . . . . . . . . . . 140 Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 ␣-Adrenergic Antagonists . . . . . . . . . . . . . . . . . . . . . . 140 ␤-Adrenergic Antagonists . . . . . . . . . . . . . . . . . . . . . . 141 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 142 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

 INTRODUCTION

metabolism to receptor activation. Then the physiologic roles of the endogenous catecholamines epinephrine, norepinephrine, and dopamine are discussed, with emphasis on the specificity of receptor expression in different organ systems.

Adrenergic pharmacology involves the study of agents that act on pathways mediated by the endogenous catecholamines norepinephrine, epinephrine, and dopamine. The sympathetic nervous system is the major source of endogenous catecholamine production and release. Signaling through catecholamine receptors mediates diverse physiologic effects, including: increasing the rate and force of cardiac contraction, modifying the peripheral resistance of the arterial system, inhibiting the release of insulin, stimulating hepatic release of glucose, and increasing adipocyte release of free fatty acids. Drugs that target the synthesis, storage, release, and reuptake of norepinephrine and epinephrine or that directly target the postsynaptic receptors for these transmitters are frequent therapies for many major diseases, including hypertension, shock, asthma, and angina. This chapter examines the biochemical and physiologic basis for adrenergic action and then discusses the action of the different classes of adrenergic drugs.

 BIOCHEMISTRY AND PHYSIOLOGY OF ADRENERGIC FUNCTION The autonomic nervous system contributes to homeostasis through the concerted action of its sympathetic and parasympathetic branches. Catecholamines are the major effectors of sympathetic signaling. The following discussion presents the biochemistry of catecholamine action, from synthesis to 132

Catecholamine Synthesis, Storage, and Release Catecholamines are synthesized by sequential chemical modifications of the amino acid tyrosine. This synthesis occurs primarily at sympathetic nerve endings and in chromaffin cells. Epinephrine is predominately synthesized in chromaffin cells of the adrenal medulla; sympathetic neurons produce norepinephrine as their primary neurotransmitter (Fig. 10-1). Tyrosine, the precursor for catecholamine synthesis, is transported into neurons via an aromatic amino acid transporter that uses the Na⫹ gradient across the neuronal membrane to concentrate tyrosine (as well as phenylalanine, tryptophan, and histidine). The first step in catecholamine synthesis, the oxidation of tyrosine to dihydroxyphenylalanine (DOPA), is mediated by the enzyme tyrosine hydroxylase (TH). TH is the rate-limiting enzyme in catecholamine synthesis. DOPA is converted to dopamine by a relatively nonspecific aromatic amino acid decarboxylase. Dopamine is then hydroxylated by dopamine-␤-hydroxylase to yield norepinephrine. In tissues that produce epinephrine, norepinephrine is then methylated on its amino group by phenylethanolamine Nmethyltransferase (PNMT). Expression of PNMT in the adrenal medulla is largely dependent on the high concentrations

134 Principles of Autonomic and Peripheral Nervous System Pharmacology Aromatic L-amino acid transporter

A Normal uptake of norepinephrine from synaptic cleft and concentration of NE in synaptic vesicle Tyrosine

Na+ Tyrosine

Adrenergic neuron

Cytoplasm

Tyrosine hydroxylase

H+

ATP

ADP

Dihydroxyphenylalanine (L-DOPA)

VMAT

Action potential

NE NE

Dopamine

Na+

NE

NE NE

VMAT

NE transporter (NET)

H+

Aromatic L-amino acid decarboxylase

Synaptic cleft

NE

NE transporter

NE

NE

Na+ Ca2+

H+ Dopamine Dopamine β−hydroxylase NE

NE

B Cocaine inhibits NE transporter NE

α2 (autoreceptor)

adrenergic receptor

MAO

H+

ATP

ADP

NE

Cocaine

DOPGAL NET

VMAT H+

NE

NE

NE

Synaptic cleft

NE

NE NE

NE NE

α1

β1

β2

Postsynaptic adrenergic receptors

C Reserpine inhibits VMAT

Postsynaptic cell ATP

H+

ADP

VMAT

FIGURE 10-1.

Catecholamine synthesis, storage, release, and reuptake pathways. The endogenous catecholamines dopamine, norepinephrine, and epinephrine are all synthesized from tyrosine. The rate-limiting step in catecholamine synthesis, the oxidation of cytoplasmic tyrosine to dihydroxyphenylalanine (L-DOPA), is catalyzed by the enzyme tyrosine hydroxylase. Aromatic L-amino acid decarboxylase then converts L-DOPA to dopamine. Vesicular monoamine transporter (VMAT) translocates dopamine (and other monoamines) into synaptic vesicles. In adrenergic neurons, intravesicular dopamine-␤-hydroxylase converts dopamine to norepinephrine (NE). Norepinephrine is then stored in the vesicle until release. In adrenal medullary cells, norepinephrine returns to the cytosol, where phenylethanolamine N-methyltransferase (PNMT) converts norepinephrine to epinephrine. The epinephrine is then transported back into the vesicle for storage (not shown). ␣-Methyltyrosine inhibits tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis (not shown). Released norepinephrine can stimulate postsynaptic ␣1-, ␤1-, or ␤2-adrenergic receptors or presynaptic ␣2-adrenergic autoreceptors. Released norepinephrine can also be taken up into presynaptic terminals by the selective NE transporter. NE in the cytoplasm of the presynaptic neuron can be further taken up into synaptic vesicles by VMAT (not shown) or degraded to 3, 4-dihydroxyphenylglycoaldehyde (DOPGAL; see Fig. 10-3) by mitochondrion-associated monoamine oxidase (MAO).

NET

Na+

NE NE NE

NE

Reserpine

FIGURE 10-2.

Mechanisms of action of cocaine and reserpine. A. Norepinephrine (NE) that has been released into the synaptic cleft can be taken up into the cytoplasm of the presynaptic neuron by the selective NE transporter (NET), a Na⫹-NE co-transporter. Cytoplasmic NE is concentrated in synaptic vesicles by the nonselective vesicular monoamine transporter (VMAT), an H⫹-monoamine antiporter. An H⫹-ATPase uses the energy of ATP hydrolysis to concentrate protons in synaptic vesicles, and thereby generates a transmembrane H⫹ gradient. This H⫹ gradient is used by VMAT to drive monoamine transport into the synaptic vesicle. B. Cocaine inhibits the NE transporter, allowing released NE to remain in the synaptic cleft for a longer period of time. By this mechanism, cocaine potentiates neurotransmission at adrenergic synapses. C. Reserpine inhibits the vesicular monoamine transporter, preventing the refilling of synaptic vesicles with NE and eventually depleting the adrenergic terminal of neurotransmitter. By this mechanism, reserpine inhibits neurotransmission at adrenergic synapses.

Neurotransmitter OH HO

Catechol-O-methyl transferase (COMT)

NH2

Monoamine oxidase (MAO)

HO

Norepinephrine

OH HO

H

OH O

NH2

Aldehyde reductase

O

HO

DOPGAL

HO

Normetanephrine

Aldehyde dehydrogenase

OH HO

OH OH

MAO

HO

OH

DOPEG

OH O

HO O

HO

H

DOMA COMT

O

HO

MOPGAL

Aldehyde reductase

COMT

OH O

OH OH O

HO

MOPEG HO

Aldehyde dehydrogenase

OH O

Vanillylmandelic acid (VMA) Major metabolite (excreted in urine)

CHAPTER 10 / Adrenergic Pharmacology 137

action has led to speculation that ␤3-agonists may be useful in the treatment of obesity, noninsulin-dependent diabetes mellitus, and other potential indications, but such selective pharmacologic agents remain to be developed for clinical use.

Regulation of Receptor Response The ability of receptor agonists to initiate downstream signaling is proportional to the number of receptors activated. Accordingly, changes in the density of receptors on the cell surface will alter the apparent efficacy of an agonist. Thus, both short-term (desensitization) and long-term (down-regulation) changes in the number of functional adrenoceptors are important in regulating tissue response (see Fig. 1-10). When an agonist activates an adrenoceptor, the dissociation of heterotrimeric G proteins leads to downstream signaling as well as a negative feedback mechanism that limits tissue responses. The accumulation of ␤␥ subunits in the membrane recruits a G protein receptor kinase (GRK), which phosphorylates the receptor at residues in the C-terminus that are important targets for inactivator proteins. Alternatively, protein kinase A and protein kinase C can phosphorylate G proteins. The phosphorylated state of a G protein can bind to another protein called ␤-arrestin that sterically inhibits the receptor–G protein interaction, effectively silencing receptor signaling. On a longer time scale, the receptor–␤-arrestin complex is sequestered, in a clathrin-dependent manner, into an endocytic compartment for internalization, a process called down-regulation. Each of these processes is important in regulating tissue responsiveness on a short- or long-term basis. Somewhat paradoxically, recent evidence suggests that ␤-arrestins can turn on (rather than off) novel signaling pathways involving activation of tyrosine kinases and small GTP-binding proteins.

Physiologic and Pharmacologic Effects of Endogenous Catecholamines The endogenous catecholamines epinephrine and norepinephrine act as agonists at both ␣- and ␤-adrenoceptors. At supraphysiologic concentrations, dopamine can also act as an agonist at ␣- and ␤-receptors. The overall effect of each catecholamine is complex and depends on the concentration of the agent and on tissue-specific receptor expression. Epinephrine Epinephrine is an agonist at both ␣- and ␤-adrenoceptors. At low concentrations, epinephrine has predominantly ␤1 and ␤2 effects, while at higher concentrations, its ␣1 effects become more pronounced. Acting at ␤1-receptors, epinephrine increases cardiac contractile force and cardiac output, with consequent increases in cardiac oxygen consumption and systolic blood pressure. Vasodilation mediated by ␤2-receptors causes a decrease in peripheral resistance and a decrease in diastolic blood pressure. Stimulation of ␤2receptors also increases blood flow to skeletal muscle, relaxes bronchial smooth muscle, and increases the concentrations of glucose and free fatty acids in the blood. These ␤1 and ␤2 effects are all components of the “fight-or-flight” response. Epinephrine was used to treat acute asthmatic attacks shortly after its discovery more than 100 years ago; other drugs with greater selectivity for ␤2-receptors are now more often used in the treatment of asthma. Epinephrine remains a drug of choice for the treatment of anaphylaxis. Locally injected epinephrine

causes vasoconstriction and prolongs the action of local anesthetics; for example, it is often used in combination with a local anesthetic in dentistry. It is ineffective orally due to extensive first-pass metabolism. Epinephrine has a rapid onset and a brief duration of action when injected intravenously. Adverse consequences of rapid intravenous infusions include increased cardiac excitability that may lead to cardiac arrhythmias and excessive increases in blood pressure. Norepinephrine Norepinephrine is an agonist at ␣1- and ␤1-receptors, but has relatively little effect at ␤2-receptors. Because of the lack of action at ␤2-receptors, systemic administration of norepinephrine increases not only systolic blood pressure (␤1 effect) but also diastolic blood pressure and total peripheral resistance. Norepinephrine is used in the pharmacologic treatment of hypotension in patients with distributive shock, most frequently due to sepsis. Dopamine Although dopamine is a prominent CNS neurotransmitter, systemic administration has few CNS effects because it does not readily cross the blood–brain barrier. Dopamine activates one or more subtypes of catecholamine receptor in peripheral tissues, and the predominant effect is dependent on the local concentration of the compound. At low doses (⬍2 ␮g/kg per min), a continuous intravenous infusion of dopamine acts predominately on D1 dopaminergic receptors in renal, mesenteric, and coronary vascular beds. D1 dopaminergic receptors activate adenylyl cyclase in vascular smooth muscle cells, leading to increased cAMP levels and vasodilation. At higher rates of infusion (2–10 ␮g/kg per min), dopamine is a positive inotrope via its activation of ␤1-adrenergic receptors. At still higher rates of infusion (⬎10 ␮g/kg per min), dopamine acts on vascular ␣1-adrenergic receptors to cause vasoconstriction. Dopamine is used in the treatment of shock, particularly in states of shock caused by low cardiac output and accompanied by compromised renal function leading to oliguria. However, efficacy in protecting the kidneys has not been clearly demonstrated.

 PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic intervention is possible at each of the major steps in catecholamine synthesis, storage, reuptake, metabolism, and receptor activation. The following discussion presents the various classes of agents in the order of their action on adrenergic pathways, from neurotransmitter synthesis to receptor activation.

Inhibitors of Catecholamine Synthesis Inhibitors of catecholamine synthesis have limited clinical utility because such agents nonspecifically inhibit the formation of all catecholamines (see Fig. 10-1). ␣-Methyltyrosine is a structural analogue of tyrosine that is transported into nerve terminals, where it inhibits tyrosine hydroxylase, the first enzyme in the catecholamine biosynthesis pathway. This agent is used occasionally in the treatment of hypertension associated with pheochromocytoma (a tumor of the enterochromaffin cells of the adrenal medulla that produces norepinephrine and epinephrine). Its clinical use is limited, however, because it causes significant orthostatic hypotension and sedation, and many other antihypertensive drugs are available for this indication.

A Acute effect of indirect sympathomimetic

NE NE G

NE NE

G

NET

VMAT

G

NE

G G

NE NE

NE NE

NE NE

MAO Mitochondrion

NE

DOPGAL

B Chronic effect of indirect sympathomimetic

G

NET

VMAT

G

G

NE

G NE

G

NE

MAO Mitochondrion

DOPGAL

140 Principles of Autonomic and Peripheral Nervous System Pharmacology

supporting its capacity to decrease adverse cardiovascular outcomes in patients with hypertension is quite limited. Clonidine has limited utility in ameliorating symptoms of withdrawal from ethanol and opioid drugs. Adverse effects include bradycardia caused by decreased sympathetic activity and increased vagal activity, as well as dry mouth and sedation. Because sympathetic nervous system activation is an important mechanism in maintaining blood pressure on standing, postural hypotension may also complicate therapy with this drug. Other centrally acting ␣2-agonists include the seldom-used agents guanabenz and guanfacine. These agents have adverse effect profiles similar to that of clonidine. Dexmedetomidine is an ␣2-receptor agonist whose capacity to cause sedation has been exploited as a beneficial effect in surgical patients, because sedation is induced by this drug without additional respiratory depression. Suppression of sympathetic nervous system activity by this drug helps to avoid swings in blood pressure in surgical patients, who are carefully monitored by anesthetists during surgical procedures. Dexmedetomidine may also possess analgesic properties. Note that the ␣2-mediated effects of sedation and decreased sympathetic activity are adverse effects of clonidine in the setting of outpatient treatment for hypertension but beneficial effects of dexmedetomidine in the controlled setting of the surgical patient. ␣-Methyldopa is a precursor (prodrug) to the ␣2-agonist ␣-methylnorepinephrine. Endogenous enzymes catalyze the metabolism of methyldopa to methylnorepinephrine, and the ␣-methylnorepinephrine is then released by the adrenergic nerve terminal, where it can act presynaptically as an ␣2agonist. This action results in decreased sympathetic outflow from the CNS and consequent lowering of blood pressure in hypertensive patients. Because ␣-methyldopa use can be associated with rare hepatotoxicity, autoimmune hemolytic anemia, and adverse CNS effects, this drug is very rarely used in the treatment of hypertension in the United States, with one exception—there is considerable experience with methyldopa as an antihypertensive drug in pregnancy, and it is still used as a preferred drug in that context. ␤-Adrenergic Agonists Stimulation of ␤1-adrenergic receptors causes an increase in heart rate and the force of cardiac muscle contraction, resulting in increased cardiac output, while stimulation of ␤2-adrenergic receptors causes relaxation of vascular, bronchial, and gastrointestinal smooth muscle. Isoproterenol is a nonselective ␤-agonist. This drug lowers peripheral vascular resistance and diastolic blood pressure (a ␤2 effect), while systolic blood pressure remains unchanged or slightly increased (a ␤1 effect). Because isoproterenol is a positive inotrope (increases cardiac contractility) and chronotrope (increases heart rate), cardiac output is increased. Isoproterenol can be used to relieve bronchoconstriction in asthma (␤2 effect). However, because isoproterenol is a nonselective activator of ␤1- and ␤2adrenoceptors, its use for relief of bronchoconstriction is often accompanied by adverse cardiac effects. Use of this drug in asthma has therefore been supplanted by newer ␤2-selective agonists (see below). Isoproterenol may occasionally be used to stimulate heart rate in emergency situations of profound bradycardia, typically in anticipation of the placement of an electrical cardiac pacemaker. The overall effect of dobutamine depends on the differential effects of the two stereoisomers contained in the racemic

mixture (see Chapter 1 for a discussion of stereoisomers). The (⫺) isomer acts as both an ␣1-agonist and a weak ␤1-agonist, whereas the (⫹) isomer acts as both an ␣1-antagonist and a potent ␤1-agonist. The ␣1-agonist and antagonist properties effectively cancel each other out when the racemic mixture is administered, and the observed clinical result is that of a selective ␤1-agonist. This agent has more prominent inotropic than chronotropic effects, resulting in increased contractility and cardiac output. Dobutamine can be used intravenously in the urgent treatment of severe heart failure. It is also used as a diagnostic agent, in conjunction with imaging of the heart, in the investigation of ischemic heart disease. ␤2-selective agonists are valuable in the treatment of asthma. These drugs represent pharmacologic improvements over epinephrine (an agonist at all adrenergic receptors) and isoproterenol (an agonist at ␤1- as well as ␤2-receptors) in that their effects are more limited at nontarget tissues. It is particularly important that these selective drugs have limited capacity to stimulate ␤1-adrenoceptors in the heart and, therefore, limited capacity to produce adverse cardiac effects. Specificity for the lung rather than the heart or other peripheral tissues has been further enhanced by generally delivering these drugs via aerosols inhaled into the lungs. Administration of these drugs directly into the lungs lowers the amount of drug that reaches the systemic circulation, again limiting the activation of cardiac ␤1-receptors and skeletal muscle ␤2-receptors. The most important effects of these agents are relaxation of bronchial smooth muscle and decrease in airway resistance. ␤2-selective agonists are not completely specific for airway ␤2-receptors, however, and adverse effects can include skeletal muscle tremor (through ␤2-stimulation) and tachycardia (through ␤1-stimulation). Metaproterenol is the prototype ␤2-selective agonist. This drug is used to treat obstructive airway disease and acute bronchospasm. Terbutaline and albuterol are two other agents in this class that have similar efficacy and duration of action. Salmeterol is a long-acting ␤2-agonist; its effects last for about 12 hours. The clinical utility of ␤2-selective agonists is discussed more fully in Chapter 47, Integrative Inflammation Pharmacology: Asthma.

Receptor Antagonists There is a wide spectrum of disease states that respond to modulation of adrenoceptor activity, and antagonists at ␣and ␤-adrenoceptors are among the most widely used drugs in clinical practice. ␣-Adrenergic Antagonists ␣-Adrenergic antagonists block endogenous catecholamines from binding to ␣1- and ␣2-adrenoceptors. These agents cause vasodilation, decreased blood pressure, and decreased peripheral resistance. The baroreceptor reflex usually attempts to compensate for the fall in blood pressure, resulting in reflex increases in heart rate and cardiac output. An important laboratory tool since the 1950s, phenoxybenzamine is an alkylating agent that blocks both ␣1- and ␣2-receptors irreversibly. In addition, phenoxybenzamine inhibits catecholamine uptake into both adrenergic nerve terminals and extraneuronal tissues. Because of its many direct and indirect effects on the sympathetic nervous system and target tissues, phenoxybenzamine, once used in the treatment of hypertension and benign prostatic hyperplasia (BPH), is

142 Principles of Autonomic and Peripheral Nervous System Pharmacology

have not been developed clinically as there is no obvious indication for selective ␤2-receptor antagonism. Propranolol, nadolol, and timolol do not distinguish between ␤1- and ␤2-receptors in their binding affinities. This is the origin of the term “nonselective ␤-blockers.” At clinical doses, these drugs do not block ␣-receptors. Nonselective ␤-blockers have been used for many years in the treatment of hypertension and angina. Although nonselective ␤-blockers are relatively contraindicated in patients with asthma, these drugs are often well tolerated in patients with chronic obstructive pulmonary disease (COPD) and may be initiated cautiously in many such patients if they have a compelling indication (e.g., coronary artery disease). Nadolol is also efficacious in the prevention of bleeding from esophageal varices in patients with cirrhosis. It is pharmacologically attractive for this indication because it has a long half-life, allowing once-daily administration, and, because the drug is excreted primarily by renal elimination without hepatic metabolism, no dosing adjustments are needed on account of hepatic insufficiency. Penbutolol is an additional drug in this class. An ocular formulation of timolol is used in the treatment of glaucoma; even when administered to the eye, systemic absorption of the drug may be sufficiently high to cause adverse effects in susceptible patients. Levobunolol and carteolol are additional nonselective ␤-blockers that are indicated for administration via eye drops in the treatment of glaucoma. Labetalol and carvedilol block ␣1-, ␤1-, and ␤2-receptors. Labetalol has two chiral centers; the clinically used drug is a combination of four stereoisomers that have differing pharmacologic properties. Because the effect and metabolism of these isomers may vary among individual patients, the relative proportion of ␣1- versus ␤-blockade is variable. The ␣1-receptor blockade tends to lower peripheral resistance; ␤-blockade also contributes to a decrease in blood pressure, as indicated above. An intravenous formulation of labetalol is available for the lowering of blood pressure in patients with hypertensive emergencies. An unpredictable and idiosyncratic adverse effect of labetalol is drug-induced hepatitis. While carvedilol is also efficacious in the outpatient management of hypertension, much interest in this drug has been due to its efficacy in the management of heart failure with decreased systolic function. Pindolol is a partial agonist at ␤1- and ␤2-receptors. The drug blocks the action of endogenous norepinephrine at ␤1-receptors and is useful in treating hypertension. As a partial agonist, pindolol also causes partial stimulation of ␤1-receptors, leading to overall smaller decreases in resting heart rate and blood pressure than those caused by pure ␤-antagonists. Acebutolol is a partial agonist at ␤1-adrenoceptors but has no effect at ␤2-receptors. This agent is also used to treat hypertension. While it has been suggested that partial agonists may be less likely to cause adverse effects in patients with bradycardia, the clinical advantages of drugs in this category remain unclear. Esmolol, metoprolol, atenolol, and betaxolol are ␤1selective adrenergic antagonists. The elimination half-life is the main feature that distinguishes among these agents. Esmolol has an extremely short half-life (3–4 minutes); metoprolol and atenolol have intermediate half-lives (4–9 hours). Because of its short half-life, esmolol may be safer in unstable patients requiring ␤-blockade. Esmolol is

rapidly metabolized by esterases. Clinical trials have shown that some ␤-blockers, including metoprolol, prolong life expectancy in patients with mild to moderate heart failure and in patients who have survived a first myocardial infarction (see Chapter 25, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure). Nebivolol is a novel ␤1-selective adrenergic antagonist that has the ancillary property of promoting vasodilation via nitric oxide release from endothelial cells. Many of the major adverse effects of ␤-adrenergic antagonists are a predictable extension of their pharmacologic effects. Such effects include worsening of bronchoconstriction in patients with asthma, decreased cardiac output in patients with decompensated heart failure, or potentially impaired recovery from hypoglycemia in diabetic patients receiving insulin. While ␤1-selective adrenergic antagonists may have less propensity to block ␤2-receptors in bronchial smooth muscle, the selectivity of these drugs is modest and may not be a clinically reliable safeguard against adverse effects. With chronic administration of ␤-receptor antagonists, pharmacologic adaptations may occur that leave cells hypersensitive to catecholamines if the drug is stopped suddenly.

 CONCLUSION AND FUTURE DIRECTIONS Adrenergic pharmacology encompasses drugs that act at essentially every step of adrenergic neurotransmission, from synthesis of catecholamines to stimulation of ␣- and ␤-receptors. Additional drugs, such as L-channel Ca2⫹ blockers, interfere with effector responses activated by these receptors. Novel drugs are being developed that selectively inhibit the downstream effector pathways activated by adrenergic receptors. The drugs discussed in this chapter are mainstays of therapy for hypertension, angina, heart failure, shock, asthma, pheochromocytoma, and other conditions. The beneficial pharmacologic actions of these drugs, as well as many of their important adverse effects, can be anticipated from knowledge of their molecular and cellular mechanisms of action and how these actions affect the processes of adrenergic neurotransmission. While nine subtypes of adrenergic receptor have been identified—three in each of the major classes—the pharmacologic implications of these discoveries, in terms of the development of novel subtype-selective drugs, may not have been fully exploited. Therefore, the clinical relevance of these subtypes has not yet been fully determined; the development of more selective agonists and antagonists may lead to more effective (and less toxic) therapies.

Acknowledgment We thank Timothy J. Turner for his valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK. Beta-arrestins and cell signaling. Annu Rev Physiol 2007;69:483–510. (Review of novel mechanisms of signaling via seven transmembrane receptors.) Rosenbaum DM, Rasmussen SG, Kobilka BK. The structure and function of G protein-coupled receptors. Nature 2009;459:356–363. (Detailed review of the structure of adrenergic receptors.)

11 Local Anesthetic Pharmacology Joshua M. Schulman and Gary R. Strichartz

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 147-148 PHYSIOLOGY OF NOCICEPTION . . . . . . . . . . . . . . . . . . . . . . 147 Transmission of Pain Sensation . . . . . . . . . . . . . . . . . . . . 149 First Pain and Second Pain . . . . . . . . . . . . . . . . . . . . . 149 Pain Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Analgesia and Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . 150 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 151 Chemistry of Local Anesthetics . . . . . . . . . . . . . . . . . . . . 151 Aromatic Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Amine Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Mechanism of Action of Local Anesthetics . . . . . . . . . . . . 152 Anatomic Considerations . . . . . . . . . . . . . . . . . . . . . . 152 Voltage-Gated Sodium Channel. . . . . . . . . . . . . . . . . . 153 Other Receptors for Local Anesthetics . . . . . . . . . . . . . 155 Pharmacokinetics of Local Anesthetics . . . . . . . . . . . . . . 155 Systemic Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Metabolism and Excretion . . . . . . . . . . . . . . . . . . . . . . 156 Administration of Local Anesthetics . . . . . . . . . . . . . . . . . 156 Topical Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Infiltration Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . 156 Peripheral Nerve Blockade . . . . . . . . . . . . . . . . . . . . . 156 Central Nerve Blockade . . . . . . . . . . . . . . . . . . . . . . . 157 Intravenous Regional Anesthesia . . . . . . . . . . . . . . . . . 157 Major Toxicities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Individual Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Ester-Linked Local Anesthetics . . . . . . . . . . . . . . . . . . 158 Amide-Linked Local Anesthetics . . . . . . . . . . . . . . . . . 158 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 159 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

 INTRODUCTION

transmission of information to and from the central nervous system (CNS). LA actions are not selective for pain fibers; they can also block other sensory fibers as well as motor and autonomic fibers and action potentials in skeletal and cardiac muscle. This nonselective blockade can serve other useful functions (see Chapter 23, Pharmacology of Cardiac Rhythm) or can be a source of toxicity.

The word anesthesia comes directly from the Greek: “an” meaning without, and “aisthesis” meaning feeling or sensation. Local anesthetics (LAs) are a set of locally applied chemicals, with similar molecular structures, that can both inhibit the perception of sensations (importantly pain) and prevent movement. They are used in a variety of situations, ranging from topical application for burns and small cuts, to injections during dental care, to epidural and intrathecal (“spinal”) blocks during obstetric procedures and major surgery. Cocaine, the first local anesthetic, comes from the leaves of the coca shrub (Erythroxylon coca). It was first isolated in 1860 by Albert Niemann, who noted its numbing powers. In 1886, Carl Koller introduced cocaine into clinical practice as a topical ophthalmic anesthetic. However, its addictive properties and toxicity prompted the search for substitutes. Procaine, the first of these substitutes, was synthesized in 1905. Known as Novocain®, it is still used today, although less frequently than some more recently developed LAs. Local anesthetics exert their effect by blocking voltagegated sodium channels, thus inhibiting the propagation of action potentials along neurons (see Chapter 7, Principles of Cellular Excitability and Electrochemical Transmission). By inhibiting action potential propagation, LAs prevent

 PHYSIOLOGY OF NOCICEPTION Nociception is the activation of primary sensory nerve fibers (nociceptors) by noxious stimuli, that is, stimuli that are potentially tissue damaging. These include high temperatures, intense mechanical perturbations, and harsh chemicals. Nociceptors have free nerve endings located in the skin, deep soma, and viscera. Nociceptor cell bodies are located in the dorsal root ganglia close to the spinal cord, or in the trigeminal ganglion for innervation of the face (Fig. 11-1). Nociceptors transmit impulses from the periphery to the dorsal horn of the spinal cord, where the information is subsequently processed through synaptic circuitry and transmitted to various parts of the brain. Thus, nociceptors are the first in a chain of neurons responsible for pain perception. Because nociceptors, among other sensory fibers, transmit information toward the brain, they are termed afferent neurons. 147

Dorsal root ganglion To brain

1a Thermal Mechanical Chemical

Free nerve endings

2 Nociceptor activation

1b Nearby cell damage

Spinal cord

Bradykinin Serotonin

Axon

Prostaglandins 3

Serotonin

CGRP and substance P released by activated nociceptors

4b Mast cell degranulation 4a Blood vessel dilation

150 Principles of Autonomic and Peripheral Nervous System Pharmacology

A First and second pain (no block)

Second pain

Pain intensity

First pain

Painful stimulus

Time (sec)

Pain intensity

B Effect of Aδ fiber block

peripheral sensory (including pain), motor, and autonomic pathways. Analgesics have actions at specific receptors on primary nociceptors and in the CNS (see Chapter 17). For example, opioid analgesics activate opiate receptors, which signal cells to increase potassium conductance in postsynaptic neurons and to decrease calcium entry into presynaptic neurons. By these mechanisms, postsynaptic excitability and presynaptic transmitter release are reduced, and pain sensations are not transmitted as effectively to the brain (or within it). Importantly, the transmission of other sensations and motor information is not affected. Local anesthetics act by a different mechanism. These agents inhibit conduction of action potentials in all afferent and efferent nerve fibers, usually in the peripheral nervous system. Thus, pain and other sensory modalities are 3˚ neuron projects to various regions of the brain

Painful stimulus

Time (sec)

Primary somatic sensory cortex

Time (sec)

Cerebrum

2˚ neuron synapses with 3˚ neuron in thalamus

Pain intensity

C Effect of C fiber block

Painful stimulus

Ventral posterior lateral nucleus of thalamus

FIGURE 11-2.

First and second pain. First pain, which is transmitted by A␦-fibers, is sharp and highly localizable. Second pain, which is transmitted by C-fibers, is slower in arriving, duller, and longer lasting (A). First pain can be prevented by selective blockade of A␦-fibers (B), and second pain can be prevented by selective blockade of C-fibers (C). Because A␦-fibers are more susceptible than C-fibers to blockade by local anesthetics, first pain often disappears at concentrations of anesthetic lower than those required to eliminate second pain.

Midbrain

Pons

lateral areas of the spinal cord and project mainly to the thalamus, a gray-matter structure just superior to the brainstem. The thalamus has cells that project to the somatosensory cortex of the parietal lobe and to other areas of the cortex (Fig. 11-3). Pain perception is a complex process that normally results from the activation of non-nociceptive as well as nociceptive afferents, and can be altered depending on the situation, the person’s state of mind, and other factors. The CNS uses efferent projections within the brain and spinal cord to modulate the incoming nociceptive signals and thus to modify pain perception (see Chapter 17, Pharmacology of Analgesia). For example, an athlete focused on an important game might not feel the pain of an injury intensely until after the game is over. Her brain modulates the effect of the input so that the same stimulus is less painful at certain times than at others.

Analgesia and Anesthesia The terms “analgesic” and “anesthetic” have different meanings. Analgesics are specific inhibitors of pain pathways, whereas local anesthetics are nonspecific inhibitors of

Medulla 2˚ neurons

Dorsal root ganglion 1˚ neuron (nociceptor)

Cervical spinal cord 1˚ and 2˚ neurons synapse in dorsal horn of spinal cord

FIGURE 11-3.

Pain pathways. Primary (1°) nociceptors have cell bodies in the dorsal root ganglion and synapse with secondary (2°) afferent neurons in the dorsal horn of the spinal cord. Primary afferents use the neurotransmitter glutamate. The 2° afferents travel in the lateral areas of the spinal cord and eventually reach the thalamus, where they synapse with tertiary (3°) afferent neurons. The processing of pain is complex, and 3° afferents have many destinations including the somatosensory cortex (localization of pain) and the limbic system (emotional aspects of pain).

CHAPTER 11 / Local Anesthetic Pharmacology 151

not transmitted effectively to the brain, and motor and autonomic impulses are not transmitted effectively to muscles and end-organs in the periphery.

 PHARMACOLOGIC CLASSES AND AGENTS Local anesthetics can be structurally classified as ester-linked LAs or amide-linked LAs. Because all LAs share similar properties, the next section highlights the general principles of LA pharmacology. Specific LA agents are discussed at the end of the chapter.

Chemistry of Local Anesthetics All local anesthetics have three structural domains: an aromatic group, an amine group, and an ester or amide linkage connecting these two groups (Fig. 11-4). As discussed below, the structure of the aromatic group influences the hydrophobicity of the drug, and the nature of the amine group influences the charge on the drug. Both features define the rate of onset, potency, duration of action, and adverse effects of an individual local anesthetic. Aromatic Group All local anesthetics contain an aromatic group that gives the molecule much of its hydrophobic character. Adding alkyl substituents on the aromatic ring, or on the amino nitrogen, increases the hydrophobicity of these drugs. Biological membranes have a hydrophobic interior because of their lipid bilayer structure. The hydrophobicity of an LA drug affects the ease with which the drug passes through nerve cell membranes to reach its target site, which is the cytoplasmic side of the voltage-gated sodium channel (Fig. 11-5). Molecules with low hydrophobicity partition very poorly into the membrane because their solubility in the lipid bilayer is so low; such molecules are largely restricted to the polar aqueous environment. As the hydrophobicity of a series of drugs increases, the concentration in the membrane and the correlated permeability of the drugs through the cell membrane also increase. However, at a certain hydrophobicity, this relationship reverses, and a further increase in hydrophobicity results in a decrease in permeability. This somewhat paradoxical behavior occurs because molecules that are very hydrophobic partition so strongly into the cell membrane that they remain there. The same strong hydrophobic forces that concentrate such molecules in the cell membrane cause them to dissociate very slowly from that compartment. To be effective, a local anesthetic must partition into, diffuse across, and finally dissociate from the membrane into the cytoplasm; the compounds most likely to do so have moderate hydrophobicity. The LA binding site on the sodium channel also contains hydrophobic residues. Therefore, more hydrophobic drugs bind more tightly to the target site, increasing the potency of the drug. However, as noted above, because of the practical need for the drug to diffuse across several membranes in order to reach the target site, LAs with moderate hydrophobicity are the clinically most effective forms. In addition, excessively hydrophobic drugs have limited solubility in the aqueous environment around a nerve, and even the molecules that do dissolve remain in the first membrane that is encountered, never reaching the target site (despite their high affinity for that site).

Amine Group The amine group of a local anesthetic molecule can exist in either the protonated (positively charged) form or the deprotonated (neutral) or base form. The pKa is the pH at which the concentrations of a base and its conjugate acid are equal. LAs are weak bases; their pKa values range from about 8 to 10. Thus, at the physiologic pH of 7.4, substantial amounts of both the protonated form and the neutral form coexist in solution. As the pKa of a drug increases, a greater fraction of molecules exists in solution in the protonated form at physiologic pH (see Chapter 1, Drug–Receptor Interactions). Protonation and deprotonation reactions are very A Ester-linked local anesthetic (procaine) Aromatic group (R)

Ester linkage

Tertiary amine (R')

O N O H+

H2N H+

Basic form

O NH + O H2N Protonated (acidic) form B Amide-linked local anesthetic (lidocaine) Aromatic group (R)

Amide linkage

Tertiary amine (R')

H N N

H+

O Basic form

H+ H N

H+ N O

Protonated (acidic) form

FIGURE 11-4. Prototypical local anesthetics. Procaine (A) and lidocaine (B) are prototypical ester-linked and amide-linked local anesthetics, respectively. Local anesthetics have an aromatic group on one end and an amine on the other end of the molecule; these two groups are connected by an ester (-RCOOR⬘) or amide (-RHNCOR⬘) linkage. In solution at high pH, the equilibrium between the basic (neutral) and acidic (charged) forms of a local anesthetic favors the basic form. At low pH, the equilibrium favors the acidic form. At intermediate (physiologic) pH, nearly equal concentrations of the basic and acidic forms are present. Generally, esterlinked local anesthetics are easily hydrolyzed to a carboxylic acid (RCOOH) and an alcohol (HOR⬘) in the presence of water and esterases. In comparison, amides are far more stable in solution. Consequently, amide-linked local anesthetics generally have a longer duration of action than do ester-linked anesthetics.

152 Principles of Autonomic and Peripheral Nervous System Pharmacology A Poorly hydrophobic local anesthetic LA

1

Linker region

B Moderately hydrophobic local anesthetic Voltage-gated Na+ channel LA 4

1

H+

Extracellular

LA

Local anesthetic binding site

2 LA

LA

of the voltage-gated sodium channel and is accessible from the intracellular, cytoplasmic entrance of the channel. This is why moderately hydrophobic weak bases are so effective as local anesthetics. At physiologic pH, a significant fraction of the weak base molecules are in the neutral form that, because of its moderate hydrophobicity, can rapidly cross membranes to enter nerve cells. Once the drug is inside the cell, it can then readily gain a proton, become positively charged, and bind to the sodium channel. Surprisingly, the major path by which protons reach local anesthetics is through the Na⫹ channel’s pore. As the extracellular pH becomes more acidic, there is a higher chance that the drug will become protonated at its binding site in the channel. Once protonated, the drug dissociates much more slowly from the channel. The pH inside the cell does not have an important effect on the protonation state of drug molecules that are already bound to the channel; this lack of effect is thought to be attributable to the orientation of the drug, which effectively blocks H⫹ access from within the cell. Some nonionizable drugs, such as benzocaine, are permanently neutral but are still able to block sodium channels. For these drugs, however, the block is weak and rapidly reversible and does not depend on extracellular pH.

Intracellular 3

C Extremely hydrophobic local anesthetic LA

1

LA

2

FIGURE 11-5. Local anesthetic hydrophobicity, diffusion, and binding. Local anesthetics (LAs) act by binding to the cytoplasmic (intracellular) side of the voltage-gated Na⫹ channel. The hydrophobicity of a local anesthetic determines how efficiently it diffuses across lipid membranes and how tightly it binds to the Na⫹ channel, and therefore governs its potency. A. Poorly hydrophobic LAs are unable to cross the lipid bilayer efficiently: (1) Neutral LA cannot adsorb to or enter the neuronal cell membrane because the LA is very stable in the extracellular solution and has a very high activation energy for entering the hydrophobic membrane. B. Moderately hydrophobic LAs are the most effective agents: (1) Neutral LA adsorbs to the extracellular side of the neuronal cell membrane; (2) LA diffuses through the cell membrane to the cytoplasmic side; (3) LA diffuses and binds to its binding site on the voltage-gated sodium channel; and (4) once bound, LA can switch between its neutral and protonated forms by binding and releasing protons. C. Extremely hydrophobic LAs become trapped in the lipid bilayer: (1) Neutral LA adsorbs to the neuronal cell membrane (2) where it is so stabilized that it cannot dissociate from or translocate across the membrane.

rapid in solution (103 sec⫺1), but drugs in membranes or bound to proteins are protonated and deprotonated more slowly. The neutral forms of LAs cross membranes much more easily than do the positively charged forms. However, the positively charged forms bind with much higher affinity to the drugs’ target binding site. This site is located in the pore

Mechanism of Action of Local Anesthetics Anatomic Considerations The peripheral nerve is composed of a collection of different types of nerve fibers (A-, B-, and C-fibers) surrounded by three protective membranes, or “sheaths”: the epineurium, perineurium, and endoneurium. Local anesthetic molecules must pass through these sheaths, which present the same permeation-limiting barriers as the nerve cell membranes, considered above, before they can reach the neuronal membranes to block conduction (Fig. 11-6). The sheaths are made up of connective tissue and cellular membranes. LAs are injected outside the most external sheath, the epineurium, to avoid mechanical needle damage to the nerve, but the major barrier to LA penetration into the nerve is the perineurium, an epithelium-like tissue that bundles axons into separate fascicles. Recall that LAs affect not only nociceptors but also other afferent and efferent, somatic and autonomic nerve fibers. All of these fibers may be contained within a peripheral nerve, and conduction in all fibers can be blocked by local anesthetics. If the peripheral nerve is considered to represent a multilane road, then each fiber type can be considered a lane in this road. A blockade across the whole road (the blockade by a local anesthetic) will stop traffic in all lanes in both directions. This is why, in the introductory case, EM experienced not only loss of pain sensation, but also a more complete block of all sensation in his digits. In general, more proximal regions of the body (shoulder, thigh) are innervated by axons traveling relatively superficially in a peripheral nerve, while more distal regions (hands, feet) are innervated by axons traveling closer to the core of the nerve. Because local anesthetics are applied to the outside of a peripheral nerve, external to the epineurium, the axons innervating more proximal areas are usually reached first by the local anesthetic diffusing radially into the nerve. Consequently, in the anatomic progression of functional block, proximal areas are numbed before distal areas. For example, if a nerve block is applied in the brachial plexus,

Epineurium Perineurium Endoneurium Schwann cell

1

2

3

Needle injecting LA Unmyelinated fiber bundle (C fibers) Myelinated fiber (A fibers)

154 Principles of Autonomic and Peripheral Nervous System Pharmacology A

Intermediate closed conformation

Resting conformation

Open conformation

S4 regions

Extracellular

+ + +

+ Voltage

+ + +

Intracellular

Voltage

Inactivated conformation

Na+

+ + +

+ + +

+ + +

+ + +

After 1 ms

+ + +

+ + +

Linker region Voltage ("refractory period")

B

Intermediate closed conformation (high affinity for LA)

Resting conformation (low affinity for LA)

+ + +

+ + +

+ Voltage Voltage

+ + +

+ + + LA

Open conformation (high affinity for LA)

+ + +

+ + + LA

Inactivated conformation (high affinity for LA)

+ + +

+ + + LA

Stabilized conformation

Voltage (longer "refractory period")

FIGURE 11-7. Local anesthetic binding to different conformations (states) of the sodium channel. A. The sodium channel is composed of one polypeptide chain that has four repeating units. One region, known as the S4 region, has many positively charged amino acids (lysine and arginine). These residues give the channel its voltage dependence. At rest, the pore is closed. When the membrane is depolarized, the charged residues move in response to the change in the electric field. This results in several conformational changes (intermediate closed states) that culminate in channel opening. After about 1 ms (the channel open time), the 3–4 amino acid “linker region” plugs the open channel, yielding the inactivated conformation. The inactivated conformation returns to the resting state only when the membrane is repolarized; this conformational change involves the return of the S4 region to its original position and the expulsion of the linker region. The time required for the channel to return from the inactivated state to the resting state is known as the refractory period ; during this period, the sodium channel is incapable of being activated. B. The binding of local anesthetic (LA) alters the properties of the intermediate forms assumed by the sodium channel. Sodium channels in any of the conformations (resting, closed, open, or inactivated) can bind local anesthetic molecules, although the resting state has a low affinity for LA, while the other three states have a high affinity for LA. LA can dissociate from the channel–LA complex in any conformational state, or the channel can undergo conformational changes while associated with the LA molecule. Ultimately, the channel–LA complex must dissociate, and the sodium channel must return to the resting state to become activated. LA binding extends the refractory period, including both the time required for dissociation of the LA molecule from the sodium channel and the time required for the channel to return to the resting state.

bound channels increases with each successive impulse; the inhibition is said to be phasic, or use-dependent (Fig. 11-8). Tonic inhibition occurs when the time between action potentials is long compared to the time for dissociation of the LA from the sodium channel. Assume, for example, that before an action potential arrives, an equilibrium has been established where 5% of the sodium channels are bound by local anesthetic molecules. When an action potential arrives, the other 95% of the channels are available to open and subsequently to inactivate. During the brief impulse, some of these channels become bound by local anesthetic molecules. However, in the relatively long time before the next impulse arrives at the LA-exposed region, the bound LA can dissociate from the sodium channel, allowing those channels to return to the resting state. Thus, before the next action potential arrives, the 5% binding equilibrium is reestablished. The next action potential will, therefore, be blocked to the same extent as the previous one.

Phasic inhibition occurs when there is not enough time between action potentials for this equilibrium to be reestablished. Rapidly arriving action potentials cause resting sodium channels to open and then inactivate, and some of these channels will be bound by LAs. However, because there is not enough time between impulses for all the newly formed LA–sodium channel complexes to dissociate, only some of the channels are able to return to the resting state. With each arriving action potential, more and more channels are blocked, until a new steady state of LA–sodium channel binding is reached. This is the phenomenon of phasic, or usedependent, inhibition. As more of the channels are bound by LA, fewer and fewer channels are available to open when the next action potential arrives. Consequently, action potential conduction is increasingly inhibited at higher frequencies of impulses. The clinical importance of this phenomenon is that tissue injury or trauma causes nociceptors in the area of injury

A Tonic block (low frequency stimulation)

Depolarized

Voltage Resting

0.5 Fraction of bound channels 0 LA in equilibrium with sodium channels

Equilibrium reestablished

Time B Phasic block (high frequency stimulation)

Depolarized

Voltage Resting

0.5 Fraction of bound channels 0 LA in equilibrium with sodium channels

New baseline established

Time

158 Principles of Autonomic and Peripheral Nervous System Pharmacology

Local anesthetics have complex effects on the peripheral vasculature. Lidocaine, for example, initially causes vasoconstriction, but at later times, it produces vasodilation. Such biphasic actions may be attributable to separate effects on the vascular smooth muscle and on sympathetic nerves that innervate resistance arterioles. Bronchial smooth muscle is also affected in a biphasic manner. Initially, local anesthetics cause bronchoconstriction, but at later times, they cause bronchorelaxation. The early event may reflect LA-induced release of calcium ions into the cytoplasm from intracellular stores, while the later effect may be caused by LA inhibition of plasma membrane sodium and calcium channels (see below). The cardiac effects of LAs are complex due to their actions on different molecular targets, including Na⫹, K⫹, and Ca2⫹ channels. An early effect is to reduce the conduction velocity of the cardiac action potential through both conducting and nodal tissues. At very low concentrations, LAs can act as antiarrhythmic drugs because of their ability to prevent ventricular tachycardia and ventricular fibrillation (this is an example of use-dependent block; see above). Lidocaine, for example, acts as both a local anesthetic and a class I antiarrhythmic (see Chapter 23). Local anesthetics also cause a dose-dependent decrease in cardiac contractility (a negative inotropic effect). The mechanism of this effect is not entirely understood but may be caused by LA-mediated slow release of calcium from the sarcoplasmic reticulum, with a consequent reduction in the stores of calcium available to drive subsequent contractions. LAs can also directly inhibit calcium channels in the plasma membrane. The combination of reduced intracellular calcium storage and decreased calcium entry may lead to decreased myocardial contractility. It has recently been shown that lipid emulsions injected into the circulation can rapidly reverse the cardiac toxicity from systemic LAs such as bupivacaine. This finding has been demonstrated in animal models of local anesthetic toxicity and in several clinical case reports of successful resuscitation after cardiac arrest related to local anesthetic overdose. Hypersensitivity to local anesthetics is rare. This adverse effect is usually manifested as allergic dermatitis or asthma. LA-induced hypersensitivity occurs almost exclusively with ester-linked LAs. For example, a metabolite of procaine, paraaminobenzoic acid (PABA), is a known allergen (as well as the active agent in many sunscreens).

Individual Agents Having discussed the general properties of local anesthetics, this section presents the individual anesthetics in current clinical use, with an emphasis on the agents’ differences in potency and half-life.

site on the sodium channel, accounting for the low potency of this agent. Procaine’s primary uses are in infiltration anesthesia and in dental procedures. Occasionally it is used in diagnostic nerve blocks. Procaine is rarely used for peripheral nerve block because of its low potency, slow onset, and short duration of action. However, the rapidly hydrolyzed, short-acting homologue of procaine, 2-chloroprocaine (Nesacaine®), is popular as an obstetric anesthetic that is sometimes given epidurally just before delivery to control pain. One of the metabolites of procaine is PABA, a compound required by some bacteria for purine and nucleic acid synthesis. The antibacterial sulfonamides are structural analogues of PABA that competitively inhibit the synthesis of an essential metabolite in folate biosynthesis (see Chapter 32, Principles of Antimicrobial and Antineoplastic Pharmacology). Excess PABA can reduce the effectiveness of sulfonamides and therefore exacerbate bacterial infections. As mentioned above, PABA is also an allergen. Tetracaine

Tetracaine is a long-acting, highly potent, ester-linked LA. Its long duration of action is caused by its high hydrophobicity—it has a butyl group attached to its aromatic group—which allows tetracaine to remain in the tissue surrounding a nerve for an extended period of time. Tetracaine’s hydrophobicity also promotes prolonged interaction with its binding site on the sodium channel, accounting for its greater potency than lidocaine and procaine. It is mainly used in spinal and topical anesthesia. Its effective metabolism is slow, despite the potential for rapid hydrolysis by esterases, because it is released only gradually from tissues into the bloodstream. Cocaine

Cocaine, the prototypical and only naturally occurring LA, is ester-linked. It has a medium potency (one-half that of lidocaine) and a medium duration of action. Cocaine’s structure is slightly unusual for local anesthetics; its tertiary amine is part of a complex cyclic structure to which a secondary ester group is attached. Cocaine’s primary therapeutic uses are in ophthalmic anesthesia and as part of the topical anesthetic TAC (tetracaine, adrenaline, cocaine; see above). Like prilocaine (see below), cocaine has a marked vasoconstrictive action that results from its inhibition of catecholamine uptake in synaptic terminals of both the peripheral and central nervous systems (see Chapter 10, Adrenergic Pharmacology). Inhibition of this uptake system is also the mechanism for cocaine’s profound cardiotoxic potential, and for the “high” associated with cocaine use. Cardiotoxicity and euphoria limit the value of cocaine as a local anesthetic. Amide-Linked Local Anesthetics

Ester-Linked Local Anesthetics

Lidocaine and Prilocaine

Procaine

Lidocaine, the most commonly used LA and the one used in EM’s case, is an amide-linked drug of moderate hydrophobicity. It has a rapid onset of action and a medium duration of action (about 1–2 hours) and is moderately potent. Lidocaine has two methyl groups on its aromatic ring, which enhance its hydrophobicity relative to procaine and slow its rate of hydrolysis. Lidocaine has a relatively low pKa, and a large fraction of the drug is present in neutral form at physiologic pH. This

Procaine (Novocain®) is a short-acting, ester-linked LA. Its low hydrophobicity allows for rapid removal of drug from the site of administration via the circulation and results in little sequestration of drug in the local tissue surrounding the nerve. In the bloodstream, procaine is degraded rapidly by plasma pseudocholinesterases, and the metabolites are subsequently excreted in the urine. Procaine’s low hydrophobicity also causes it to dissociate rapidly from its binding

160 Principles of Autonomic and Peripheral Nervous System Pharmacology

that seen in patients with diabetic neuropathy, postherpetic neuralgia, burns, cancer, and strokes. The development of ultralong-acting LAs (whose effects could last for days) is continuing to be investigated; these studies involve altering LA structure at the molecular level, using a variety of drug delivery systems, and discovering new classes of neuronal impulse blockers. Lastly, a promising area of current discovery involves nociceptor-specific LAs. Some of these experimental agents bind to particular sodium channel subtypes that are expressed preferentially on A␦- or C-fibers. Others are charged anesthetics that typically cannot diffuse through neuronal cell membranes; co-administration of these anesthetics with agents that activate ion channels found preferentially on nociceptors (such as TRPV1) allows the anesthetic molecules to cross the nociceptor membrane through these open channels in a modality-specific fashion. Nociceptor-specific LAs

have the potential to block pain perception without affecting motor, autonomic, or other neuronal signaling and may therefore be useful in a variety of clinical settings.

Suggested Reading Berde CB, Strichartz GR. Local anesthetics. In: Miller RD, et al, eds. Miller’s anesthesia. 7th ed. Philadelphia: Elsevier Churchill Livingstone; 2009. (A more complete mechanistic and, primarily, clinical summary.) Crystal CS, McArthur TJ, Harrison B. Anesthetic and procedural sedation techniques for wound management. Emerg Med Clin North Am 2007;25:41–71. (A clinically oriented review that discusses how to administer LAs at various anatomic sites.) McLure HA, Rubin AP. Review of local anaesthetic agents. Minerva Anestesiol 2005;71:59–74. (A clear discussion of both general concepts and individual agents.) Suzuki S, Gerner P, Colvin AC, Binshtok AM. C-fiber-selective peripheral nerve blockade. Open Pain J 2009;2:24–29. (Recent research on agents that may have selectivity for C-fibers.)

IIC Principles of Central Nervous System Pharmacology

12 Pharmacology of GABAergic and Glutamatergic Neurotransmission Stuart A. Forman, Janet Chou, Gary R. Strichartz, and Eng H. Lo

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 164-165 OVERVIEW OF GABAERGIC AND GLUTAMATERGIC NEUROTRANSMISSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 PHYSIOLOGY OF GABAERGIC NEUROTRANSMISSION. . . . . 165 GABA Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 GABA Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Ionotropic GABA Receptors: GABAA and GABAC . . . . . . 166 Metabotropic GABA Receptors: GABAB. . . . . . . . . . . . . 168 PHARMACOLOGIC CLASSES AND AGENTS AFFECTING GABAERGIC NEUROTRANSMISSION . . . . . . . . . . . . . . . . . . 168 Inhibitors of GABA Metabolism . . . . . . . . . . . . . . . . . . . . . 169 GABAA Receptor Agonists and Antagonists . . . . . . . . . . . . 170 GABAA Receptor Modulators. . . . . . . . . . . . . . . . . . . . . . . 170 Benzodiazepines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 Barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Etomidate, Propofol, and Alphaxalone . . . . . . . . . . . . . 175 GABAB Receptor Agonists and Antagonists . . . . . . . . . . . . 175

Nonprescription Uses of Drugs That Alter GABA Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Chloral Hydrate, ␥-Hydroxybutyric Acid, and Flunitrazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 PHYSIOLOGY OF GLUTAMATERGIC NEUROTRANSMISSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Glutamate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Glutamate Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Ionotropic Glutamate Receptors . . . . . . . . . . . . . . . . . 176 Metabotropic Glutamate Receptors . . . . . . . . . . . . . . . 177 PATHOPHYSIOLOGY AND PHARMACOLOGY OF GLUTAMATERGIC NEUROTRANSMISSION . . . . . . . . . . . 178 Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . 178 Stroke and Trauma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Hyperalgesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 180 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

 INTRODUCTION

 OVERVIEW OF GABAERGIC AND GLUTAMATERGIC NEUROTRANSMISSION

Inhibitory and excitatory neurotransmitters regulate almost every behavioral process, including consciousness, sleep, learning, memory, and all sensations. Inhibitory and excitatory neurotransmitters are also implicated in pathologic processes such as epilepsy and the neurotoxicity associated with stroke. The interactions among ion channels, the receptors that regulate these channels, and amino acid neurotransmitters in the central nervous system (CNS) constitute the molecular basis for these processes. This chapter discusses the physiology, pathophysiology, and pharmacology of ␥-aminobutyric acid (GABA) and glutamate neurotransmission. Together, these molecules are the two most important amino acid neurotransmitters in the CNS. 164

The CNS has high concentrations of certain amino acids that bind to postsynaptic receptors and thereby act as inhibitory or excitatory neurotransmitters. Of the two main classes of neuroactive amino acids, ␥-aminobutyric acid (GABA) is the major inhibitory amino acid, and glutamate is the primary excitatory amino acid. Amino acid neurotransmitters elicit inhibitory or excitatory responses by altering the conductance of one or more ion-selective channels. Inhibitory neurotransmitters induce a net outward current, generally hyperpolarizing the membrane. For example, inhibitory neurotransmitters may open K⫹ channels or Cl⫺ channels to induce K⫹ efflux or Cl⫺ influx, respectively. Either type of ion movement—the loss

Direct effects

Indirect effects

Cl-

Intracellular

Extracellular

A Effects of inhibitory neurotransmitters Ca2+

K Cl-

+

+

channel

K channel

Ca2+ channel (closed)

Intracellular

Extracellular

B Effects of excitatory neurotransmitters Direct effects +

Na

Indirect effects

Ca2+

Ca2+

+

K Na+ channel

Ca2+ channel

K+ channel (closed)

Ca2+ channel

166 Principles of Central Nervous System Pharmacology

GABA Metabolism The synthesis of GABA is mediated by glutamic acid decarboxylase (GAD), which catalyzes the decarboxylation of glutamate to GABA in GABAergic nerve terminals (Fig. 12-2A). Thus, the amount of GABA in brain tissue correlates with the amount of functional GAD. GAD requires pyridoxal phosphate (vitamin B6) as a cofactor. GABA is packaged into presynaptic vesicles by a transporter (VGAT), which is the same transporter expressed in nerve terminals that release glycine, another inhibitory neurotransmitter. In response to an action potential and the presynaptic elevation of intracellular Ca2⫹, GABA is released into the synaptic cleft by fusion of GABAcontaining vesicles with the presynaptic membrane. Termination of GABA action at the synapse depends on the removal of GABA from the extracellular space. Neurons and glia take up GABA via specific GABA transporters (GATs). Four GATs have been identified, GAT-1 through GAT-4, each with a characteristic distribution in the CNS. Within cells, the widely distributed mitochondrial enzyme GABA-transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde (SSA), which is subsequently oxidized to succinic acid by SSA dehydrogenase

and then enters the Krebs cycle to become ␣-ketoglutarate. GABA-T then regenerates glutamate from ␣-ketoglutarate (Fig. 12-2A).

GABA Receptors GABA mediates its neurophysiologic effects by binding to GABA receptors. There are two types of GABA receptors. Ionotropic GABA receptors (GABAA and GABAC) are multisubunit membrane proteins that bind GABA and open an intrinsic chloride ion channel. Metabotropic GABA receptors (GABAB) are heterodimeric G protein-coupled receptors that affect neuronal ion currents through second messengers. Ionotropic GABA Receptors: GABAA and GABAC The most abundant GABA receptors in the CNS are ionotropic GABAA receptors, which are members of the superfamily of fast neurotransmitter-gated ion channels. This superfamily includes peripheral and neuronal nicotinic acetylcholine receptors (nAChRs), serotonin type 3A/B (5-HT3A/B) receptors, and glycine receptors. Like other members of this superfamily, GABAA receptors are pentameric transmembrane

O

A

O

O

HO

O OH

HO

OH

NH2

O

Via the Krebs cycle

α-ketoglutarate

Glutamate

O

GABA-T GAD O

O

O

OH

SSADH

H

HO

O

HO

O

O

Succinic acid

C

NH2

HO GABA

Succinic semialdehyde Vigabatrin

B

Presynaptic neuron

Glial cell Gln

Glutamine synthetase

Gln Glu

Glutaminase Glu

Gt(g)

Gt(n) Glu Glu Glu

Glutamate receptors Postsynaptic cell

FIGURE 12-2. Glutamate and GABA synthesis and metabolism. A. Glutamate synthesis and metabolism are intertwined with GABA synthesis and metabolism. In one pathway for glutamate synthesis, ␣-ketoglutarate produced by the Krebs cycle serves as a substrate for the enzyme GABA transaminase (GABA-T), which reductively transaminates intraneuronal ␣-ketoglutarate to glutamate. The same enzyme also converts GABA to succinic semialdehyde. Alternatively, glutamate is converted to GABA by the enzyme glutamic acid decarboxylase (GAD), changing the major excitatory neurotransmitter to the major inhibitory transmitter. GABA-T is irreversibly inhibited by vigabatrin; by blocking the conversion of GABA to succinic semialdehyde, this drug increases the amount of GABA available for release at inhibitory synapses. GABA-T, GABA transaminase; SSADH, succinic semialdehyde dehydrogenase; GAD, glutamic acid decarboxylase. B. Glutamate transporters present in neurons [Gt(n)] and glial cells [Gt(g)] sequester glutamate (Glu) from the synaptic cleft into their respective cells. In the glial cell, the enzyme glutamine synthetase transforms glutamate into glutamine (Gln). Glutamine is then transferred to the neuron, which converts it back to glutamate via mitochondria-associated glutaminase.

CHAPTER 12 / Pharmacology of GABAergic and Glutamatergic Neurotransmission 167

N

C

GABAA receptor

B Benzodiazepines (+) Flumazenil (-)

Furosemide (-)

GABA (+) and competitive antagonists (-) Penicillin (-)

Barbiturates (+)

Neurosteroids (+) Picrotoxin (-)

FIGURE 12-3.

Schematic representation of the GABAA receptor. A. The pentameric structure of the GABAA receptor. Each of the five subunits is one of three predominant subtypes: ␣, ␤, or ␥. Activation requires the simultaneous binding of two GABA molecules to the receptor, one to each of the two binding sites at the interface of the ␣ and ␤ subunits. Each subunit of the GABAA receptor has four membrane-spanning regions and a cysteine loop in the extracellular N-terminal domain (depicted as a blue segment and a dashed line). B. Drug binding sites on the GABAA receptor. For most of the exact locations schematically indicated in this diagram, the current evidence is largely indirect. (⫹) indicates agonist or allosteric modulator action at the GABAA receptor; (⫺) indicates competitive or noncompetitive antagonist action.

glycoproteins assembled to form a central ion pore surrounded by five subunits, each of which has four membrane-spanning domains (Fig. 12-3A). Sixteen different GABAA receptor subunits (␣1–6, ␤1–3, ␥1–3, ␦, ⑀, ␲, ␪) are currently known. The number of pentameric ion channels that could be formed by potential combinations of 16 subunits is very large, but only about 20 different subunit combinations have been identified in native GABAA receptors. Importantly, receptors containing different subunit combinations display distinct distributions at the cellular and tissue levels, and evidence is accumulating that different GABAA receptor subtypes play distinct roles in specific neural circuits. Most synaptic GABAA receptors consist of two ␣, two ␤, and one ␥ subunit. “Extrasynaptic” GABAA receptors have also been identified on dendrites, axons, and neuronal cell bodies. These often contain a ␦ or ⑀ subunit instead of a ␥ subunit. The five subunits of GABAA receptors surround a central chloride-selective ion pore that opens in the presence of GABA. GABA and other agonists bind to two sites, which are located in extracellular portions of the receptor-channel

complex at the interface between the ␣ and ␤ subunits. GABAA receptors also contain a number of modulatory sites where other endogenous ligands and/or drugs bind (Fig. 12-3B). In many cases, the presence of these sites and the impact of ligand binding depend on the receptor subunit composition. GABAA receptor-channel activation follows the binding of two molecules of GABA, one to each of the receptor’s agonist sites (Fig. 12-3). Fast inhibitory postsynaptic currents (IPSCs) are responses activated by very brief (highfrequency) bursts of GABA release at synapses. Uptake by GAT removes GABA from the synapse in less than 1 ms; IPSCs deactivate over about 12–20 ms, a rate that is determined by both closure of the GABAA receptor ion channel and dissociation of GABA from the receptor. Prolonged occupation of the agonist sites by GABA also leads to GABAA receptor desensitization, a transition to an inactive agonistbound state (Fig. 12-4). During burst (or “phasic”) firing, the presynaptic nerve membrane releases “quanta” (⬃1 mM) of GABA by exocytosis of synaptic vesicles, resulting in transient, large-amplitude inhibitory postsynaptic potentials (IPSPs). Low levels of GABA can also cause a baseline inhibitory current in many neurons. Recent studies suggest that basal inhibitory currents are caused by activation of extrasynaptic GABAA receptors, which are activated by low micromolar concentrations of GABA that diffuse into cerebrospinal fluid and interstitial spaces. Because the internal chloride concentration [Cl⫺]in of mature neurons is lower than the extracellular Cl⫺ concentration [Cl⫺]out, activation of chloride-selective channels (increasing conductance) shifts the neuronal transmembrane voltage toward the Cl⫺ equilibrium potential (ECl ⬃ ⫺70 mV). This Cl⫺ flux hyperpolarizes or stabilizes the postsynaptic cell near its normal resting membrane potential (Vm ⬃ ⫺65 mV), reducing the likelihood that excitatory stimuli will initiate action potentials. Open Cl⫺ channels attenuate the change in membrane potential caused by excitatory synaptic currents, an effect called shunting. This is the molecular explanation for the inhibitory effects of GABA signaling via GABAA receptors. (In immature neurons, as found in neonates, the Cl⫺ ion gradient is reversed, due to a difference in the Cl⫺ ion transporting pumps, such that Cl⫺ ions flow out of the cell constituting an

Cl- current

1 subunit

A

0

3 ␮M GABA

30 ␮M GABA

300 ␮M GABA

Time (shaded bar = 1 sec)

FIGURE 12-4. Effects of GABA on GABAA-mediated chloride conductance. Increasing concentrations of GABA induce both greater Cl⫺ currents and more rapid receptor desensitization. The latter phenomenon can be observed as the rapid decline from the peak current during continuous exposure to 300 ␮M GABA (right panel). In each panel, the shaded bar indicates the 1-second period during which GABA was applied. Although individual presynaptic endings will release GABA for much shorter times, the cumulative GABA released from many presynaptic elements, stimulated by trains of invading action potentials, can persist for seconds.

168 Principles of Central Nervous System Pharmacology GABAB receptor

Effector protein (PLC or AC)

GABA

Ca2+

β

β

γ K

Closes Ca2+ channel

+

Opens K+ channel

γ

α GTP GTP-GDP exchange

α β

γ

β

γ

α GTP

GDP GTP-GDP exchange

Downstream signaling of the GABAB receptor. GABAB receptor activation alters cytoplasmic G proteins that then dissociate into ␣ and ␤␥ subunits, the latter of which bind directly to K⫹ or Ca2⫹ channels (leftward arrow). The released ␣ subunits are linked to second messenger systems such as adenylyl cyclase (AC) or phospholipase C (PLC) (rightward arrow). The increased K⫹ efflux leads to slow, long-lasting inhibitory postsynaptic potentials. The reduced Ca2⫹ influx may account for the ability of GABAB autoreceptors to inhibit presynaptic neurotransmitter release. The GABAB receptor functions as an obligate heterodimer of GABAB1 and GABAB2 subunits, each of which is a seven-transmembrane-spanning G protein-coupled receptor (not shown).

FIGURE 12-5.

inward current and thus depolarizing rather than hyperpolarizing the membrane. Importantly, as a consequence of this, drugs that activate or potentiate GABAA receptors will have an excitatory action in the very young instead of the inhibitory effect that they have later in development.) The molecular role of GABAA receptors in neurons is consistent with their known physiologic roles in CNS disease and their pharmacology. Drugs that inhibit GABAA receptors produce seizures in animals, and mutations in GABAA receptor subunits that impair activation at the molecular level are associated with inherited human epilepsies. Conversely, endogenous or exogenous substances that enhance the activation of GABAA receptors reduce neuronal excitability and may impair numerous CNS functions. Recent evidence indicates that GABAA receptors are also expressed in airway epithelium. Activation of these receptors may enhance smooth muscle relaxation (bronchodilation) and could represent a future therapy for asthma. Certain endogenous steroids, known as neurosteroids, allosterically modulate GABAA receptor activity. The steroid hormones deoxycorticosterone and progesterone are metabolized in the brain to produce pregnenolone, dehydroepiandrosterone (DHEA), 5␣-dihydrodeoxycorticosterone (DHDOC), 5␣-tetrahydrodeoxycorticosterone (THDOC), and allopregnanolone. Rather than acting through nuclear receptors like most steroid hormones, neurosteroids alter GABAA receptor function by binding to allosteric sites on the receptor protein, causing increased GABAA receptor activation. DHDOC and THDOC are thought to modulate brain activity during stress. Menstrual variations in allopregnanolone, a metabolite of progesterone, contribute to perimenstrual (catamenial) epilepsy. Sulfation of pregnenolone and DHEA results in neurosteroids that inhibit GABAA receptors. Another endogenous substance that enhances GABAA receptor activity is oleamide, a fatty acid amide found in the cerebrospinal fluid of sleep-deprived animals. Injection of oleamide into normal animals induces sleep, in part through potentiation of GABAA receptors. Another group of ionotropic GABA receptors, GABAC, are formed by three subunits that are not found in GABAA receptors (␳1–3). GABAC receptors are also pentameric ligand-gated chloride channels, but their distribution in the CNS is restricted primarily to the retina. GABAC receptors display distinct pharmacologic properties that differ from

those of most GABAA receptors. No drugs currently in use target GABAC receptors. Metabotropic GABA Receptors: GABAB GABAB receptors are G protein-coupled receptors that are expressed at lower levels than GABAA receptors and are found principally in the spinal cord (Fig. 12-5). They function as heterodimers of GABAB1 and GABAB2 subunits. The GABAB receptor interacts with heterotrimeric G proteins, leading to the dissociation of their ␤␥ subunit, which directly activates K⫹ channels and inhibits the opening of voltage-gated Ca⫹2 channels (Fig. 12-5). (GABAB receptor activation also leads to suppression of adenylyl cyclase and concomitant reduction in cAMP, but this seems to have only minor effects on cellular excitability.) At GABAergic synapses, GABAB receptors are expressed both presynaptically and postsynaptically. Presynaptic “autoreceptors” modulate neurotransmitter release by reducing Ca2⫹ influx, while postsynaptic GABAB receptors produce slow IPSPs through activation of G protein-activated “inward rectifier” K⫹ channels (GIRKS). The slower rates of activation and deactivation for GABAB currents in comparison with GABAA currents are due to the relatively slow second messenger signal transduction mechanisms. Activation of K⫹ channels by GABAB-coupled G proteins inhibits neuronal firing, because K⫹ has an equilibrium potential near ⫺70 mV. Thus, increased K⫹ conductance, like increased Cl⫺ conductance, drives the neuronal transmembrane voltage toward “resting” potentials, reduces the frequency of action potential initiation, and shunts excitatory currents.

 PHARMACOLOGIC CLASSES AND AGENTS AFFECTING GABAERGIC NEUROTRANSMISSION Pharmacologic agents acting on GABAergic neurotransmission affect GABA metabolism or receptor activity. The majority of pharmacologic agents affecting GABAergic neurotransmission act on the ionotropic GABAA receptor. Several drug classes can regulate GABAA receptors by interacting with the GABA binding sites or with allosteric sites (Fig. 12-3). Therapeutic agents that activate GABAA receptors are used for sedation, anxiolysis, hypnosis (general

170 Principles of Central Nervous System Pharmacology

Agonists such as muscimol and gaboxadol activate the GABAA receptor by binding directly to the GABA binding site. Muscimol, first derived from hallucinogenic Amanita muscaria mushrooms, is a full agonist at many GABAA receptor subtypes and is used primarily as a research tool. Purified muscimol (as well as other GABAA receptor agonists) does not induce hallucinations, which are probably caused by other factors from Amanita muscaria. Gaboxadol at high concentrations is a partial agonist at synaptic GABAA receptors; at low concentrations, gaboxadol selectively activates extrasynaptic receptors containing ␣4, ␤3, and ␦ subunits. Gaboxadol was initially approved for treatment of epilepsy and anxiety, but therapeutic doses were associated with ataxia and sedation. Lower gaboxadol doses, which activate extrasynaptic receptors, induce slow-wave sleep in laboratory animals. Human trials of gaboxadol for treatment of insomnia were halted in 2007 due to concerns about adverse effects such as hallucinations, disorientation, sleepwalking, and sleep-driving. Bicuculline and gabazine are competitive antagonists that bind at the GABA sites on GABAA receptors. Picrotoxin is a noncompetitive inhibitor of GABAA receptors that blocks the ion pore. All of these GABAA antagonists produce epileptic convulsions and are used exclusively for research; they also illustrate the importance of the tonic activity of GABAA receptors in maintaining a state of relatively normal excitability in the CNS.

GABAA Receptor Modulators Benzodiazepines and barbiturates are modulators of GABAA receptors that act at allosteric binding sites to enhance GABAergic neurotransmission (Fig. 12-3B). Benzodiazepines have sedative, hypnotic, muscle relaxant, amnestic, and anxiolytic effects. At high doses, benzodiazepines can cause hypnosis and stupor. However, when used alone, these drugs rarely cause fatal CNS depression. Barbiturates constitute a large group of drugs that were first introduced in the mid-twentieth century and continue to be used, albeit with diminishing frequency, for control of epilepsy, as general anesthetic induction agents, and for control of intracranial hypertension. Benzodiazepines Benzodiazepines are high-affinity, highly selective drugs that bind at a single site on GABAA receptors containing ␣1, ␣2, ␣3, or ␣5 subunits and a ␥ subunit. In molecular studies, benzodiazepine potency correlates with hydrophobicity. However, benzodiazepines are highly bound to plasma proteins such as albumin, and hydrophobicity enhances protein binding and thereby reduces the drugs’ free concentration and transport across the blood–brain barrier. Therefore, highly protein-bound benzodiazepines may appear less potent in vivo even though they display higher potency in molecular

A 100

Cl- current (% maximum)

GABAA Receptor Agonists and Antagonists

studies. Furthermore, in clinical states associated with low albumin, such as acute hemodilution or liver dysfunction, the clinical potency of benzodiazepines may be dramatically increased. Benzodiazepines act as positive allosteric modulators by enhancing GABAA receptor channel gating in the presence of GABA (Fig. 12-6). Benzodiazepines increase the frequency

Low GABA + pentobarbital

80 60 40

Low GABA + midazolam Low GABA alone

20 0 10-9

10-8

10-7

10-6

10-5

10-4

10-3

Midazolam or pentobarbital (Molar) B 100

Cl- current (% maximum)

Vigabatrin is used in the treatment of epilepsy, and it is being investigated for treatment of drug addiction, panic disorder, and obsessive-compulsive disorder. Adverse effects of ␥-vinyl GABA include drowsiness, confusion, and headache. The drug has been reported to cause bilateral visual field defects associated with irreversible diffuse atrophy of the peripheral retinal nerve fiber layer. This appears to result from accumulation of the drug in retinal nerves.

GABA + high-dose pentobarbital

80 GABA + maximal midazolam

60 40

GABA alone

20 0 10-7

10-6

10-5

10-4

10-3

GABA (Molar)

FIGURE 12-6.

Effects of benzodiazepines and barbiturates on GABAA receptor activity. A. Both benzodiazepines and barbiturates enhance GABAA receptor activation (measured experimentally by Cl⫺ current), but with different potencies and efficacies. Midazolam (a benzodiazepine) maximally enhances by about three-fold the current evoked by 10 ␮M GABA. In contrast, the anesthetic barbiturate pentobarbital increases the current evoked by 10 ␮M GABA to a much greater extent (near that of a maximal GABA response), but its maximal effect requires concentrations over 100 ␮M. Thus, benzodiazepines such as midazolam are high-potency, low-efficacy modulators of GABAA receptor activity, while barbiturates such as pentobarbital are low-potency, high-efficacy modulators. B. Another way to compare the efficacy of benzodiazepines and barbiturates is to measure the degree to which they enhance the sensitivity of GABAA receptors to GABA. Maximally effective concentrations of midazolam shift the GABA concentration–response curve modestly to the left, reducing the EC50 (increasing the potency) of GABA by about two-fold. In contrast, high-dose pentobarbital causes a much greater shift to the left, reducing the EC50 of GABA by approximately 20-fold. Pentobarbital at high concentrations also directly activates GABAA receptors, even in the absence of GABA (note the nonzero Cl⫺ current at 10⫺7 M GABA). In contrast, the benzodiazepines do not show direct agonist activity.

CHAPTER 12 / Pharmacology of GABAergic and Glutamatergic Neurotransmission 171

of channel openings in the presence of low GABA concentrations, and, at GABA concentrations similar to those in synapses, receptor deactivation is slowed. Both actions result in a net increase of Cl⫺ influx. In addition, GABAA receptors in the open state have a higher affinity for GABA than in the closed state, so the ability of benzodiazepines to favor channel openness results, secondarily, in an apparently higher agonist affinity. Benzodiazepines do not activate native GABAA receptors in the absence of GABA, but they do activate certain mutant receptors and enhance maximal activation by partial agonists, indicating that they are weak positive allosteric agonists (Fig. 12-7). This mechanism is consistent with the known location of the benzodiazepine-binding site at the interface between the external domains of the ␣ and ␥ subunits. This site is a structural homolog of the two GABA agonist sites at the interfaces between the ␤ and ␣ subunits. In GABA dose–response studies, benzodiazepines shift the response curve to the left, increasing the apparent potency of GABA up to three-fold (Fig. 12-6B). This is a smaller allosteric effect than that caused by other modulators, such as barbiturates or other general anesthetics (see etomidate, below). The limited efficacy of benzodiazepines is accompanied by a reduced potential for fatal overdose. However, the margin of safety decreases when benzodiazepines are coadministered with alcohol or other sedative/hypnotics. Clinical Applications

Benzodiazepines are used as sleep enhancers, anxiolytics, sedatives, antiepileptics, and muscle relaxants, and for treatment of ethanol withdrawal symptoms (Table 12-2). Benzodiazepines achieve an anxiolytic effect by inhibiting synapses in the limbic system, a CNS region that controls emotional behavior and is characterized by a high density of GABAA receptors. Benzodiazepines such as diazepam and alprazolam are used to mitigate chronic, severe anxiety and the anxiety associated with some forms of depression and schizophrenia. Because of the potential for the development of tolerance, dependence, and addiction, benzodiazepine use should be intermittent. In acute-care settings, such as in preparation

Cl- current (% maximum)

A

for invasive procedures, midazolam is frequently used as a rapid-onset and short-acting anxiolytic/sedative/amnestic. Benzodiazepines are often adequate as sedatives for brief, uncomfortable procedures associated with minimal sharp pain, such as endoscopy. When combined with opioids, however, a synergistic potentiation of both sedation and respiratory depression can occur. Given prior to general anesthesia, benzodiazepines reduce the requirement for hypnotic agents. Many benzodiazepines, including estazolam, flurazepam, quazepam, temazepam, triazolam, and zolpidem, are prescribed for treatment of insomnia. Benzodiazepines both facilitate sleep onset and increase the overall duration of sleep. They also alter the proportion of the various sleep stages: they increase the length of stage 2 non-rapid eye movement (NREM) sleep (the light sleep that normally comprises approximately half of sleeping time) and decrease the length of REM sleep (the period characterized by frequent dreams) and slow-wave sleep (the deepest level of sleep). After extended use, these effects may diminish because of tolerance. In a healthy individual, hypnotic doses of benzodiazepines induce respiratory changes comparable to those present during natural sleep and do not cause significant cardiovascular changes. Patients with either pulmonary or cardiovascular disease may experience significant respiratory or cardiovascular depression because of medullary depression from otherwise therapeutic doses of these drugs. Patients who have suffered brain damage from stroke or head trauma may also become profoundly sedated with these drugs. The sedative benzodiazepines differ in their rates of onset, durations of effect, and tendencies to cause rebound insomnia when withdrawn. For example, flurazepam is a long-acting benzodiazepine that facilitates sleep onset and maintenance and increases sleep duration. Although it does not cause significant rebound insomnia, its long elimination half-life (about 74 hours) and the accumulation of active metabolites may cause daytime sedation. Triazolam is a fastonset benzodiazepine that also decreases the time needed to fall asleep. Intermittent rather than chronic administration of this drug is recommended to lessen the rebound insomnia associated with its discontinuation. Zolpidem is unique

B

100 80

Maximal GABA response

P4S + midazolam

60

P4S alone 40 20

Current from spontaneously active mutant GABAA channels

Midazolam alone activates current

Picrotoxin blocks current

0

Time (sec)

FIGURE 12-7.

Evidence that benzodiazepines enhance the GABAA receptor channel opening probability. A. When GABAA receptors are activated using saturating concentrations of the partial agonist P4S, midazolam increases the peak current. This indicates that the P4S efficacy (the maximal channel opening probability) is increased by the addition of midazolam. B. GABAA receptors containing a single point mutation are spontaneously active, which can be demonstrated by the loss of current caused by picrotoxin (a noncompetitive GABAA receptor antagonist). When these mutant receptors are exposed to midazolam, the amount of current increases, indicating that midazolam directly influences the opening of GABAA receptors. This effect is not observed in wild-type channels, which exhibit only rare spontaneous openings.

N

A

1 subunit

Tetrameric structure

C B Glutamate/AMPA/Kainate Alcohols, volatile anesthetics?

Glutamate/NMDA Glycine Phencyclidine

Barbiturate

Na+

+

K

AMPA/Kainate receptor

Zn2+

Ca2+

Mg2+

+

K

NMDA receptor

CHAPTER 12 / Pharmacology of GABAergic and Glutamatergic Neurotransmission 175

Tolerance and Dependence

Repeated and extended misuse of barbiturates induces tolerance and physiologic dependence. Prolonged barbiturate use increases the activity of cytochrome P450 enzymes and accelerates barbiturate metabolism, thereby contributing to the development of tolerance to barbiturates and cross-tolerance to benzodiazepines, other sedative/hypnotics, and ethanol. Development of physiologic dependence results in a drug withdrawal syndrome characterized by tremors, anxiety, insomnia, and CNS excitability. If left untreated, these withdrawal signs may progress to seizures and cardiac arrest. Etomidate, Propofol, and Alphaxalone Etomidate, propofol, and alphaxalone are drugs used for induction of general anesthesia. Etomidate and propofol are also discussed in Chapter 16. Like barbiturates, these intravenous anesthetics act primarily on GABAA receptors. Etomidate is particularly useful during induction of anesthesia in hemodynamically unstable patients. Propofol is the most widely used anesthetic induction agent in the United States. It is used both for single-bolus induction of anesthesia and for maintenance via continuous intravenous infusion. Alphaxalone is a neuroactive steroid that is rarely used clinically. Mechanisms of Action

Like barbiturates, etomidate, propofol, and alphaxalone enhance activation of GABAA receptors by GABA and, at high concentrations, can act as agonists. For etomidate, both of these actions display similar stereoselectivity. Quantitative analysis indicates that both actions are caused by etomidate binding at a single set of two identical allosteric sites per receptor. It is unknown whether a similar mechanism accounts for the actions of propofol and alphaxalone. Etomidate and propofol act selectively at GABAA receptors that contain ␤2 and ␤3 subunits. Based on knock-in animal experiments, where ␤3 subunits are expressed as transgenes, ␤3-containing receptors are the most important for the hypnosis and muscle relaxation associated with general anesthesia. Alphaxalone shows little selectivity among synaptic GABAA receptors but is more potent at extrasynaptic receptors that contain ␦ subunits. Pharmacokinetics and Metabolism

Etomidate and propofol both induce anesthesia rapidly following bolus intravenous injection. Like barbiturates, these hydrophobic drugs cross the blood–brain barrier rapidly. The CNS effect of a bolus dose lasts for only several minutes, because redistribution to muscle and other tissues rapidly reduces the CNS drug concentrations. Propofol has an extremely large volume of distribution, and prolonged continuous infusions may be used without significant changes in the apparent clearance of the drug. Metabolism of etomidate and propofol is primarily hepatic. Adverse Effects

Etomidate inhibits the synthesis of cortisol and aldosterone. Suppression of cortisol production is thought to contribute to mortality among critically ill patients who receive prolonged etomidate infusions, and administration of exogenous glucocorticoids is effective in preventing this complication. Etomidate is generally used only for single-dose induction of anesthesia, not for anesthesia maintenance. It is also used rarely at subhypnotic doses for treatment of metastatic cortisol-producing tumors.

The major toxicity of propofol as a general anesthetic is depression of cardiac output and vascular tone. Hypotension is observed in patients who are hypovolemic or, as with many elderly patients, dependent on vascular tone to maintain blood pressure. Propofol is formulated in a lipid emulsion, and hyperlipidemia has been reported in patients receiving prolonged infusions for sedation. There is growing evidence in cellular and animal models that positive GABAA receptor modulators lead to neurotoxicity and increased neuroapoptosis. The mechanism suggested for this toxicity, which is not seen in adult animals, is that GABAA receptors are excitatory in some fetal and neonatal neurons (see above), resulting in excitotoxicity in the presence of certain drugs. These data have raised concerns about potential damage to the brains of human fetuses and neonates that are exposed to general anesthetics. Clinical studies are underway to assess the clinical relevance of this toxicity.

GABAB Receptor Agonists and Antagonists Baclofen is the only compound currently in clinical use that targets GABAB receptors. It was first synthesized as a GABA analogue and screened for antispastic action before GABAB receptors were discovered. Subsequently, it was found that baclofen is a selective GABAB receptor agonist. It is used primarily for treatment of spasticity associated with motor neuron diseases (e.g., multiple sclerosis) or spinal cord injury. Oral baclofen is effective for mild spasticity. Severe spasticity may be treated with intrathecal baclofen therapy using doses that are far lower than those required systemically. By activating metabotropic GABA receptors in the spinal cord, baclofen stimulates downstream second messengers to act on Ca2⫹ and K⫹ channels. Although baclofen is prescribed primarily for treatment of spasticity, clinical observations suggest that it also modulates pain and cognition, and it is being investigated as a therapy for drug addiction. Baclofen is absorbed slowly after oral administration; peak plasma concentrations are reached after 90 minutes. It has a modest volume of distribution and does not readily cross the blood–brain barrier. Baclofen is primarily cleared from the circulation in an unmodified form in the urine; about 15% of the drug is metabolized by the liver before excretion in bile. The elimination half-time is about 5 hours in patients with normal renal function, and dosing is typically three times daily. Following intrathecal injection and infusion, spasmolytic effects are observed after 1 hour and peak effects are observed at 4 hours. Adverse effects of baclofen include sedation, somnolence, and ataxia. These are worsened when baclofen is taken with other sedative drugs. Reductions in renal function may precipitate toxicity as drug levels rise. Baclofen overdose can produce blurry vision, hypotension, cardiac and respiratory depression, and coma. Tolerance apparently does not develop to oral baclofen. In contrast, dosing requirements after initiation of intrathecal baclofen often increase over the first 1 to 2 years. Withdrawal from baclofen therapy, especially from intrathecal infusion, can precipitate acute hyperspasticity, rhabdomyolysis, pruritus, delirium, and fever. Withdrawal has also led to multiorgan failure, coagulation abnormalities, shock, and death. If withdrawal symptoms persist, effective treatments reportedly include benzodiazepine, propofol, intrathecal opioid administration, and restarting baclofen.

176 Principles of Central Nervous System Pharmacology

Nonprescription Uses of Drugs That Alter GABA Physiology Ethanol Ethanol acts as an anxiolytic and sedative by causing CNS depression, but not without significant potential toxicity. Ethanol appears to exert its effects by acting on multiple targets, including GABAA and glutamate receptors. Ethanol increases GABAA-mediated Cl⫺ influx and inhibits the excitatory effects of glutamate at NMDA receptors. Ethanol interacts synergistically with other sedatives, hypnotics, antidepressants, anxiolytics, anticonvulsants, and opioids. Ethanol tolerance and dependence are associated with changes in GABAA receptor function. In animal models, chronic ethanol administration blunts the ethanol-mediated potentiation of GABA-induced Cl⫺ influx in the cerebral cortex and cerebellum. Acute tolerance to ethanol occurs without a change in the number of GABAA receptors, but chronic ethanol exposure alters GABAA receptor subunit expression in the cortex and cerebellum. Changes in the subunit composition of GABAA receptors may be responsible for the changes in receptor function associated with chronic ethanol use. Other mechanisms proposed for the development of tolerance to ethanol include post-translational modifications of GABAA receptors and changes in second messenger systems, for example, alterations in the expression patterns of different isoforms of protein kinase C (PKC). The up-regulation of NMDA receptor expression that occurs with prolonged ethanol use may account for the hyperexcitability associated with ethanol withdrawal. Benzodiazepines, such as diazepam and chlordiazepoxide, reduce the tremors, agitation, and other effects of acute alcohol withdrawal. Use of these medications in a patient experiencing withdrawal from chronic alcohol abuse can also prevent the development of withdrawal seizures (delirium tremens). Chloral Hydrate, ␥-Hydroxybutyric Acid, and Flunitrazepam Chloral hydrate is an older sedative–hypnotic rarely used today to alleviate insomnia. It has occasionally been employed to incapacitate individuals against their will; for example, to facilitate the commission of a crime. Gamma (␥)-hydroxybutyric acid (GHB) is a GABA isomer that has clinical utility as a sedative and treatment for narcolepsy, but finds much wider illicit use as a recreational drug and “date rape” drug. There is recent evidence that GHB acts in part by activating GABAB receptors, but it is also an endogenous molecule that may act as a neurotransmitter at other receptors that have not yet been identified. Like barbiturates, high doses of GHB can produce deep sedation and coma, and its effects are exacerbated by ethanol. Flunitrazepam (Rohypnol®) is a fast-acting benzodiazepine that can cause amnesia and thereby prevent an individual’s recall of events that occurred under the drug’s influence. This drug has also been reported to facilitate “date rape.”

 PHYSIOLOGY OF GLUTAMATERGIC NEUROTRANSMISSION Glutamatergic synapses exist throughout the CNS. The binding of glutamate to its receptors initiates excitatory neuronal responses associated with motor neuron activation; acute sensory responses, including the development of elevated pain sensation (hyperalgesia); synaptic changes involved in

certain types of memory formation; and cerebral neurotoxicity from brain ischemia as well as functional deficits from spinal cord injury. Although the clinical applications of glutamate pharmacology are currently limited, it is anticipated that glutamate pharmacology will become an increasingly important area of neuropharmacology.

Glutamate Metabolism Glutamate synthesis occurs via two distinct pathways. In one pathway, ␣-ketoglutarate formed in the Krebs cycle is transaminated to glutamate in CNS nerve terminals, a step that is directly linked to GABA conversion (Fig. 12-2A). Alternatively, glutamine produced and secreted by glial cells is transported into nerve terminals and converted to glutamate by glutaminase (Fig. 12-2B). Glutamate is released via calcium-dependent exocytosis of transmitter-containing vesicles. Glutamate is removed from the synaptic cleft by glutamate reuptake transporters located on presynaptic nerve terminals and on the plasma membrane of glial cells. These transporters are Na⫹-dependent and have a high affinity for glutamate. In glial cells, the enzyme glutamine synthetase converts glutamate to glutamine, which is recycled into adjacent nerve terminals for conversion back to glutamate. Glutamine generated in glial cells can also enter the Krebs cycle and undergo oxidation; the resulting ␣ketoglutarate enters neurons to replenish the ␣-ketoglutarate consumed during glutamate synthesis (Fig. 12-2B).

Glutamate Receptors As with GABA receptors, glutamate receptors are divided into ionotropic and metabotropic subgroups. Ionotropic Glutamate Receptors Ionotropic glutamate receptors mediate fast excitatory synaptic responses. These receptors are multisubunit, cation-selective channels that, on activation, permit the flow of Na⫹, K⫹, and, in some channels, Ca2⫹ ions across plasma membranes. Ionotropic glutamate receptors are thought to be tetramers composed of different subunits, with each subunit containing helical domains that span the membrane three times, in addition to a short sequence that forms the channel’s pore when the entire tetramer is assembled (Fig. 12-8A). There are three main subtypes of glutamate-gated ion channels, classified according to their activation by the selective agonists AMPA, kainate, and NMDA. The diversity of ionotropic receptors arises from differences in amino acid sequence because of alternative mRNA splicing and posttranscriptional mRNA editing, and from the use of different combinations of subunits to form the receptors (Table 12-5). AMPA (␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are located throughout the CNS, particularly in the hippocampus and cerebral cortex. Four AMPA receptor subunits (GluR1–GluR4) have been identified (Table 12-5). AMPA receptor activation results primarily in Na⫹ influx (as well as some K⫹ efflux), allowing these receptors to regulate fast, excitatory postsynaptic depolarization at glutamatergic synapses (Fig. 12-8B). Although most AMPA receptors in the CNS have low Ca2⫹ permeability, the absence of certain subunits (such as GluR2) in the receptor complex increases the Ca2⫹ permeability of the channel. Calcium entry through AMPA receptors may play a role in long-term changes in neuronal phenotype and in neuronal damage during stroke.

Metabotropic glutamate receptor

Neurotransmitter N binding regions Effector protein (PLC or AC)

Glutamate

Glu

Ion channel

1

Ca2+

4 3 C G protein binding regions

α GTP

β

α β

γ

2 GTP-GDP exchange

GDP

α

γ

2

GTP

GTP-GDP exchange

β

γ

β K

+

Closes K+ channel

γ Opens Ca2+ channel

CHAPTER 12 / Pharmacology of GABAergic and Glutamatergic Neurotransmission 179

Stroke and Trauma In ischemic stroke, interruption of blood flow to the brain provides the initial deficits in oxygen supply and glucose metabolism that trigger excitotoxicity (Fig. 12-10). In hemorrhagic strokes, high concentrations of glutamate are found in blood leaking into the brain. In traumatic brain injury, the direct rupture of brain cells can release high intracellular stores of glutamate and K⫹ into the restricted extracellular space. Once excitatory transmitters such as glutamate become unbalanced, widespread membrane depolarization and elevation of intracellular Na⫹ and Ca2⫹ concentrations propagate, and more glutamate is released from adjacent neurons. Increasing glutamate levels activate Ca2⫹-permeable NMDA and AMPA receptor-coupled channels. Ultimately, the resultant accumulation of intracellular Ca2⫹ activates many Ca2⫹-dependent degradation enzymes (e.g., DNAses, proteases, phosphatases, phospholipases) that lead to neuronal cell death. Although the highly Ca2⫹-permeable NMDA receptor was originally viewed as the major contributor to neuronal cell death caused by Ca2⫹ overload, AMPA receptors have also been implicated. Clinical trials of NMDA and AMPA receptor antagonists in patients with stroke have not been successful to date and, in some cases, have led to schizophrenia-like

Ischemia O2

ATP

Disrupted ion gradients

Membrane depolarization

Impaired Na+-coupled glutamate transporters

Increased synaptic glutamate

NMDA-R activation

AMPA-R activation

mGluR activation

intracellular Ca2+

Activation of DNases, proteases, phosphatases, phospholipases

Intracellular and membrane damage

FIGURE 12-10.

Damage by free radicals

Mitochondrial damage

Release of pro-apoptotic factors

Role of glutamate receptors in excitotoxicity. Although a multiplicity of damaging cellular processes occur as a consequence of the decreased ATP levels that result from impaired oxidative metabolism or from the superoxidative damage from activated neutrophils that invade an ischemic region, only glutamate-mediated processes are depicted here.

effects, memory impairment, and neurotoxic reactions. Future pharmacologic research will be directed at the development and use of drugs with fewer adverse effects, such as the noncompetitive NMDA receptor antagonist memantine or drugs targeted to specific subunits of the NMDA or AMPA receptor complex. Glutamate released during ischemic or traumatic brain damage can also activate metabotropic receptors. In animal models of stroke, pharmacologic antagonism of the mGluR1 receptor subtype facilitates recovery and survival of hippocampal neurons and prevents memory and motor loss caused by trauma. These findings suggest that the mGluR1 subunit may represent another target for future pharmacologic intervention (Figs. 12-10 and 12-11).

Hyperalgesia Hyperalgesia is the elevated perception of pain, often due to stimuli that, under normal conditions, cause little or no pain. It occurs in the presence of peripheral nerve injury, inflammation, surgery, and diseases such as diabetes. Although hyperalgesia is reversed in most cases when the underlying pathophysiology has resolved, it may persist even in the absence of an identified organic source, leading to chronic pain that is physically crippling and psychologically debilitating. There is accumulating evidence that glutamatergic transmission contributes to the development and/or maintenance of hyperalgesia. NMDA receptors enhance synaptic transmission between nociceptive afferent fibers and neurons in the dorsal horn of the spinal cord. As discussed in Chapter 17, Pharmacology of Analgesia, experimental hyperalgesia often involves a phenomenon called central sensitization, in which repeated nociceptive stimuli in the periphery lead to progressively increasing excitatory postsynaptic responses in postsynaptic pain neurons in the superficial dorsal horn. One mechanism by which this synaptic potentiation occurs involves postsynaptic NMDA receptors that, when stimulated chronically, increase the strength of excitatory connections between pre- and postsynaptic neurons in spinal pain circuits. In turn, the Ca2⫹ influx through activated NMDA receptors acts on special localized kinases to effect a phosphorylationinduced switch of the subunits of the AMPA receptor, allowing more Ca2⫹ to enter through AMPA receptors. Increased intracellular Ca2⫹ also activates Ca2⫹-sensitive transcription factors, such as CREB, and induces changes in protein synthesis via ribosomes located right at the synaptic terminals. Experimental NMDA receptor antagonists can both prevent and reverse central sensitization in patients. Many of these antagonists, however, also inhibit a wide range of fast excitatory synaptic pathways in the CNS. For this reason, current NMDA receptor drug development focuses on intraspinal or extradural administration of NMDA receptor antagonists to limit the effects of the drug to the dorsal horn of the spinal cord. The high density of kainate receptors in sensory neurons may also modulate transmitter release, providing another future pharmacologic target for the relief of chronic pain.

Epilepsy Seizures can result from overstimulation of glutamatergic pathways, beginning with overactivation of AMPA receptors and progressing to overactivation of NMDA receptors. In animal models, inhibition of AMPA receptor activation

180 Principles of Central Nervous System Pharmacology

prevents seizure onset, whereas NMDA receptor antagonists decrease seizure intensity and duration. Lamotrigine, a drug used in the treatment of refractory focal seizures (see Chapter 15), stabilizes the inactivated state of the voltage-gated Na⫹ channel, and thereby reduces membrane excitability, the number of action potentials in a burst, glutamate release, and glutamate receptor activation. Felbamate is another antiepileptic that has a variety of actions, including the inhibition of NMDA receptors. Because of associated aplastic anemia and hepatotoxicity, its use is restricted to patients with refractory seizures.

Action potential

Presynaptic neuron Na+

Ca2+

Glu u Glu

 CONCLUSION AND FUTURE DIRECTIONS

G Glu

Glu

Glu Glu

Synaptic cleft

Glu

Glu NMDA-R

NMDA-R PLC

Glu

mGluR

DAG PIP2

α β

Ca2+

PKC (active)

α

AMPA-R Mg2+ Na+

3 2 Ca2+

PKC

GTP

1

γ

GDP

Removal of Mg2+

IP3

blockade of NMDA-R Ca2+

Kinases

Na+

Depolarization

Postsynaptic neuron Release of retrograde messengers leading to increased presynaptic transmitter release

FIGURE 12-11. Interactions among metabotropic, AMPA, and NMDA classes of glutamate receptors. Action potentials depolarize the plasma membrane of presynaptic neurons, leading to opening of voltage-gated Ca2⫹ channels and ultimately to glutamate release into the synaptic cleft. Studies have proposed a “tonic” physiologic role for activation of the metabotropic glutamate receptor (mGluR) during low-frequency stimulation of postsynaptic neurons by glutamate. In contrast, high-frequency presynaptic stimulation “phasically” activates AMPA receptors (1 ) and thereby induces the prolonged membrane depolarization required to relieve the Mg2⫹ blockade of NMDA receptors (2 ). The activated, Mg2⫹-free NMDA receptor (3 ) is then able to activate downstream kinases independently of the mGluR. Kinases associated with the postsynaptic densities, which act to scaffold the ionotropic receptors to the membrane, phosphorylate AMPA receptor subunits and thereby cause a change in that receptor’s composition (not shown). AMPA-R, AMPA receptor; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; mGluR, metabotropic glutamate receptor; NMDA-R, NMDA receptor; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C.

GABA and glutamate represent the major inhibitory and excitatory neurotransmitters in the CNS, respectively. Most drugs that act on GABAergic neurotransmission enhance GABAergic activity and thereby depress CNS functions. Modulation of GABAergic transmission can occur either presynaptically or postsynaptically. Drugs acting at presynaptic sites primarily target GABA synthesis, degradation, and reuptake. Drugs acting postsynaptically affect GABA receptors directly, either by occupying the GABA binding site or by an allosteric mechanism. Each of the three main GABA receptor types has a distinct pharmacology. The GABAA receptor is targeted by the largest number of drugs, including GABA binding-site agonists, benzodiazepines, barbiturates, general anesthetics, and neuroactive steroids. GABAB receptors are currently targeted by only a few therapeutic agents, which are used to treat spasticity. Recently, GABAB receptors have been found to influence pain, cognition, and addictive behavior, and interest is growing in drugs that modulate these receptors. GABAC receptors have not yet been developed as a target of pharmacologic agents. To improve safety and reduce adverse effects, including ataxia, tolerance, and physical dependence, development of new anxiolytics and sedatives has aimed for low-efficacy compounds (e.g., benzodiazepines) as well as compounds with selective activity at GABAA receptor subtypes. Animal models with selectively mutated GABAA receptor subunits have revealed that sedation/hypnosis is produced by enhancing the activity of receptors containing ␣1 subunits. In contrast, anxiolysis is produced by modulation of ␣2- or ␣3-containing receptors, and amnesia is associated with ␣5-containing receptors. There is also evidence for distinct pharmacology and physiology of synaptic GABAA receptors containing different ␤ subunits. Because of the potential role of excitatory neurotransmission in a number of pathologic processes, such as neurodegenerative diseases, stroke, trauma, hyperalgesia, and epilepsy, glutamate receptors have become important targets for drug development. The diversity of glutamate receptors and receptor subunits constitutes a potential advantage for the development of glutamate receptor antagonists that are selective for a particular receptor subtype. In the future, highly specific antagonists for glutamate receptor subtypes could potentially protect the CNS in stroke, prevent hyperalgesia after tissue trauma, and treat epileptic seizures. Although neurotransmitter receptors comprise the traditional targets for drug development, recent experimental studies suggest that targeting scaffolding proteins may also be a promising area for treatment of stroke and other diseases.

CHAPTER 12 / Pharmacology of GABAergic and Glutamatergic Neurotransmission 181

Postsynaptic cytoskeletal proteins such as postsynaptic density protein-95 (PSD-95) comprise an important part of the dendrite scaffolding structure, and PSD-95 mediates the intracellular signaling that occurs after glutamate receptor activation. In the context of excitotoxicity, PSD-95 can amplify the initial NMDA signal into deleterious cascades of nitric oxide generation. Blockade of PSD-95 reduces ischemic brain injury after experimental stroke in rats. A clinical trial testing this approach as a therapy for ischemic stroke is now ongoing.

Suggested Reading Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 2002;298:846–850. (Scaffolding proteins as therapeutic targets for glutamate excitotoxicity and neuropathic pain.) Besancon E, Guo S, Lok J, Tymianski M, Lo EH. Beyond NMDA and AMPA glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke. Trends Pharmacol Sci 2008;29:268–275. (This review

expands on traditional concepts of excitotoxicity to include newly discovered mechanisms of cell death.) Foster AC, Kemp JA. Glutamate- and GABA-based CNS therapeutics. Curr Opin Pharmacol 2006;6:7–17. (General overview of pharmacologic strategies in GABAergic and glutamatergic neurotransmission.) Herd MD, Belelli D, Lambert JJ. Neurosteroid modulation of synaptic and extrasynaptic GABAA receptors. Pharmacol Ther 2007;116:20–34. (Reviews physiology of neurosteroids and their interactions with GABAA receptors.) Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 2003;4:399–415. (Advances in pathophysiology of excitotoxicity in stroke.) Mizuta K, Xu D, Pan Y, Comas G, Sonett JR, Zhang Y, Paettieri RA Jr, Yang J, Emala CW Sr. GABAA receptors are expressed and facilitate relaxation in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2008;294:L1206–L1216. (Points to a role for GABAA receptors in airway tone.) Olsen RW, Sieghart W. GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 2008;56:141–148. (Reviews different GABAA receptor subtypes and their physiologic and pharmacologic roles.)

13 Pharmacology of Dopaminergic Neurotransmission David G. Standaert and Ryan R. Walsh

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 186–187 BIOCHEMISTRY AND CELL BIOLOGY OF DOPAMINERGIC NEUROTRANSMISSION . . . . . . . . . . . . . . . 186 Dopamine Storage, Release, Reuptake, and Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Dopamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Central Dopamine Pathways . . . . . . . . . . . . . . . . . . . . . . 189 DOPAMINE AND CONTROL OF MOVEMENT: PARKINSON’S DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Physiology of Nigrostriatal Pathways . . . . . . . . . . . . . . . . 191 Pathophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . 193 Dopamine Precursors . . . . . . . . . . . . . . . . . . . . . . . . . 193 Dopamine Receptor Agonists . . . . . . . . . . . . . . . . . . . 194

Inhibitors of Dopamine Metabolism . . . . . . . . . . . . . . . 195 Nondopaminergic Pharmacology in Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Treatment of Patients with Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 DOPAMINE AND DISORDERS OF THOUGHT: SCHIZOPHRENIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Pathophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . 197 Typical Antipsychotic Agents . . . . . . . . . . . . . . . . . . . . 197 Atypical Antipsychotic Agents . . . . . . . . . . . . . . . . . . . 199 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 200 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

 INTRODUCTION

 BIOCHEMISTRY AND CELL BIOLOGY OF DOPAMINERGIC NEUROTRANSMISSION

Dopamine (DA) is a catecholamine neurotransmitter that is the therapeutic target for a number of important central nervous system (CNS) disorders, including Parkinson’s disease and schizophrenia. DA is also a precursor for the other catecholamine neurotransmitters norepinephrine and epinephrine. The machinery of catecholamine neurotransmission has a number of components that are shared among members of the class, including biosynthetic and metabolic enzymes. There are also components that are specialized for the individual members of the class, including reuptake pumps and presynaptic and postsynaptic receptors. This chapter presents the principles that underlie current therapies for diseases that directly or indirectly involve changes in dopaminergic neurotransmission. The chapter begins with a discussion of the biochemistry and cell biology of dopaminergic neurotransmission and the localization of the major DA systems in the brain. Following this background, the chapter explores the physiology, pathophysiology, and pharmacology of Parkinson’s disease, which results from the specific loss of neurons in one of these DA systems, and schizophrenia, which is currently treated, in part, with drugs that inhibit dopaminergic neurotransmission. 186

Dopamine belongs to the catecholamine family of neurotransmitters. In addition to dopamine, this family includes norepinephrine (NE) and epinephrine (EPI). As the name suggests, the basic structure of the catecholamines consists of a catechol (3,4-dihydroxybenzene) moiety connected to an amine group by an ethyl bridge (Fig. 13-1A). Recall from Chapter 8, Principles of Nervous System Physiology and Pharmacology, that catecholaminergic pathways in the brain have “single source-divergent” organization, in that they arise from small clusters of catecholamine neurons that give rise to widely divergent projections. CNS catecholamines modulate the function of point-to-point neurotransmission and affect complex processes such as mood, attentiveness, and emotion. The neutral amino acid tyrosine is the precursor for all catecholamines (Fig. 13-1B). The majority of tyrosine is obtained from the diet; a small proportion may also be synthesized in the liver from phenylalanine. The first step in the synthesis of DA is the conversion of tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine, or levodopa) by oxidation of the 3 position on the benzene ring. This reaction is catalyzed by the enzyme tyrosine hydroxylase (TH), a ferro

188 Principles of Central Nervous System Pharmacology A

HO

R

HO Catechol nucleus

O

B

OH

Inhibition of MAO-A, on the other hand, retards the breakdown of all central and peripheral catecholamines; as noted in Chapter 10, MAO-A inhibition may lead to life-threatening toxicity when combined with catecholamine releasers such as the indirect-acting sympathomimetic tyramine found in certain wines and cheeses. Synaptic DA that is not taken up into the presynaptic cell can either diffuse out of the synaptic cleft or be degraded by the action of COMT. COMT is expressed in the brain, liver, kidney, and heart; it inactivates catecholamines by adding a methyl group to the hydroxyl group at the 3 position of the benzene ring. In the CNS, COMT is expressed primarily by

NH2

HO Tyrosine Tetrahydrobiopterin O2, Fe2+

Aromatic L-amino acid transporter

Tyrosine hydroxylase Tyrosine

Na+

O

Tyrosine

HO

OH NH2

HO

L-DOPA

Action potential

Dopamine

L-DOPA

Pyridoxal phosphate

Aromatic L-amino acid decarboxylase

HO

DA

Dopaminergic neuron ATP

H+

Dopamine transporter

ADP

Na+

NH2

HO

DA

H+

Dopamine Ascorbic acid O2, Cu2+

DA

DA

Ca2+

VMAT

Dopamine β-hydroxylase

Dopamine autoreceptor

DA

MAO DA

DOPAC OH

HO

NH2 Synaptic cleft

HO Norepinephrine S-adenosylmethionine

Phenylethanolamine N-methyltransferase

Postsynaptic dopamine receptors

OH H N

HO

Postsynaptic cell

HO Epinephrine

FIGURE 13-1.

Catecholamine synthesis. A. Catecholamines consist of a catechol nucleus with an ethylamine side chain (R group). The R group is ethylamine in dopamine, hydroxyethylamine in norepinephrine, and N-methyl hydroxyethylamine in epinephrine. B. Dopamine is synthesized from the amino acid tyrosine in a series of step-wise reactions. In cells that contain dopamine ␤-hydroxylase, dopamine can be further converted to norepinephrine; in cells that also contain phenylethanolamine N-methyltransferase, norepinephrine can be converted to epinephrine.

FIGURE 13-2.

Dopaminergic neurotransmission. Dopamine (DA) is synthesized in the cytoplasm and transported into secretory vesicles by the action of a nonselective monoamine-proton antiporter (VMAT) that is powered by the electrochemical gradient created by a proton ATPase. Upon nerve cell stimulation, DA is released into the synaptic cleft, where the neurotransmitter can stimulate postsynaptic dopamine receptors and presynaptic dopamine autoreceptors. DA is transported out of the synaptic cleft by the selective, Na⫹-coupled dopamine transporter (DAT). Cytoplasmic DA is re-transported into secretory vesicles by VMAT or degraded by the enzyme monoamine oxidase (MAO).

CHAPTER 13 / Pharmacology of Dopaminergic Neurotransmission 189

Neurotransmitter

HO Monoamine oxidase / Aldehyde dehydrogenase (MAO / AD)

NH2

HO

Catechol-Omethyltransferase (COMT)

Dopamine

HO

OH

O HO Dihydroxyphenylacetic acid (DOPAC)

O

NH2

HO 3-Methoxytyramine

MAO / AD

COMT

O

OH O

HO Homovanillic acid (HVA) Major metabolite (excreted in urine)

FIGURE 13-3. Catecholamine metabolism. Dopamine is metabolized to homovanillic acid (HVA) in a series of reactions. Dopamine is oxidized to dihydroxyphenylacetic acid (DOPAC) by sequential action of the enzymes monoamine oxidase (MAO) and aldehyde dehydrogenase (AD). Catechol-O-methyltransferase (COMT) then oxidizes DOPAC to HVA. Alternatively, dopamine is methylated to 3-methoxytyramine by COMT, and then oxidized to HVA by MAO and AD. HVA, the most stable dopamine metabolite, is excreted in the urine.

neurons. The sequential action of COMT and MAO degrades DA to the stable metabolite homovanillic acid (HVA), which is excreted in the urine (Fig. 13-3).

Dopamine Receptors Dopamine receptors are members of the G protein-coupled family of receptor proteins. The properties of dopamine receptors were originally classified by their effect on the formation of cyclic AMP (cAMP): activation of D1 class receptors leads to increased cAMP, while activation of D2 class receptors inhibits cAMP generation (Fig. 13-4). Subsequent studies led to cloning of the receptor proteins, revealing five distinct receptors, each encoded by a separate gene. All known DA receptors have the typical structure of G protein-coupled receptors, with seven transmembrane domains. The D1 class contains two dopamine receptors (D1 and D5), while the D2 class contains three receptors (D2, D3, and D4). There are two alternative forms of the D2 protein, D2S (i.e., short) and D2L (i.e., long), which represent alternate splice variants of the same gene; their difference lies in the third cytoplasmic loop, which affects G protein interaction but not dopamine binding. The five different dopamine receptor proteins have distinct distributions in the brain (Fig. 13-5). Both D1 and D2 receptors are expressed at high levels in the striatum (caudate and putamen), where they play a role in motor control by the basal

ganglia, as well as in the nucleus accumbens (see Chapter 18, Pharmacology of Drugs of Abuse) and olfactory tubercle. D2 receptors are also expressed at high levels on anterior pituitary gland lactotrophs, where they regulate prolactin secretion (see Chapter 26, Pharmacology of the Hypothalamus and Pituitary Gland). D2 receptors are thought to play a role in schizophrenia because many antipsychotic medications have high affinity for these receptors (see below), although the localization of the D2 receptors involved remains to be elucidated. D3 and D4 receptors are structurally and functionally related to D2 receptors and may also be involved in the pathogenesis of schizophrenia. High levels of D3 receptors are expressed in the limbic system, including the nucleus accumbens and olfactory tubercle, while D4 receptors have been localized to the frontal cortex, diencephalon, and brainstem. D5 receptors are distributed sparsely and expressed at low levels, mainly in the hippocampus, olfactory tubercle, and hypothalamus. Regulation of cAMP formation is the defining characteristic of the dopamine receptor classes, but dopamine receptors can also affect other aspects of cellular function depending on their localization and linkage to second messenger systems. Most dopamine receptors are expressed on the surface of postsynaptic neurons at dopaminergic synapses. The density of these receptors is tightly controlled through regulated insertion and removal of dopamine receptor proteins from the postsynaptic membrane. DA receptors are also expressed presynaptically on the terminals of dopaminergic neurons. Presynaptic dopamine receptors, most of which are of the D2 class, serve as autoreceptors. These autoreceptors sense dopamine overflow from the synapse and reduce dopaminergic tone, both by decreasing DA synthesis in the presynaptic neuron and by reducing the rate of neuronal firing and dopamine release. Inhibition of DA synthesis occurs through cAMP-dependent down-regulation of TH activity, while the inhibitory effect on DA release and neuronal firing is due, in part, to a separate mechanism involving the modulation of K⫹ and Ca2⫹ channels. Increased K⫹ channel opening results in a larger current that hyperpolarizes the neuron, so that a larger depolarization is needed to reach the firing threshold. Decreased Ca2⫹ channel opening results in decreased levels of intracellular Ca2⫹. Because Ca2⫹ is required for synaptic vesicle trafficking to and fusion with the presynaptic membrane, decreases in intracellular Ca2⫹ levels result in decreased dopamine release.

Central Dopamine Pathways Most central dopaminergic neurons originate in discrete areas of the brain, as shown in Figure 13-6 (see also Fig. 8-8), and have divergent projections. Three major pathways can be distinguished. The largest DA tract in the brain is the nigrostriatal system, which contains about 80% of the brain’s DA. This tract projects rostrally from cell bodies in the pars compacta of the substantia nigra to terminals that richly innervate the caudate and putamen, two nuclei that are collectively called the striatum. The striatum is named for the striped appearance of the white fiber tracts that run through it; the substantia nigra is named for the dark pigmentation that results from the decomposition of DA to melanin. Dopaminergic neurons of the nigrostriatal system are involved in the stimulation of purposeful movement. Their degeneration results in the abnormalities of movement that are characteristic of Parkinson’s disease. Medial to the substantia nigra is an area of dopaminergic cell bodies in the midbrain called the ventral tegmental area (VTA).

190 Principles of Central Nervous System Pharmacology

D1 Receptor Family

D2 Receptor Family N

N

Schematic structure

C C

cAMP (via Gi) K+ currents Voltage-gated Ca2+ currents

cAMP (via Gs) PIP2 hydrolysis Ca2+ mobilization (via IP3) PKC activation

Second messenger systems

D1

D5

Striatum Neocortex

Distribution in CNS

D2

Hippocampus Hypothalamus

Striatum Substantia nigra Pituitary gland

D3

D4

Olfactory tubercle Nucleus accumbens Hypothalamus

Frontal cortex Medulla Midbrain

FIGURE 13-4. Dopamine receptor families. The five dopamine receptor subtypes (D1–D5) can be classified into two major families of receptors. The D1 receptor family has a long C-terminal tail and a short cytoplasmic loop between transmembrane helices 5 and 6, whereas the D2 receptor family has a short C-terminal tail and a long cytoplasmic loop between helices 5 and 6. Stimulation of the D1 family is excitatory, increasing cAMP and intracellular Ca2⫹ levels and activating protein kinase C (PKC). Stimulation of the D2 family is inhibitory, decreasing cAMP and intracellular Ca2⫹ levels and hyperpolarizing the cell. The five receptor subtypes exhibit distinctive patterns of distribution in the central nervous system. Within the D2 receptor subtype, there are D2S and D2L isoforms (not shown). IP3, inositol trisphosphate; PIP2, phosphatidylinositol-4,5-bisphosphate. The VTA has widely divergent projections that innervate many forebrain areas, most notably the cerebral cortex, the nucleus accumbens, and other limbic structures. These systems play an important and complex (as yet poorly understood) role in motivation, goal-directed thinking, regulation of affect, and positive reinforcement (reward). Derangement of these pathways may

D1

D5

be involved in the development of schizophrenia; as discussed below, the blocking of dopaminergic neurotransmission can lead to a remission in psychotic symptoms. (See Chapter 18 for a more complete discussion of the reward pathway.) DA-containing cell bodies in the arcuate and paraventricular nuclei of the hypothalamus project axons to the median

D2

D3

D4

Cx

Cx

C nAc

C nAc

P

OT

nAc

P

AM

AM OT

AM

OT

OT

H

H

H

VTA HIPP

HIPP

SN

HIPP

SN

HIPP

FIGURE 13-5. Location of dopamine receptors in the brain. The location of the five dopamine receptor subtypes in the human brain, as determined by localization of receptor mRNAs in corresponding regions of the rat brain, is shown in orange in coronal section. Both D1 and D2 receptors are localized in the caudate and putamen (the striatum), nucleus accumbens, amygdala, olfactory tubercle, and hippocampus. In addition, D1 receptors are present in the cerebral cortex, whereas D2 receptors are present in the substantia nigra, ventral tegmental area, and hypothalamus. Abbreviations: AM, amygdala; C, caudate; Cx, cerebral cortex; H, hypothalamus; HIPP, hippocampus; nAc, nucleus accumbens; OT, olfactory tubercle; P, putamen; SN, substantia nigra; VTA, ventral tegmental area.

Hypothalamus Area postrema Ventral tegmental area

Substantia nigra

192 Principles of Central Nervous System Pharmacology

Normal Balanced activity of direct and indirect pathways Motor cortex

Glutamatergic input from cortex

Putamen D1

Direct pathway (enables movement)

ACh

D2

Indirect pathway (inhibits movement)

Caudate

Thalamus

Direct pathway Putamen

Parkinson's disease Direct pathway inhibited and indirect pathway activated, both leading to reduced movement

Indirect pathway STN SNc

GPi GPe SNr

Dopaminergic input from SNc

Glutamatergic input from cortex

Putamen Direct pathway Activity reduced, because of loss of D1 stimulation

D1 ACh

D2

Movement inhibited Indirect pathway To spinal motor neurons

Activity increased, because of release of D2 inhibition

Dopaminergic input from SNc

Movement inhibited

FIGURE 13-7. Effect of Parkinson’s disease on dopaminergic pathways that regulate movement. Two principal pathways in the basal ganglia regulate movement: the indirect pathway, which inhibits movement, and the direct pathway, which enables movement. Dopamine inhibits the indirect pathway and stimulates the direct pathway, yielding a net bias that allows purposeful movement. Excitatory pathways are shown in blue, and inhibitory pathways are shown in black. The direct pathway signals from putamen to GPi to thalamus to cortex, while the indirect pathway signals from putamen to GPe to STN to GPi to thalamus to cortex. GPi, internal segment of the globus pallidus; GPe, external segment of the globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulate; STN, subthalamic nucleus. Inset: Both direct and indirect pathway neurons in the putamen receive inputs from the nigrostriatal dopaminergic system (dotted blue arrow) and from cortical glutamatergic systems (solid blue arrow), process these inputs in the context of local cholinergic influences (ACh), and transmit a GABAergic output (not shown). Degeneration of dopaminergic neurons in the substantia nigra results in understimulation of the direct (movement-enabling) pathway and underinhibition of the indirect (movement-inhibiting) pathway. The net result is a paucity of movement. Dotted gray arrow indicates decreased activity caused by understimulation, and thick black arrow indicates increased activity caused by underinhibition.

This model of basal ganglia function is greatly simplified, of course, but it has been useful in developing a deeper understanding of how the basal ganglia work. An important prediction of the model is that, in Parkinson’s disease, the indirect pathway (and, in particular, the subthalamic nucleus) should be overactive. This prediction has been proven directly by in vivo electrical recordings in patients with Parkinson’s disease. Furthermore, surgical therapies that target the subthalamic nucleus, such as deep brain stimulation in this location, are now often used to treat Parkinson’s disease when pharmacologic treatments are inadequate.

Pathophysiology In Parkinson’s disease, there is a selective loss of dopaminergic neurons in the substantia nigra pars compacta (Fig. 13-7). The extent of loss is profound, with at least 70% of the neurons destroyed at the time symptoms first appear; often, 95%

of the neurons are missing at autopsy. The destruction of these neurons results in the core motor features of the disease: bradykinesia, or slowness of movement; rigidity, a resistance to passive movement of the limbs; impaired postural balance, which predisposes to falling; and a characteristic tremor when the limbs are at rest. The mechanisms underlying the destruction of DA neurons in the substantia nigra in Parkinson’s disease are not fully understood. Both environmental factors and genetic influences have been implicated. In 1983, the unexpected development of Parkinson’s disease in abusers of the synthetic opioid meperidine (see Chapter 17, Pharmacology of Analgesia) yielded the first agent known to produce Parkinson’s disease directly and the strongest evidence that environmental factors can cause Parkinson’s disease. These individuals, who tended to be young and otherwise healthy, suddenly developed severe, levodopa-responsive parkinsonian symptoms. The cases were all linked to a single contaminated batch of meperidine that

Periphery

Brain 3MT

3-O-MD Entacapone Tolcapone

COMT LNAA L-DOPA

Carbidopa

AADC

DA Blood-brain barrier

Tolcapone

COMT AADC L-DOPA

DA MAOB

DOPAC

Selegiline Rasagiline

CHAPTER 13 / Pharmacology of Dopaminergic Neurotransmission 195

Dopamine receptor agonists may also trigger symptoms of the dopamine dysregulation syndrome, in which patients exhibit impaired impulse control. Common manifestations include pathological gambling, overspending, compulsive eating, and hypersexuality. These behaviors may be socially destructive and require discontinuation of the medications. Recent studies have examined the use of pramipexole and ropinirole as initial monotherapy for Parkinson’s disease. It was thought that, because the dopamine agonists have longer half-lives than levodopa, they might be less likely to induce “off” periods. These studies show that use of the dopamine receptor agonists as initial treatment for Parkinson’s disease does delay the onset of “off” periods and dyskinesias, but there is also an increased rate of adverse effects compared to initial treatment with levodopa. At present, many practitioners use dopamine agonists as the initial treatment for Parkinson’s disease, especially in younger individuals. Inhibitors of Dopamine Metabolism A third strategy that has been employed to treat Parkinson’s disease involves the inhibition of DA breakdown. Inhibitors of both MAO-B (the isoform of MAO that predominates in the striatum) and COMT have been used as adjuvants to levodopa in clinical practice (Fig. 13-8). Selegiline is an MAO inhibitor that, in low concentrations, is selective for MAO-B. It does not interfere with the peripheral metabolism of monoamines by MAO-A, and it avoids the toxic effects of dietary tyramine and other sympathomimetic amines that are associated with nonselective MAO blockade (see Chapter 14, Pharmacology of Serotonergic and Central Adrenergic Neurotransmission). A drawback of selegiline is that this drug forms a potentially toxic metabolite, amphetamine, which can cause sleeplessness and confusion, especially in the elderly. Rasagiline, a newer MAO-B inhibitor that does not form toxic metabolites, has recently been approved in the United States. Both rasagiline and selegiline improve motor function in Parkinson’s disease when used alone, and both can augment the effectiveness of levodopa therapy. There has also been interest in the question of whether MAO inhibitors can limit the formation of reactive free radicals associated with dopamine catabolism and thereby alter the rate of disease progression. Early studies of the potential protective properties of selegiline were inconclusive. More recent studies with rasagiline have been promising but have not yet proven a disease-modifying effect. Tolcapone and entacapone inhibit COMT and thereby inhibit the degradation of levodopa as well as DA. Tolcapone is a highly lipid-soluble agent that can cross the BBB, while entacapone distributes only to the periphery. Both drugs decrease the peripheral metabolism of levodopa and thereby make more levodopa available to the CNS. Tolcapone has the additional property of crossing the blood-brain barrier effectively and inhibiting central as well as peripheral COMT. In clinical trials, both tolcapone and entacapone have been shown to reduce the “off” periods that are associated with decreasing plasma levodopa levels. Although the central effect of tolcapone is an advantage (Fig. 13-8), there have been several reports of fatal hepatic toxicity associated with tolcapone, and it must be used with great care. In practice, therefore, entacapone is the most widely used COMT inhibitor. Nondopaminergic Pharmacology in Parkinson’s Disease Amantadine, trihexyphenidyl, and benztropine are all drugs that do not clearly affect dopaminergic pathways but are

nonetheless effective in the treatment of Parkinson’s disease. Amantadine was developed and is marketed primarily as an antiviral that reduces the length and severity of influenza A infections (see Chapter 37, Pharmacology of Viral Infections). In patients with Parkinson’s disease, however, amantadine is used to treat levodopa-induced dyskinesias that develop late in the course of the disease. The mechanism by which amantadine reduces dyskinesia is thought to involve blockade of excitatory NMDA receptors. Trihexyphenidyl and benztropine are muscarinic receptor antagonists that reduce cholinergic tone in the CNS. They reduce tremor more than bradykinesia and are therefore more effective in treating patients for whom tremor is the major clinical manifestation of Parkinson’s disease. These anticholinergic drugs are thought to act by modifying the actions of striatal cholinergic interneurons, which regulate the interactions of direct and indirect pathway neurons. They also cause a range of anticholinergic adverse effects, which may include dry mouth, urinary retention, and most importantly, impairment of memory and cognition.

Treatment of Patients with Parkinson’s Disease The treatment of patients with Parkinson’s disease is an individualized process that must take into account not only the extent of symptoms but also the patient’s age, occupation, activities, and perceived disabilities. There is at present no laboratory test that can specifically confirm the diagnosis; instead, diagnosis is based on history and physical examination, along with laboratory studies to exclude other possible diagnoses. In patients with early disease, it may be appropriate to recommend a nonpharmacologic approach to treatment that emphasizes exercise and lifestyle modification. Almost all patients eventually require treatment with medication. In patients with mild symptoms, MAO-B inhibitors, amantadine, or anticholinergic medications may be considered. When symptoms are more advanced, a dopaminergic therapy is indicated. Levodopa is the most effective therapy, but many younger patients are treated first with a dopamine agonist in the hope of delaying the onset of motor fluctuations. Advanced disease with fluctuations requires polypharmacy, often including levodopa, dopamine agonists, entacapone, MAO-B inhibitors, and amantadine. It is important to be vigilant for the development of cognitive symptoms and adverse effects, which may require modification of the therapeutic approach.

 DOPAMINE AND DISORDERS OF THOUGHT: SCHIZOPHRENIA Pathophysiology Schizophrenia is a thought disorder characterized by one or more episodes of psychosis (impairment in reality testing). Patients may manifest disorders of perception, thinking, speech, emotion, and/or physical activity. Schizophrenic symptoms are divided into two broad categories. Positive symptoms involve the development of abnormal functions; these symptoms include delusions (distorted or false beliefs and misinterpretation of perceptions), hallucinations (abnormal perceptions, especially auditory), disorganized speech, and catatonic behavior. Negative symptoms involve the reduction or loss of normal functions; these symptoms include affective flattening (decrease in the range or intensity of emotional expression), alogia (decrease in the

CHAPTER 13 / Pharmacology of Dopaminergic Neurotransmission 197

hyperactivity is responsible for the positive symptoms of schizophrenia. This hypothesis is supported by positron emission tomography (PET) scans of the brains of patients displaying the earliest signs of schizophrenia; these PET images show changes in blood flow to the mesolimbic system that represent changes in the level of functioning of this system. Dopaminergic neurons of the mesocortical system originate in the ventral tegmental area and project to regions of the cerebral cortex, particularly the prefrontal cortex. Because the prefrontal cortex is responsible for attention, planning, and motivated behavior, the hypothesis has been advanced that the mesocortical system plays a role in the negative symptoms of schizophrenia. All of the evidence implicating DA in the pathogenesis of schizophrenia is circumstantial, however, and much of it is conflicting. Changes in DA levels, particularly in the mesolimbic and mesocortical systems, could simply reflect downstream consequences of a pathologic process in a heretofore undiscovered pathway. One hypothesis involving such an upstream process suggests that an imbalance in glutamatergic neurotransmission plays an important role in schizophrenia. This model is supported by the observation that phencyclidine (PCP) (see Chapter 18), an antagonist at NMDA receptors, causes symptoms similar to those of schizophrenia. In fact, the syndrome seen in patients taking PCP chronically—consisting of psychotic symptoms, visual and auditory hallucinations, disorganized thought, blunted affect, withdrawal, psychomotor retardation, and an amotivational state—has components of both the positive and negative symptoms of schizophrenia. Dopaminergic neurons and excitatory glutamatergic neurons often form reciprocal synaptic connections, which could account for the efficacy of DA receptor antagonists in schizophrenia. Even if this hypothesis is correct, at present, there are no useful therapies for schizophrenia that act at glutamate receptors. Glutamate is the primary excitatory transmitter in the brain, and further research will be required to identify drugs that are selective enough for use in schizophrenia and that have an acceptable adverse effect profile.

Pharmacologic Classes and Agents Although the biological basis of schizophrenia remains controversial, a number of drugs are effective in treating the illness. When successful, these medications can lead to a remission of psychosis and allow the patient to integrate into society. Patients only rarely return completely to their premorbid state, however. D rugs used in the management of psychosis are often called neuroleptics or antipsychotics. Although these terms are frequently used interchangeably, they have slight yet important differences in connotation. The term neuroleptic emphasizes the drugs’ neurological actions that are commonly manifested as adverse effects of treatment. These adverse effects, often called extrapyramidal effects, result from DA receptor blockade in the basal ganglia and include the parkinsonian symptoms of slowness, stiffness, and tremor. The term antipsychotic denotes the ability of these drugs to abrogate psychosis and alleviate disordered thinking in schizophrenic patients. The antipsychotics may be further divided into typical antipsychotics, older drugs with prominent actions at the D2 receptor, and atypical antipsychotics, a newer generation of drugs with less prominent D2 antagonism and consequently fewer extrapyramidal effects. Typical Antipsychotic Agents The history of the typical antipsychotic drugs dates back to the approval of chlorpromazine in 1954. Approval was

based on observations of the drug’s effectiveness in schizophrenia, but there was little understanding of its mechanism of action. In the 1960s, as the role of DA in the brain became better understood, the ability of the typical antipsychotic drugs to block dopaminergic neurotransmission in the CNS was first elucidated. Affinity binding studies performed in the 1980s demonstrated that both therapeutic efficacy and extrapyramidal adverse effects of the typical antipsychotics correlate directly with the affinity of these drugs for D2 receptors. As shown in Figure 13-9, drugs with higher affinity for D2 receptors, as represented by lower dissociation constants, tend to require smaller doses to control psychotic symptoms and alleviate schizophrenia. Mechanism of Action

Although the typical antipsychotics block D2 receptors in all of the CNS dopaminergic pathways, their mechanism of action as antipsychotics appears to involve antagonism of mesolimbic, and possibly mesocortical, D2 receptors. As described above, one hypothesis holds that the positive symptoms of schizophrenia correlate with hyperactivity of the mesolimbic system, and antagonism of mesolimbic dopamine receptors could alleviate these symptoms. The typical antipsychotics are relatively less effective at controlling the negative symptoms of schizophrenia. This relative lack of efficacy at treating the negative symptoms could relate to the hypothesis that the negative symptoms correlate with hypoactivity of mesocortical neurons, because the antagonist action of the antipsychotics would not be expected to correct dopaminergic hypoactivity. Many of the adverse effects of the typical antipsychotics are likely mediated by binding of these drugs to D2 receptors in the basal ganglia (nigrostriatal pathway) and pituitary gland (see below). The typical antipsychotics fall into several structural classes, of which the most prominent are the phenothiazines and the butyrophenones (Fig. 13-10). Chlorpromazine is the prototypical phenothiazine, and haloperidol is the most widely used butyrophenone. Despite differences in structure and D2 receptor affinity, all typical antipsychotics have similar clinical efficacy at their standard doses. In general, aliphatic phenothiazines (such as chlorpromazine) are less potent antagonists at D2 receptors than are butyrophenones, thioxanthenes (phenothiazines in which a nitrogen in the phenothiazine nucleus is substituted by a carbon), or phenothiazines functionalized with a piperazine derivative (such as fluphenazine). For all of these drugs, the clinical dose can be adjusted to account for the in vitro D2 receptor binding affinity, so that efficacy is unaffected by potency at clinically useful doses. However, the potency of the typical antipsychotics is critical in determining the drugs’ adverse effects profiles. Adverse Effects

The adverse effects of typical antipsychotic drugs can be divided into two broad categories: those caused by antagonist action at dopamine D2 receptors outside the mesolimbic and mesocortical systems (on-target effects) and those caused by nonspecific antagonist action at other receptor types (off-target effects). Given the broad distribution of dopamine receptors, it is not surprising that dopamine receptor antagonists have a wide range of on-target adverse effects. As noted above, the most prominent of these effects are often referred to as extrapyramidal effects. Because endogenous stimulation of

198 Principles of Central Nervous System Pharmacology

1000 Remoxipride

Dissociation constant at D2 receptor (nM)

Clozapine

100

Sulpiride Thioridazine

10

Prochlorperazine Trifluoperazine Olanzapine

Chlorpromazine

Moperone

Haloperidol Raclopride Fluphenazine Butaclamol Flupentixol Thiothixene Trifluperidol Droperidol Pimozide

1.0

Benperidol

0.10

Spiroperidol

0.01 0.1

1

10

100

1000

10000

Antipsychotic drug dose (mg/day)

FIGURE 13-9.

Antipsychotic potency of dopamine receptor antagonists. Over at least three orders of magnitude, the clinically effective dose of the typical antipsychotics is proportional to the dissociation constant of the drugs at D2 receptors. (Note that a higher dissociation constant represents a lower binding affinity.) Atypical antipsychotics such as clozapine and remoxipride (blue diamonds) are exceptions to this rule; these agents have clinical effects at a dose lower than that predicted by their dissociation constants. Data points represent the mean dissociation constant (averaged over multiple studies) at the most common clinically effective dose. The dotted line represents the best fit to the data for all of the typical antipsychotics (blue circles).

dopamine D2 receptors inhibits the indirect pathway within the basal ganglia, antagonism of D2 receptors by typical antipsychotic drugs can disinhibit the indirect pathway and thereby induce parkinsonian symptoms. Such symptoms can sometimes be treated with the nondopaminergic therapies for Parkinson’s disease, such as amantadine and anticholinergic drugs. Dopaminergic drugs are often ineffective because of the high affinity of the antagonists for the D2 receptor and because, when used in this setting, dopaminergic drugs could cause a relapse of schizophrenic symptoms. The most severe adverse effect of the typical antipsychotics is the so-called neuroleptic malignant syndrome (NMS), a rare but life-threatening syndrome characterized by catatonia, stupor, fever, and autonomic instability; myoglobinemia and death occur in about 10% of these cases. NMS is most commonly associated with the typical antipsychotic drugs that have a high affinity for D2 receptors, such as haloperidol. It can also be seen in patients with Parkinson’s disease who abruptly discontinue dopaminergic medications, emphasizing the importance of dopamine in the causation of NMS. The symptoms are thought to arise at least in part from the actions of the antipsychotics on the dopaminergic systems in the hypothalamus, which are essential for the body’s ability to control temperature. Treatment with antipsychotics and other dopamine antagonists can also cause abnormal movements, a condition known as tardive dyskinesia. This condition is observed most frequently after prolonged treatment with drugs that have a high affinity for the D2 receptor, such as haloperidol. It is

occasionally seen in patients after only short-term treatment and has been reported to occur after a single dose of a D2 receptor antagonist. The syndrome is characterized by repetitive, involuntary, stereotyped movements of the facial musculature, arms, and trunk. The exact mechanism is unknown, but it is believed to involve adaptive hypersensitivity of D2 receptors in the striatum, which, in turn, results in excessive dopaminergic activity. Antiparkinsonian drugs can exacerbate tardive dyskinesia, and discontinuation of antiparkinsonian drugs can ameliorate the symptoms. Administration of high doses of high-potency typical antipsychotics can temporarily suppress the disorder, presumably by overcoming the adaptive response in striatal neurons, but may in the long run lead to worsening of symptoms. In many cases, cessation of all typical antipsychotic medications will lead to slow reversal of the striatal adaptations, with eventual improvement in the symptoms of tardive dyskinesia. Some patients, however, are left with a permanent and irreversible movement disorder. Some adverse effects of typical antipsychotics are thought to be caused by antagonist action at dopamine receptors in the pituitary gland, where dopamine tonically inhibits prolactin secretion. Antagonism of D2 receptors increases prolactin secretion, leading to amenorrhea, galactorrhea, and false-positive pregnancy tests in women, and to gynecomastia and decreased libido in men. Other adverse effects of the typical antipsychotics result from nonspecific antagonism of muscarinic and ␣-adrenergic receptors. Antagonism of peripheral muscarinic pathways

CHAPTER 13 / Pharmacology of Dopaminergic Neurotransmission 199

during chronic antipsychotic use, it is considered an adverse effect. In the acutely psychotic patient, however, sedation may be part of the drug’s intended spectrum of action. The adverse effect profiles of the typical antipsychotics depend on their potency. High-potency drugs (whose clinical doses are only a few milligrams) tend to have fewer sedative effects and cause less postural hypotension than drugs with lower potency (i.e., drugs that require high doses to achieve a therapeutic effect). On the other hand, lower potency typical antipsychotics tend to cause fewer extrapyramidal adverse effects. These observations can be rationalized by the fact that high-potency drugs have high affinity for D2 receptors and are therefore more selective in their action. Thus, these drugs are more likely to cause adverse effects mediated by D2 receptors (i.e., extrapyramidal effects) and fewer adverse effects mediated by muscarinic and ␣-adrenergic receptors (i.e., anticholinergic effects, sedation, and postural hypotension). Conversely, low-potency typical antipsychotics do not bind D2 receptors as tightly and cause fewer extrapyramidal effects, while their lower selectivity results in more prominent anticholinergic and antiadrenergic effects.

S

N

R2

R1

Phenothiazine skeleton

S

N

Cl N

Chlorpromazine

S

N

CF3

Pharmacokinetics, Metabolism, and Drug Interactions

N N OH Fluphenazine S

C

R2

H R1

Thioxanthene skeleton

OH O N

F Haloperidol (a butyrophenone)

FIGURE 13-10.

Chemical structures of the typical antipsychotics. The structure of the phenothiazines is based on a common skeleton, with two variable functional groups. Chlorpromazine, the first approved antipsychotic, has substituted aminopropyl (R1) and chloride (R2) side groups. Piperazine (in blue box)substituted phenothiazines, such as fluphenazine, are significantly more potent than aliphatic-substituted phenothiazines, such as chlorpromazine. The fourth structure represents the skeleton of a thioxanthene, which substitutes a carbon (in blue box) for the phenothiazine nitrogen. As illustrated by the structure of haloperidol, butyrophenones (in blue box) are structurally distinct from phenothiazines and thioxanthenes.

causes anticholinergic effects, including dry mouth, constipation, difficulty urinating, and loss of accommodation (see Chapter 9, Cholinergic Pharmacology). ␣-Adrenergic antagonism can cause orthostatic hypotension and, in men, failure to ejaculate. Sedation can also occur because of inhibition of central ␣-adrenergic pathways in the reticular activating system. When sedation interferes with normal functioning

As with many drugs active in the CNS, the typical antipsychotics are highly lipophilic. In part because of this lipophilicity, typical antipsychotics tend to be metabolized in the liver and to exhibit both high binding to plasma proteins and high first-pass metabolism. The drugs are generally formulated as oral or intramuscular dosage forms. The latter are useful in treating acutely psychotic patients who may be a danger to themselves or others, while the oral formulations are generally used for chronic therapy. Elimination half-lives of the typical antipsychotics are erratic because their kinetics of elimination typically follow a multiphasic pattern and are not strictly first-order. In general, however, the half-lives of most typical antipsychotics are on the order of 1 day, and it is common practice to follow a once-daily dosing regimen. Two drugs, haloperidol and fluphenazine, are available as the decanoate esters. These highly lipophilic drugs are injected intramuscularly, where they are slowly hydrolyzed and released. The decanoate ester dosage forms provide a long-acting formulation that can be administered every 3 to 4 weeks. These formulations are particularly useful for treating poorly compliant patients. Because typical antipsychotics are antagonists at dopamine receptors, it is logical that these drugs should interact prominently with antiparkinsonian drugs that act either by increasing synaptic dopamine concentrations (levodopa) or through direct stimulation of dopamine receptors (dopamine agonists). Antipsychotics inhibit the action of both of these drug classes, and the administration of typical antipsychotics to patients with Parkinson’s disease often leads to a marked worsening of parkinsonian symptoms. In addition, typical antipsychotics potentiate the sedative effects of benzodiazepines and centrally active antihistamines. Because the latter are pharmacodynamic effects that result from the nonspecific binding of typical antipsychotics to cholinergic and adrenergic receptors, the low-potency typical antipsychotics tend to manifest more pronounced sedative effects than their high-potency counterparts. Atypical Antipsychotic Agents The so-called atypical antipsychotics have efficacy and adverse effect profiles that differ from those of the typical

200 Principles of Central Nervous System Pharmacology

antipsychotics. The six principal atypical antipsychotics are risperidone, clozapine, olanzapine, quetiapine, ziprasidone, and aripiprazole. All of these drugs are more effective than the typical antipsychotics at treating the “negative” symptoms of schizophrenia. In addition, direct comparisons of risperidone and haloperidol have shown that risperidone is more effective at combating the positive symptoms of schizophrenia and preventing a relapse of the active phase of the disease. Atypical antipsychotics cause significantly milder extrapyramidal symptoms than typical antipsychotics. The atypical antipsychotics have a relatively low affinity for D2 receptors; unlike the typical antipsychotics, their affinity for D2 receptors does not correlate with their clinically effective dose (Fig. 13-9). Three main hypotheses have emerged to explain this discrepancy. The 5-HT2 hypothesis states that antagonist action at the serotonin 5-HT2 receptor (see Chapter 14), or antagonist action at both 5-HT2 and D2 receptors, is critical for the antipsychotic effect of the atypical antipsychotics. This hypothesis is based on the finding that the Food and Drug Administration (FDA)-approved atypical antipsychotics are all high-affinity 5-HT2 receptor antagonists. It is not clear, however, how 5-HT2 antagonism contributes to the antipsychotic effect. The second model, the D4 hypothesis, is based on the finding that many of the atypical antipsychotics are also dopamine D4 receptor antagonists. This model suggests that selective D4 antagonism, or a combination of D2 and D4 antagonism, is critical to the mechanism of action of the atypical antipsychotics. Quetiapine does not act as a D4 receptor antagonist, however, so the D4 hypothesis cannot account for the mechanism of action of all atypical antipsychotics. The final hypothesis states that the atypical antipsychotics exhibit a milder adverse effect profile because of their relatively rapid dissociation from the D2 receptor. As described in Chapter 2, Pharmacodynamics, the binding affinity (Kd) of a drug is equal to the ratio of its rate of dissociation from the receptor (koff) to its rate of association to the receptor (kon): kon koff æææÆ æææÆ DRæ DR DR æ Kd 

koff kon

Equation 13-1

Because of their rapid off-rates, atypical antipsychotics bind D2 receptors more transiently than do typical antipsychotics. This could allow the atypical antipsychotics to inhibit the low-level, tonic dopamine release that may occur in the mesolimbic system. However, the drugs would be displaced by a surge of dopamine, as would occur in the striatum during the initiation of movement. Thus, extrapyramidal adverse effects would be minimized. The atypical antipsychotics comprise a structurally diverse set of drugs. Their receptor-binding profiles also differ, as summarized in the Drug Summary Table. As noted above, these agents all show combined antagonist properties at dopamine D2 and serotonin 5-HT2 receptors, and most of the drugs are also dopamine D4 receptor antagonists. Clozapine has a distinct pharmacology; it binds D1–D5 receptors and 5-HT2 receptors and it blocks ␣1-adrenergic, H1, and muscarinic receptors as well. Clozapine has been used therapeutically in patients who have failed other antipsychotic drugs, whether for lack of efficacy or intolerable adverse effects.

Clozapine has not been used as a first-line agent because of a small but significant risk of agranulocytosis (approximately 0.8% per year) and seizures. The administration of clozapine requires frequent monitoring of white blood cell counts and close follow-up. Although the atypical antipsychotics are primarily approved for use in schizophrenia and other primary psychotic disorders, they have also been used in the management of psychosis associated with Parkinson’s disease and dementia. In Parkinson’s disease, quetiapine has proved particularly useful because it does not seem to worsen the motor features of the disease. The atypical agents can also be used in managing patients with dementia, although epidemiological studies have shown that this use is associated with an increased risk of stroke and cerebral vascular disease; therefore, the risks and benefits of the therapies in this setting must be weighed carefully.

 CONCLUSION AND FUTURE DIRECTIONS Treatments for both Parkinson’s disease and schizophrenia modulate dopaminergic neurotransmission in the CNS. In Parkinson’s disease, the degeneration of dopaminergic neurons that project to the striatum is responsible for motor symptoms, including resting tremor, rigidity, and bradykinesia. In this disease, the direct pathway—which enables movement—is understimulated, whereas the indirect pathway—which inhibits movement—is disinhibited. Pharmacologic treatment of Parkinson’s disease depends on agents that increase dopamine release or activate dopamine receptors in the caudate and putamen, and thereby help restore the balance between the direct and indirect pathways. Schizophrenia is treated by inhibiting dopamine receptors at various sites in the limbic system. The pathophysiology of schizophrenia is not fully understood, and this lack of knowledge about etiology limits rational drug development. The clinical effectiveness of the various antipsychotic agents has provided useful clues, however. In particular, the pharmacology of the typical antipsychotic agents has formed the basis of the dopamine model of schizophrenia, which argues that dysregulated levels of dopamine in the brain play a role in the pathophysiology of the disease. The effectiveness of the atypical antipsychotic agents, which affect the function of several different receptor types, has highlighted the fact that the dopamine hypothesis is a simplification. The atypical agents represent an attractive new modality for treating schizophrenia because they have fewer extrapyramidal effects and are more effective for some disease symptoms than the typical antipsychotics. Future developments in the treatment of Parkinson’s disease and schizophrenia are focused on creating more selective agents within the current drug classes and on better elucidating the underlying pathophysiology of the disorders. New dopamine receptor agonists with higher selectivity, particularly those that bind D1 receptors, may one day provide more effective treatment for Parkinson’s disease with less severe adverse effects. The development of newer antipsychotics with increased receptor selectivity may similarly expand the therapeutic options for treating schizophrenia. Because Parkinson’s disease involves the death of dopaminergic neurons, much effort is currently directed at neuroprotective drugs that may slow the progression of the disease. Further research into a potential role for a glutamate deficit in the

CHAPTER 13 / Pharmacology of Dopaminergic Neurotransmission 201

pathophysiology of schizophrenia may yield new therapeutics for this disorder. For example, the development of selective glutamate receptor agonists may one day complement or even replace the use of dopamine receptor antagonists. Another important advance in the treatment of schizophrenia will likely result from the elucidation of models for the mechanism of the atypical antipsychotics, which will allow rational development of more effective drugs.

Acknowledgment We thank Joshua M. Galanter for his valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Albin RL, Young AB, Penney JB. The functional anatomy of basal ganglia disorders. Trends Neurosci 1989;12:366–375. (A classic article that describes the concept of “direct” and “indirect” pathways.)

Farrer MJ. Genetics of Parkinson disease: paradigm shifts and future prospects. Nat Rev Genet 2006;7:306–318. (A review of the rapidly evolving genetics of Parkinson’s disease.) Kellendonk C, Simpson EH, Polan HJ, et al. Transient and selective overexpression of dopamine D2 receptors in the striatum causes persistent abnormalities in prefrontal cortex functioning. Neuron 2006;49:603–615. (A new mouse model for schizophrenia suggesting a role for D2 receptors in cognitive impairment.) Langston JW. The Parkinson’s complex: parkinsonism is just the tip of the iceberg. Ann Neurol 2006;59:591–596. (A review that emphasizes the many aspects of Parkinson’s disease beyond the motor abnormalities.) Spooren W, Riemer C, Meltzer H. NK3 receptor antagonists: the next generation of antipsychotics? Nat Rev Drug Discov 2005;4:967–975. (Discusses pathophysiologic basis of potential antipsychotic agents.) Suchowersky O, Reich S, Perlmutter J, et al. Practice parameter: diagnosis and prognosis of new onset Parkinson disease (an evidence-based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 2006;66:968–975. (This “parameter,” as well as several others published in the same issue, represents the product of a careful review of the evidence for the effectiveness of various treatments for Parkinson’s disease.)

14 Pharmacology of Serotonergic and Central Adrenergic Neurotransmission Miles Berger and Bryan Roth

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 207-208 BIOCHEMISTRY AND PHYSIOLOGY OF SEROTONERGIC AND CENTRAL ADRENERGIC NEUROTRANSMISSION . . . . . 208 Serotonin Synthesis and Regulation . . . . . . . . . . . . . . . . . 208 Serotonin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 PATHOPHYSIOLOGY OF AFFECTIVE DISORDERS . . . . . . . . . 211 Clinical Characteristics of Affective Disorders . . . . . . . . . . 211 The Monoamine Theory of Depression . . . . . . . . . . . . . . . 212 Limitations of the Monoamine Theory . . . . . . . . . . . . . 213 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 213 Inhibitors of Serotonin Storage . . . . . . . . . . . . . . . . . . . . . 213 Inhibitors of Serotonin Degradation . . . . . . . . . . . . . . . . . 214 Reuptake Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Tricyclic Antidepressants (TCAs) . . . . . . . . . . . . . . . . . 215

Selective Serotonin Reuptake Inhibitors (SSRIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Norepinephrine-Selective Reuptake Inhibitors (NRIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Atypical Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . 217 Serotonin Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . 217 Serotonin Receptor Antagonists . . . . . . . . . . . . . . . . . . . . 217 Mood Stabilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Lithium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 219 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

 INTRODUCTION

of MDD is approximately 17%, whereas that of BPAD is 1–2%. There is a particularly strong heritable risk in BPAD, even though environmental factors are often triggers for the manic or depressive episodes. Although mania is characteristic of BPAD, bipolar patients spend significant periods of their lives depressed, and the mortality of the disorder derives primarily from suicide. MDD can occur as an isolated illness or can be comorbid with other diseases such as stroke, dementia, diabetes, cancer, and coronary artery disease. Although there is some genetic predisposition for MDD, stress is a greater predictor of depressive episodes than any single genetic variant. Aging and cerebral microvascular atherosclerosis are also associated with late-onset depression in the elderly. In addition to genetic and environmental triggers, many classes of drugs can exacerbate depression (e.g., interferon and chemotherapeutic agents). Both MDD and BPAD are major causes of morbidity worldwide, resulting in lost productivity and substantial use of medical resources. Affective illnesses are associated with a substantially increased risk of suicide. In the majority of suicides, a physician (not necessarily a psychiatrist) will have seen the patient less than 1 month before the suicide.

This chapter introduces the neurotransmitter serotonin (5hydroxytryptamine; 5-HT), which is the target for many of the drugs used to treat psychiatric disorders such as depression. Many of these medications also affect norepinephrine (NE) neurotransmission, and both neurotransmitter pathways are believed to be central to the modulation of mood. The different mechanisms by which medications can alter serotonin and norepinephrine signaling are discussed. Although many of the drugs presented function as antidepressants, medications in this pharmacologic group are also effective treatments for migraine headache, irritable bowel syndrome, and other conditions. Lithium and other drugs used to treat bipolar affective disorder are also briefly discussed. The major mood disorders are defined by the presence of depressive and/or manic episodes. Patients who have experienced at least one manic episode, with or without an additional history of depressive episodes, are said to have bipolar affective disorder (BPAD); patients with recurrent depressive episodes and no history of mania are said to have major depressive disorder (MDD). The lifetime prevalence

207

CHAPTER 14 / Pharmacology of Serotonergic and Central Adrenergic Neurotransmission 209

A

HN

O

B

O

OH

OH NH2

NH2

HO

Tyrosine Tryptophan Tryptophan hydroxylase

Tyrosine hydroxylase

O

O HN

OH

HO

OH NH2

NH2

HO L-DOPA

OH 5-Hydroxytryptophan

Aromatic L-amino acid decarboxylase

Aromatic L-amino acid decarboxylase

HO

For review, the synthesis of norepinephrine is summarized in Figure 14-1B, and the metabolic cycle of norepinephrine is summarized in Figure 14-3. For all monoamines, the first synthetic step is rate-limiting. Thus, DA and NE synthesis is rate-limited by tyrosine hydroxylase (TH), and 5-HT synthesis is rate-limited by tryptophan hydroxylase (TPH). Both enzymes are tightly regulated by inhibitory feedback via autoreceptors. 5-HT presynaptic autoreceptors respond to locally increased 5-HT concentrations by Gi protein signaling, which decreases TPH activity and serotonergic neuron firing. This autoregulatory loop could be one explanation for the observed time course of clinical action of the antidepressants, which is discussed below (The Monoamine Theory of Depression). 5-HT is transported into vesicles by the vesicular monoamine transporter (VMAT). The transporter is a nonspecific monoamine transporter that is important for the vesicular packaging of dopamine (DA) and epinephrine (EPI) as well as 5-HT. Reserpine binds irreversibly to VMAT and thereby inhibits the packaging of DA, NE, EPI, and 5-HT into vesicles. Selective serotonin reuptake transporters recycle 5-HT from the extracellular space back into the presynaptic

NH2

NH2

HN

Aromatic L-amino acid transporter

HO Dopamine Dopamineβ-hydroxylase

OH 5-Hydroxytryptamine (Serotonin; 5HT)

Na+

Tryptophan Tryptophan hydroxylase (rate limiting)

Tryptophan

OH HO

5-Hydroxytryptophan

NH2

Aromatic L-amino acid decarboxylase

Action potential CO2

HO

Serotonin

5HT

Serotonergic neuron

Norepinephrine

H+ 5HT transporter

Phenylethanolamine N-methyltransferase

Na+

VMAT

OH H N

HO

Na+ 5HT

5HT

Ca2+

5 5H 5HT 5HT 5HT

HO Epinephrine

FIGURE 14-1. Synthesis of serotonin and norepinephrine. A. 5Hydroxytryptamine (serotonin) is synthesized from the amino acid tryptophan in two steps: the hydroxylation of tryptophan to form 5-hydroxytryptophan by tryptophan hydroxylase, and the subsequent decarboxylation of this intermediate to produce 5-hydroxytryptamine (5-HT) by aromatic L-amino acid decarboxylase. Tryptophan hydroxylase is the rate-limiting enzyme in this pathway. B. Norepinephrine is synthesized from the amino acid tyrosine in a three-step process similar to the synthetic pathway for serotonin. Tyrosine is first oxidized to L-DOPA by the enzyme tyrosine hydroxylase and then decarboxylated to dopamine. After dopamine is transported into the synaptic vesicle, it is hydroxylated by the enzyme dopamine -hydroxylase to form norepinephrine. The same enzyme decarboxylates 5-hydroxytryptophan and L-DOPA; it is known generically as aromatic L-amino acid decarboxylase. Tyrosine hydroxylase is the rate-limiting enzyme in this pathway.

5HT1B receptor (autoreceptor)

MAO 5HT

5-hydroxyindole acetaldehyde 5HT 5HT

FIGURE 14-2. Presynaptic regulation of serotonin neurotransmission. Serotonin (5-HT) is synthesized from tryptophan in a two-reaction pathway: the rate-limiting enzyme is tryptophan hydroxylase. Both newly synthesized 5-HT and recycled 5-HT are transported from the cytoplasm into synaptic vesicles by the vesicular monoamine transporter (VMAT). Neurotransmission is initiated by an action potential in the presynaptic neuron, which eventually causes synaptic vesicles to fuse with the plasma membrane in a Ca2-dependent manner. 5-HT is removed from the synaptic cleft by a selective 5-HT transporter as well as by nonselective reuptake transporters (not shown). 5-HT can stimulate 5-HT1B autoreceptors on the presynaptic membrane to provide feedback inhibition. Cytoplasmic 5-HT is either sequestered in synaptic vesicles by VMAT or degraded by mitochondrial monoamine oxidase (MAO).

210 Principles of Central Nervous System Pharmacology Aromatic L-amino acid transporter

space is another important degradation enzyme for monoamines, although COMT plays a less significant role in the CNS than it does peripherally.

Tyrosine

Na+

Serotonin Receptors

Tyrosine L-DOPA Action potential

Dopamine DA

Adrenergic neuron

ATP

H+

ADP

NE transporter Na+ NE

DA

Ca2+

DA NE

H+ VMAT

α2 (autoreceptor) adrenergic receptor

NE

MAO NE

DOPGAL

FIGURE 14-3.

Presynaptic regulation of norepinephrine neurotransmission. Norepinephrine in the synaptic vesicle is derived from two sources. First, dopamine synthesized from tyrosine is transported into the vesicle by the vesicular monoamine transporter (VMAT). Inside the vesicle, dopamine is converted to norepinephrine by dopamine -hydroxylase. Second, recycled NE is transported from the cytoplasm into the vesicle, also by VMAT. Neurotransmission is initiated by an action potential in the presynaptic neuron, which eventually causes synaptic vesicles to fuse with the plasma membrane in a Ca2-dependent manner. NE is removed from the synaptic cleft by a selective norepinephrine transporter (NET) as well as by nonselective reuptake transporters (not shown). NE can stimulate 2-adrenergic autoreceptors to provide feedback inhibition. Cytoplasmic NE that is not sequestered in synaptic vesicles by VMAT is instead degraded to 3,4-dihydroxyphenylglycoaldehyde (DOPGAL) by monoamine oxidase (MAO) on the outer mitochondrial membrane.

Fifteen 5-HT receptors have been characterized, and all but one are G protein-coupled (Table 14-1). In general, the 5-HT1 class of receptors inhibits cellular activity via the Gi pathway (thus decreasing adenylyl cyclase activity and opening K channels), the 5-HT2 class increases signaling through the Gq pathway to cause phosphatidylinositol turnover, and the 5-HT4, 5-HT6, and 5-HT7 classes signal through the Gs pathway to stimulate adenylyl cyclase. The only known ligandgated ion channel is the 5-HT3 receptor. 5-HT1A receptors are expressed both on serotonergic cell bodies in the raphe nuclei (autoreceptors) and on postsynaptic neurons in the hippocampus and act to hyperpolarize neurons via the Gi pathway (as described above). Presynaptic 5-HT1B receptors are expressed on serotonergic nerve terminals, where they autoinhibit 5-HT neurotransmission. 5-HT2A and 5-HT2C signaling is excitatory and lowers the threshold for neuronal firing. The various serotonin receptors are expressed differentially throughout the brain and are differentially innervated by raphe projections. For example, a subset of 5-HT projections to the cortex stimulates postsynaptic 5-HT2A receptors, while other projections to the limbic system stimulate postsynaptic 5-HT1A receptors. There is considerable overlap of receptor subtype expression, however, and the physiologic significance of this overlap is unclear. The signaling mechanisms of norepinephrine (adrenergic) receptor subtypes are discussed in Chapter 10 and reviewed in Table 14-1.

TABLE 14-1 Signaling Mechanisms of Norepinephrine and Serotonin Receptor Subtypes NE RECEPTOR SUBTYPE SIGNALING MECHANISMS

neuron. Selective monoamine reuptake transporters are 12transmembrane–spanning proteins that couple neurotransmitter transport to the transmembrane sodium gradient. Unlike VMAT, which is a nonspecific monoamine transporter, the individual monoamine reuptake transporters show selectivity, high affinity, and low capacity for each individual monoamine. The selective monoamine transporters, which include the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT), are also capable of transporting the other monoamines, although less efficiently. Once 5-HT is returned to the neuronal cytoplasm, the neurotransmitter is transported into vesicles via VMAT or degraded by the monoamine oxidase (MAO) system. MAOs are mitochondrial enzymes that regulate the levels of monoamines in neural tissues and inactivate circulating and dietary monoamines (such as tyramine) in the liver and gut. The two isoforms, MAO-A and MAO-B, differ according to substrate specificity; MAO-A oxidizes 5-HT, NE, and DA, and MAO-B preferentially oxidizes DA. Monoamine oxidases inactivate monoamines by oxidative deamination, using a functional flavin moiety as an electron acceptor. Catechol-O-methyltransferase (COMT) in the extracellular

1

↑ IP3, DAG

2*

↓ cAMP

1,2

↑ cAMP

5-HT RECEPTOR SUBTYPE 5-HT1A,B*,D,E,F

↓ cAMP, ↑ K channel opening

5-HT2A,B,C

↑ IP3, DAG

5-HT3

Ligand-gated ion channel

5-HT4,6,7

↑ cAMP

Abbreviations: cAMP, cyclic AMP; DAG, diacylglycerol; IP3, inositol 1,4, 5-trisphosphate. *2-Adrenergic and 5-HT1B are presynaptic autoreceptors important for feedback inhibition.

214 Principles of Central Nervous System Pharmacology Neurotransmitter Synthesis

Neurotransmitter Release

Postsynaptic Effect

(NE and/or 5HT)

A Before treatment

Postsynaptic receptor

Low level of signaling

Presynaptic autoreceptor

NE and/or 5HT transporter

B Acute treatment

Low level of signaling

FIGURE 14-4. Postulated mechanism of the delay in onset of the therapeutic effect of antidepressant medications. A. Before treatment, neurotransmitters are released at pathologically low levels and exert steady-state levels of autoinhibitory feedback. The net effect is an abnormally low baseline level of postsynaptic receptor activity (signaling). B. Short-term use of antidepressant medication results in increased release of neurotransmitter and/or increased duration of neurotransmitter action in the synaptic cleft. Both effects cause increased stimulation of inhibitory autoreceptors, with increased inhibition of neurotransmitter synthesis and increased inhibition of exocytosis. The net effect is to dampen the initial effect of the medication, and postsynaptic receptor activity remains at pretreatment levels. C. Chronic use of antidepressant medication results in desensitization of the presynaptic autoreceptors. Thus, the inhibition of neurotransmitter synthesis and exocytosis is reduced. The net effect is enhanced postsynaptic receptor activity, leading to a therapeutic response. NE, norepinephrine; 5-HT, serotonin; TCA, tricyclic antidepressant; SSRI, selective serotonin reuptake inhibitor; SNRI, serotonin-norepinephrine reuptake inhibitor. and blood pressure and can induce tremors. These drugs have substantial potential for substance abuse; because the inactive prodrug lisdexamfetamine is converted relatively slowly to the active compound dextroamphetamine by ratelimiting hepatic metabolism, it may have less abuse potential than other amphetamine derivatives. Fenfluramine and dexfenfluramine are halogenated amphetamine derivatives that are modestly selective for 5-HT storage vesicles. These drugs were used briefly in the United States for appetite suppression, but severe cardiac toxicity led to their withdrawal. Another amphetamine derivative, methylenedioxymethamphetamine (MDMA), is both a selective serotonin storage inhibitor and a 5-HT receptor ligand. It is not approved for use in medical practice but is a significant drug clinically due to its illicit use (as “Ecstasy”).

Inhibitors of Serotonin Degradation TCA, SSRI, or SNRI

C Long-term treatment

Therapeutic level of signaling

TCA, SSRI, or SNRI

it may seem counterintuitive that a disorder such as ADHD could be treated by drugs that increase catecholamine levels, this finding makes sense in light of the differing roles of central versus peripheral NE. In the prefrontal cortex, increased NE promotes attention and higher cognitive processes, while peripheral increases in NE increase heart rate

The major pathway for serotonin degradation is mediated by MAO; accordingly, MAOIs have significant effects on serotonergic neurotransmission. The MAOIs are classified according to their specificity for the MAO-A and MAO-B isoenzymes and according to the reversibility or irreversibility of their binding. The older MAOIs are nonselective, and most older MAOIs, such as iproniazid, phenelzine, and isocarboxazid, are irreversible inhibitors. Newer MAOIs, such as moclobemide, befloxatone, and brofaromine, are selective for MAO-A and bind reversibly. Selegiline, a selective MAO-B inhibitor at low doses (see Chapter 13, Pharmacology of Dopaminergic Neurotransmission), also inhibits MAO-A at higher doses. MAOIs block the deamination of monoamines by binding to and inhibiting the functional flavin group of MAO (Fig. 14-5). By inhibiting the degradation of monoamines, MAOIs increase the 5-HT and NE available in the cytoplasm of presynaptic neurons. The increase in cytoplasmic levels of these monoamines leads not only to increased uptake and storage of 5-HT and NE in synaptic vesicles, but also to some constitutive leakage of the monoamines into the extracellular space. As noted in Chapter 10, the most toxic adverse effect of MAOI use is systemic tyramine toxicity. Because GI and hepatic MAO metabolizes tyramine, consumption of foods that contain tyramine, such as processed meats, aged hard cheeses, and red wine, can lead to excess levels of circulating tyramine. Tyramine is an indirect sympathomimetic that can stimulate the release of large amounts of

CHAPTER 14 / Pharmacology of Serotonergic and Central Adrenergic Neurotransmission 215

A Reserpine

Dopamine DA

Serotonin-norepinephrine reuptake inhibitors (SNRIs)

H+

Tricyclic antidepressants (TCAs)

VMAT

Na+ NE

DA NE NE

MAOI NE

DOPGAL

Reserpine

B Serotonin-norepinephrine reuptake inhibitors (SNRIs)

Serotonin 5HT

Tricyclic antidepressants (TCAs)

H+

Selective serotonin reuptake inhibitors (SSRIs)

VMAT

Na+ 5HT

5HT 5H 5HT 5HT

MAOI 5HT1B receptor (autoreceptor)

5HT

5HT

5-hydroxyindole acetaldehyde 5HT 5HT

FIGURE 14-5. Sites and mechanisms of action of antidepressant drugs. The sites of action of antidepressant drugs and of reserpine (which can induce depression) are indicated in noradrenergic neurons (A) and serotonergic neurons (B). Monoamine oxidase inhibitors (MAOIs) inhibit the mitochondrial enzyme monoamine oxidase (MAO); the resulting increase in cytosolic monoamines leads to increased vesicular uptake of neurotransmitter and to increased release of neurotransmitter during exocytosis. Tricyclic antidepressants (TCAs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) inhibit both the norepinephrine transporter (NET) and the serotonin transporter (SERT), causing increased levels of both NE and 5-HT in the synaptic cleft. Selective serotonin reuptake inhibitors (SSRIs) specifically inhibit the SERTmediated reuptake of 5-HT. TCAs, SNRIs, and SSRIs increase the duration of neurotransmitter action in the synaptic cleft, leading to increased downstream signaling. Reserpine, which can induce depression in humans and in animal models, blocks the VMAT-mediated uptake of monoamines into synaptic vesicles, which ultimately destroys the vesicles.

stored catecholamines by reversing the reuptake transporters. This uncontrolled catecholamine release can induce a hypertensive crisis characterized by headache, tachycardia, nausea, cardiac arrhythmia, and stroke. The older MAOIs are no longer considered first-line therapy for depression because of the potential for systemic tyramine toxicity; they should be prescribed only to patients able to commit to a tyramine-free diet. The newer MAOIs (i.e., the reversible inhibitors of MAO-A [RIMAs] that bind reversibly to MAO) are displaced by high concentrations of tyramine, resulting in significantly more tyramine metabolism and hence less tyramine toxicity. Selegiline has been approved as a transdermal patch, thus bypassing the GI system. Transdermal selegiline can maximally inhibit brain MAO-A (and MAO-B) at doses that reduce gastrointestinal MAO-A activity by only 30–40%, thus reducing the risk of a tyramine-induced hypertensive crisis and allowing patients greater dietary freedom. MAOIs, like other antidepressants, can precipitate manic or hypomanic episodes in some bipolar patients. All antidepressant drugs, including MAOIs, are hydrophobic and cross the blood–brain barrier. They are well absorbed orally and are metabolized to active metabolites by the liver. These metabolites are subsequently inactivated by acetylation, also in the liver. Excretion is primarily via renal clearance. The older, irreversibly binding MAOIs are cleared from the circulation as complexes with MAO and are effectively inactivated only when a new enzyme is synthesized. Because of the extensive effects of MAOIs on cytochrome P450 enzymes in the liver, they can cause numerous drug– drug interactions. All members of a patient’s medical team must prescribe other drugs with caution when a patient is taking an MAOI.

Reuptake Inhibitors Serotonergic tone is maintained at steady state by the balance between transmitter release and reuptake. Thus, inhibitors of the serotonin reuptake transporter decrease the reuptake rate, resulting in a net increase in the concentration of 5-HT in the extracellular space. These drugs alleviate the symptoms of a variety of common psychiatric conditions, including depression, anxiety, and obsessive-compulsive disorder. Four classes of reuptake inhibitors are in use: the nonselective tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and the newer norepinephrine-selective reuptake inhibitors (NRIs). Each class is discussed below, followed by a discussion of atypical antidepressants that do not fall clearly into one of these four categories. Tricyclic Antidepressants (TCAs) The TCAs derive their name from their common chemical backbone, consisting of three rings that include two aromatic rings attached to a cycloheptane ring. The prototype TCA is imipramine, and other members of this class include amitriptyline, desipramine, nortriptyline, and clomipramine (which is a first-line agent for obsessive-compulsive disorder). TCAs with secondary amines preferentially affect the NE system, whereas those with tertiary amines primarily affect the 5-HT system. Tetracyclic antidepressants, which include maprotiline, have also been developed, but they are not widely used. Tetracyclic antidepressants tend to be more selective for the NE system.

CHAPTER 14 / Pharmacology of Serotonergic and Central Adrenergic Neurotransmission 217

approved for the treatment of depression as well as neuropathic pain and other pain syndromes. Milnacipran is a selective NE and 5-HT reuptake inhibitor that has recently been approved for the treatment of fibromyalgia, based on clinical trials in which it improved symptoms of pain and dysphoria. Norepinephrine-Selective Reuptake Inhibitors (NRIs) Atomoxetine is a NE-selective reuptake inhibitor that is used in the treatment of ADHD. It is thought to improve ADHD symptoms by blocking NE reuptake and thereby increasing NE levels in the prefrontal cortex. (Note that methylphenidate and amphetamines are thought to improve ADHD symptoms by increasing NE levels in the prefrontal cortex via increased NE release.) Atomoxetine has several advantages over the amphetamines, including a lower abuse/ addiction potential and a longer plasma half-life that allows for once daily dosing. Atomoxetine increases peripheral as well as central NE levels, and thus increases heart rate and blood pressure.

Atypical Antidepressants Several drugs that interact with multiple targets and are indicated for the treatment of depression are sometimes referred to as “atypical antidepressants.” These agents include bupropion, mirtazapine, nefazodone, and trazodone. They are categorized together here only because they do not fit conveniently into other categories. These agents are newer than the TCAs and act by several different mechanisms, although some have unknown or incompletely characterized mechanisms of action. Bupropion appears to act mechanistically like the amphetamines and is particularly useful for the treatment of atypical depression because it increases both serotonin and dopamine levels in the brain. Bupropion is the antidepressant with the fewest sexual adverse effects. It is also believed to induce less switching into mania than the other antidepressants. The principal contraindication to the use of bupropion is a predisposition to seizures, since it lowers the seizure threshold. Thus, bupropion is contraindicated in patients with seizure disorders, electrolyte abnormalities, or eating disorders (since these can cause electrolyte imbalances). Mirtazapine blocks postsynaptic 5-HT2A and 5-HT2C receptors and the 2-adrenergic autoreceptor, and presumably decreases neurotransmission at 5-HT2 synapses while increasing NE neurotransmission. Mirtazapine is an effective somnorific as well as an appetite stimulant, making it a particularly useful antidepressant for the elderly population (who often present with insomnia and weight loss) and for other patients with weight loss and depression. Nefazodone and trazodone also block postsynaptic 5-HT2A and 5-HT2C receptors and are discussed below. Overall, the atypical antidepressants have relatively few adverse effects and demonstrate similar clinical efficacies despite their widely heterogeneous mechanisms of action and molecular targets.

Serotonin Receptor Agonists Ergots are naturally occurring serotonin receptor agonists. Several dozen structurally similar ergots are elaborated by the rye rust fungus Claviceps purpurea. Many naturally occurring ergot alkaloids produce intense vasoconstriction by acting as agonists of serotonin receptors

in vascular smooth muscle. This action was responsible for ergotism—described during the Middle Ages as “St. Anthony’s Fire”—in which consumers of fungus-infected grains experienced severe peripheral vasoconstriction leading to necrosis and gangrene. In modern times, a number of ergot alkaloids have been employed clinically. The semisynthetic ergot lysergic acid diethylamide (LSD) produces hallucinations and sensory dysfunction at doses as small as 50 g in humans. 5-HT receptor subtype-selective agonists have become therapeutics of increasing interest in the past decade. These agents are used primarily to treat anxiety and migraine headaches. Buspirone is a nonbenzodiazepine anxiolytic that does not bind to GABA receptors, but rather acts as a 5-HT1A-selective partial agonist. It is nonsedating with moderate anxiolytic properties. Although it is often not as clinically effective as a benzodiazepine, buspirone is nonetheless attractive because it is nonaddictive, does not have abuse potential, and is nonsedating. Migraine headaches are believed to be precipitated by cerebral vasodilation with subsequent activation of small pain fibers. A class of selective serotonin agonists (5-HT1 agonists) has been found to be particularly effective in the treatment of migraine headaches, presumably because of their potent vasoconstrictive effects. Sumatriptan is the prototype 5-HT1D agonist of this group, known collectively as the triptans, which also includes rizatriptan, almotriptan, frovatriptan, eletriptan, and zolmitriptan. The triptans, as well as the less selective ergot alkaloid ergotamine, act on 5-HT1 receptors in the vasculature to alter intracranial blood flow. These agents are most useful for acute migraine attacks when taken at the onset of an episode, rather than as prophylaxis. They must be taken early in a migraine (ideally, at the time of the aura) to effectively block the activation of pain receptors. The triptans are thought to activate both 5-HT1D and 5-HT1B receptors. In the CNS, both receptor subtypes are present on presynaptic endings of a variety of neurons in the vasculature. There are relatively few 5-HT2 agonists used clinically. Trazodone is a prodrug that is converted into metachlorophenylpiperazine (mCPP), a selective 5-HT2A/2C agonist used in the treatment of depression and insomnia. Trazodone is used principally as a somnorific because the higher doses required for antidepressant effects are usually oversedating. The ergot derivative methysergide is a partial agonist of 5-HT2 that also has adrenergic and muscarinic effects; this agent is no longer available in the United States. Serotonin and serotonin receptors are abundant in the GI tract. Serotonin is a critical regulator of GI motility, mediated in large part by 5-HT4 receptors. Cisapride, a 5-HT4 agonist that also enhances acetylcholine release from the myenteric plexus, induces gastric motility. However, cisapride has been withdrawn in the United States due to safety concerns; it can cause QT prolongation and cardiac arrhythmias as a consequence of hERG K channel blockade.

Serotonin Receptor Antagonists Serotonin receptor antagonists are increasingly important therapeutics. Like many receptor ligands, these drugs show varying degrees of receptor subtype selectivity and often cross-react with adrenergic, histamine, and muscarinic

CHAPTER 14 / Pharmacology of Serotonergic and Central Adrenergic Neurotransmission 219

increased lithium reabsorption in the proximal tubule and elevation of plasma lithium concentration to toxic levels. Lithium’s inhibition of K entry into myocytes leads to abnormalities in membrane repolarization, resulting in abnormal T waves seen by ECG. In addition, the transmembrane electrical potential is shifted because inhibition of K entry into cells leads to extracellular hyperkalemia and intracellular hypokalemia. This shift in transmembrane potential exposes patients to a greater risk of sudden cardiac arrest from small changes in potassium balance. Antidiuretic hormone and thyroid-stimulating hormone both activate adenylyl cyclase, which is inhibited by lithium. By this mechanism, lithium treatment can also lead to nephrogenic diabetes insipidus and to hypothyroidism and/or goiter. Given the wide range of adverse effects that may accompany lithium treatment and the euphoria that may be associated with manic or hypomanic episodes, many patients are hesitant to begin treatment. Careful serum monitoring and lithium dose titration can help to avoid some, if not all, of the adverse effects discussed above, although this requires peripheral blood draws on a regular basis. Despite its drawbacks, lithium is the most effective agent for treating bipolar disorder. Lithium and a limited number of other mood-stabilizing drugs (see Drug Summary Table) help to prevent depressive episodes as well as mania, and lithium is the only drug shown in clinical trials to reduce suicide risk in patients with bipolar disorder.

 CONCLUSION AND FUTURE DIRECTIONS This chapter discusses central monoamine neurotransmission—primarily serotonin pathways, but also norepinephrine and, to a lesser extent, dopamine pathways. Serotonin is a critical mediator of mood and anxiety and is also involved in the pathophysiology of migraine headache and IBS. The focus of the chapter is on the antidepressant class of medications. The monoamine theory of depression forms an intellectual framework for conceptualizing the pathophysiology and treatment of MDD, although this theory is clearly an oversimplification. Therapy with drugs that increase synaptic concentrations of 5-HT and NE is effective in many cases of MDD and forms the basis of treatment for this disorder. The delay between initiation of treatment and onset of clinical improvement may occur because of slow changes in presynaptic autoreceptor sensitivity and/or changes in postsynaptic neural circuitry (such as increased neurogenesis). TCAs, SSRIs, MAOIs, and other antidepressants have similar clinical efficacies when tested on groups of patients, although individual patients may respond to one drug and not to another. TCAs nonselectively inhibit 5-HT and NE reuptake transporters (in addition to other receptors); SSRIs selectively block 5-HT reuptake transporters; SNRIs selectively block 5-HT and NE reuptake transporters; and MAOIs

inhibit the degradation of both 5-HT and NE. The choice of antidepressant medication for an individual patient depends on the two goals of finding an effective agent for that patient and minimizing adverse effects. The type of depressive symptoms may suggest one treatment modality over another. SSRIs have become the most commonly prescribed antidepressants because of their favorable therapeutic index and are the first-line choice for treatment of MDD, anxiety, obsessive-compulsive disorder, and post-traumatic stress disorder. BPAD is less well understood than MDD in terms of both its pathophysiology and the mechanisms underlying effective therapies. Agents used to treat BPAD include lithium, antiepileptics, and antipsychotics. Lithium and valproic acid are referred to as mood stabilizers because they limit the extremes of both mania and depression; however, their mechanisms of action are not well understood. Recent advances in drug development for the treatment of MDD have focused on a deeper understanding of the mechanism of action of current drugs and the physiology of their molecular targets. Currently approved antidepressants are administered as racemic mixtures, and the isolation of active stereoisomers, such as S-citalopram, may yield drugs that are better tolerated. Pharmacogenomic approaches have uncovered genetic variants that affect the likelihood of SSRI treatment response. Thus, pharmacogenomics may lead to better matching of drugs to patients by identifying patients who are particularly likely or particularly unlikely to respond to or tolerate a specific drug. Other drug targets beyond the monoamine systems are also promising, including substance P and corticotropin-releasing hormone.

Acknowledgment We thank Mireya Nadal-Vicens, Jay H. Chyung, and Timothy J. Turner for their valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Berger M, Gray J, Roth BL. The expanded biology of serotonin. Annu Rev Med 2009;60:355–366. (Broad review of serotonin’s role in modulating physiologic processes.) Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature 2008;455:894–902. (Current understanding of mood disorders and targets for new antidepressant drugs.) Phiel CJ, Klein PS. Molecular targets of lithium action. Annu Rev Pharmacol Toxicol 2001;41:789–813. (Review of lithium’s likely mechanism(s) of action.) Richelson E. Pharmacology of antidepressants. Mayo Clin Proc 2001; 76:511–527. (Broad and thorough overview of the molecular mechanisms and cellular targets of antidepressant medications.) Tkachev D, Mimmack ML, Ryan MM, et al. Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet 2003;362:798–805. (Research article on BPAD.)

15 Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System Susannah B. Cornes, Edmund A. Griffin, Jr., and Daniel H. Lowenstein

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 225-226 PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Pathophysiology of Focal Seizures . . . . . . . . . . . . . . . . . . 227 Pathophysiology of Secondary Generalized Seizures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Pathophysiology of Primary Generalized Seizures. . . . . . . 229 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 231 Drugs That Enhance Naⴙ Channel-Mediated Inhibition . . . . . . . . . . . . . . . . . . . . . . 231 Phenytoin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Carbamazepine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Lamotrigine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 Lacosamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Drugs That Inhibit Calcium Channels . . . . . . . . . . . . . . . . 233 Ethosuximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Valproic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Gabapentin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Pregabalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Drugs That Enhance GABA-Mediated Inhibition . . . . . . . . 234 Benzodiazepines (Diazepam, Lorazepam, Midazolam, Clonazepam) . . . . . . . . . . . . . . . . . . . . . . 234 Barbiturates (Phenobarbital) . . . . . . . . . . . . . . . . . . . . 234 Vigabatrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Drugs That Inhibit Glutamate Receptors . . . . . . . . . . . . . . 235 Felbamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Rufinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 235 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

 INTRODUCTION

symptoms and loss of consciousness (as seen in tonic–clonic seizures), paroxysmal alterations in nonmotor functions (e.g., sensation, olfaction, vision), or changes in higher-order functions (e.g., emotion, memory, language, insight). This chapter explores the molecular mechanisms by which the brain maintains precise control over the spread of electrical activity and how various abnormalities can undermine these physiologic mechanisms and lead to seizures. The various classes of antiepileptic drugs are then discussed, with an emphasis on molecular mechanisms for restoring inhibitory function in the brain and suppressing seizure activity.

With over 10 billion neurons and an estimated 1014 synaptic connections, the human brain boasts unparalleled electrical complexity. Unlike myocardial tissue, where electrical signals spread synchronously through a syncytium of cells, proper functioning of the brain requires distinct isolation of electrical signals and thus demands a far higher level of regulation. Control of this complex function begins at the level of the ion channel and is further maintained through the effects of these ion channels on the activity of highly organized neuronal networks. Abnormal function of ion channels and neural networks can result in rapid, synchronous, and uncontrolled spread of electrical activity, which is the basis of a seizure. A seizure can present with a variety of symptoms and result from a variety of causes. A single seizure should be distinguished from epilepsy, which refers to the condition in which an individual has a tendency toward recurrent seizures (i.e., a patient who has had a single seizure does not necessarily have epilepsy). Seizure symptoms vary according to the location of seizure activity and may include prominent motor

 PHYSIOLOGY The normal human brain, in the absence of any lesions or genetic abnormalities, is capable of undergoing a seizure. Acute changes in the availability of excitatory neurotransmitters (e.g., caused by ingestion of the toxin domoate, which is a structural analogue of glutamate) or changes in the effect of inhibitory neurotransmitters (e.g., caused by injection of penicillin, a GABAA antagonist) can result in massive seizure activity in the 225

1 Resting state (closed)

2 Activated state (open)

3 Inactivated state (closed)

Na+

+ + +

+ + +

S4 regions

+ + +

Extracellular + + +

+ + +

Intracellular

Na+

Linker region

Membrane potential (mV)

+ + +

Vr

Time (ms)

Activated circuit Inhibitory surround

A C

D B

A Focal seizure Seizure focus

B Secondary generalized seizure Seizure focus

Thalamus

C Primary generalized seizure

Membrane voltage (mV)

Thalamus (seizure focus)

Tonic phase 0 -20 -40 -60 -80

Channel activity

Sodium (AMPA-R) Chloride (GABAA-R) Calcium (NMDA-R) Calcium (gCa)

Clonic phase

Postictal phase Action potentials

1 sec

230 Principles of Central Nervous System Pharmacology

generalized seizure emanates from central brain regions and then spreads rapidly to both hemispheres. These seizures do not necessarily begin with an aura (which is an important method of clinically distinguishing primary generalized seizures from focal seizures that secondarily generalize). Currently, the best understood of the primary generalized seizures is the absence seizure (also known as the petit mal seizure). Absence seizures are characterized by sudden interruptions in consciousness that are often accompanied by a blank stare and occasional motor symptoms, such as rapid blinking and lip smacking. Absence seizures are thought to result from abnormal synchronization of thalamocortical and cortical cells. The underlying pathophysiology of absence seizures is based on the observation that patients experiencing absence seizures have EEG readings somewhat similar to the patterns generated during slow-wave (stage 3) sleep. Relay neurons connecting the thalamus to the cortex exist in two different states depending on the level of wakefulness (Fig. 15-5A). During the awake state, these neurons function in transmission mode, whereby incoming sensory signals

are faithfully transmitted to the cortex. During sleep, however, the transient, bursting activity of a unique, dendritic T-type calcium channel alters incoming signals so that output signals to the cortex have an oscillatory firing rate, which, on an EEG, has a characteristic “spike and wave” readout. In this slow-wave sleep state, sensory information is not transmitted to the cortex. For reasons not yet understood, absence seizures are associated with activation of the T-type calcium channel during the awake state (Fig. 15-5B). Because this channel is active only when the cell is hyperpolarized, several factors can activate the channel during the awake state. These factors include an increase in intracellular K⫹, an increase in GABAergic input from the reticular nucleus, or a loss of excitatory input. A variety of studies have shown that the activity of the T-type calcium channel in the relay neurons is essential to the 3-per-second spike-and-wave activity observed in absence seizures. Because of its important pathophysiologic role, the T-type calcium channel is a primary target in the pharmacologic treatment of absence seizures.

A

1. Awake

2. Slow-wave sleep

3. Typical absence seizure (EEG)

EEG

Bursts 50 mV

50 mV

Single spikes

Thalamic firing 100 ms

100 ms

T-type Ca2+ channel activity

Voltage-gated Na+ channel activity

Cerebral cortex

B

FIGURE 15-5.

2

1

3

Thalamus

Mechanism of absence seizure. A. EEG recordings of patients experiencing absence seizures are similar to “sleep spindle” patterns generated during slowwave sleep. The 3-per-second oscillatory pattern is generated by the burst activity of a dendritic T-type calcium channel in the thalamus. 1. During the awake state, relay neurons of the thalamus are in “transmission mode,” in which incoming signals are faithfully transmitted to the cortex as single spikes. These signals to the cortex register on the EEG as small, desynchronized, low-voltage waves. 2. During slow-wave sleep, signals relayed through the thalamus are altered because of the bursting activity of a dendritic T-type calcium channel (see below). During this stage, called “burst mode,” sensory information is not transmitted to the cortex. 3. Absence seizures result from abnormal activation of the T-type calcium channel during the awake state, resulting in a similar spike-and-wave EEG pattern. B. The absence seizure is generated by a self-sustaining cycle of activity between the thalamus and the cortex. Synchronicity is initiated by hyperpolarization of the thalamic relay neurons (white). This occurs normally during slow-wave sleep and is caused by GABAergic input from the reticular thalamic nucleus (purple). The factors that cause hyperpolarization in relay neurons during an absence seizure are poorly understood. 1. Hyperpolarization of relay neurons induces burst activity of the T-type calcium channel, resulting in synchronous depolarization in the cortex via excitatory connections. This large depolarization in the cortex registers as a spike-and-wave pattern on the EEG. 2. Excitatory input from the cortex (light yellow) activates the reticular thalamic neurons (dark yellow). 3. The activated GABAergic reticular neurons hyperpolarize the thalamic relay neurons and reinitiate the cycle.

CHAPTER 15 / Abnormal Electrical Neurotransmission in the Central Nervous System 231

 PHARMACOLOGIC CLASSES AND AGENTS

a slow firing rate, such that Na⫹ channel blockers do not have a use-dependent effect on the Na⫹ channels in these cells.

The current approach to treating a patient with epilepsy depends in part on the type of seizure(s) experienced by the patient. An appropriate antiepileptic drug regimen will take into account whether a patient is having focal seizures, with or without secondary generalization, or primary generalized seizures. In addition, for patients with focal seizures, there is also an attempt to determine whether the seizures are caused by an identifiable focal lesion that can be removed surgically or ablated by other means. Mechanistically, the efficacy of antiepileptic drugs (AEDs) centers on manipulation of ion channel activity. As discussed above, physiologic protection against repetitive firing occurs via inhibition at two levels: the cellular level (e.g., Na⫹ channel inactivation), and the network level (e.g., GABAmediated inhibition). Accordingly, currently available AEDs fall into four main categories: (1) drugs that enhance Na⫹ channel-mediated inhibition, (2) drugs that inhibit calcium channels, (3) drugs that enhance GABA-mediated inhibition, and (4) drugs that inhibit glutamate receptors. Although AEDs fall into several different mechanistic classes, it is important to keep in mind that the therapeutic efficacy of many of the AEDs is only partially explained by the known mechanisms described below, primarily because the AEDs act pleiotropically. Valproic acid, for example, stabilizes Na⫹ channels, but the drug also has an effect on T-type calcium channels and may have effects on GABA metabolism as well. Thus, although in vitro studies may suggest that a drug is best suited for the treatment of one particular type of seizure, other seizure types may also respond to the drug. (One benefit of this pleiotropy is that many of the drugs are interchangeable, to the extent that minimization of adverse effects is often the main clinical criterion underlying the choice of AED.) The classification below is shown only for simplicity and is based on the primary target of the drug. A list of the main drugs discussed here and their multiple mechanisms of action is provided in Table 15-2.

Phenytoin Phenytoin acts directly on the Na⫹ channel to slow the rate of channel recovery from the inactivated state to the closed state. As described above, the Na⫹ channel exists in three conformations—closed, open, and inactivated—and the probability of a channel existing in each state depends on the membrane potential (Fig. 15-1; see also Fig. 11-7). By slowing the rate of recovery from the inactivated state to the closed state, phenytoin increases the threshold for action potentials and prevents repetitive firing. This has the effect of stabilizing the seizure focus by preventing the paroxysmal depolarizing shift (PDS) that initiates the focal seizure. In addition, phenytoin prevents the rapid spread of seizure activity to other neurons, accounting for its efficacy in secondarily generalized seizures. Importantly, phenytoin targets Na⫹ channels in a usedependent manner (see Fig. 11-8). Thus, only channels that are opened and closed at high frequency (i.e., those involved in the PDS) are likely to be inhibited. This use-dependency lessens the effects of phenytoin on spontaneous neuronal activity and avoids many of the adverse effects observed with GABAA potentiators (which are not use-dependent). Because of its use-dependent blockade, as well as its ability to prevent sudden rapid firing, phenytoin is a major drug of choice for focal seizures and tonic–clonic seizures. It is not used in absence seizures. The complex pharmacokinetics and drug interactions of phenytoin play a decisive role in the choice between phenytoin and similarly acting drugs such as carbamazepine. Phenytoin is more than 95% bound to plasma albumin. Phenytoin is inactivated by metabolism in the liver and, at typical doses, has a plasma half-life of about 24 hours. Phenytoin metabolism shows properties of saturation kinetics, whereby small increases in dose can cause large and often unpredictable increases in plasma drug concentration (see Chapter 3, Pharmacokinetics). These increases in plasma phenytoin concentration increase the risk of adverse effects, including ataxia, nystagmus, incoordination, confusion, gingival hyperplasia, megaloblastic anemia, hirsutism, facial coarsening, and a systemic skin rash. Phenytoin inactivation by the hepatic microsomal P450 enzyme system is susceptible to alteration by several drugs. Drugs that inhibit the P450 system, such as chloramphenicol, cimetidine, disulfiram, and isoniazid, increase phenytoin plasma concentration. Carbamazepine, an antiepileptic drug that induces the hepatic P450 system, increases the metabolism of phenytoin, thereby lowering phenytoin plasma concentration when these drugs are used concurrently. Similarly, phenytoin, because of its ability to induce the hepatic P450 system, increases the metabolism of drugs that are inactivated by this system. Some of these drugs include oral contraceptives, quinidine, doxycycline, cyclosporine, methadone, and levodopa.

Drugs That Enhance Na⫹ Channel-Mediated Inhibition Each neuron in the brain is equipped with the machinery to prevent rapid, repetitive firing. As discussed above, depolarization of the neuronal membrane results in sodium channel inactivation. This inactivation of the Na⫹ channel provides a key checkpoint in the prevention of repetitive firing within a potential seizure focus. The AEDs phenytoin, carbamazepine, lamotrigine, lacosamide, and valproic acid act directly on the Na⫹ channel (Fig. 15-6A) to increase channel inactivation, thereby enhancing inhibition at the single-cell level. In general, antiepileptic drugs that act on Na⫹ channels show strong specificity for the treatment of focal and secondary generalized seizures. This is consistent with their molecular profile. The Na⫹ channel blockers act in a use-dependent manner, much like the action of lidocaine on peripheral nerves (see Chapter 11, Local Anesthetic Pharmacology). Thus, neurons that fire rapidly are particularly susceptible to inhibition by this class of drug. Conversely, many Na⫹ channel blockers (particularly those that act only at the Na⫹ channel, such as phenytoin) have little effect on absence seizures. Presumably, the thalamocortical cells activated during an absence seizure have

Carbamazepine Although structurally unrelated to phenytoin, carbamazepine appears to exert its antiseizure activity in a manner similar to phenytoin. That is, carbamazepine is a Na⫹ channel blocker that slows the rate of recovery of Na⫹ channels from the inactivated state to the closed state. This has the effect of suppressing a seizure focus (by preventing the PDS) as well as preventing rapid spread of activity from the seizure focus. A metabolite of carbamazepine, 10,11-epoxycarbamazepine,

CHAPTER 15 / Abnormal Electrical Neurotransmission in the Central Nervous System 233

A Seizure focus

Drug treatment

Action potentials (transmission inhibited)

1 3

Felbamate NMDA-R (closed)

Barbiturate or benzodiazepine

+ + +

2

Cl-

+ + +

Loss of inhibitory surround

GABAA channel (open)

Gabapentin

Phenytoin, carbamazepine, or lamotrigine

Voltage-gated Na+ channel (inactivated)

HVA Ca2+ channel (closed)

Cl-

B

Benzodiazepine (clonazepam)

GABAA channel (open)

Ethosuximide or valproic acid

1

T-type Ca2+ channel (blocked)

Thalamus (seizure focus)

2

3

FIGURE 15-6.

Mechanisms of pharmacotherapy for seizures. A. The focal seizure (1) results from rapid, uncontrolled neuronal firing and a loss of surround inhibition (2). Antiepileptic drugs act at four molecular targets to enhance inhibition and prevent spread of synchronous activity (3). Barbiturates and benzodiazepines prevent seizure spread by acting on the GABAA receptor to potentiate GABA-mediated inhibition. Na⫹ channel inhibitors such as phenytoin, carbamazepine, and lamotrigine prevent rapid neuronal firing by selectively prolonging Na⫹ channel inactivation in rapidly firing neurons (see Figs. 11-7 and 11-8). Felbamate suppresses seizure activity by inhibiting the NMDA receptor and thereby decreasing glutamate-mediated excitation. Gabapentin decreases release of excitatory neurotransmitter by inhibiting the high-voltageactivated (HVA) calcium channel. B. The absence seizure (1) is caused by a self-sustaining cycle of activity generated between thalamic and cortical cells (2). Antiepileptic drugs prevent this synchronous thalamocortical cycle (3) by acting at two molecular targets. Clonazepam, a benzodiazepine, potentiates GABAA channels in the reticular thalamic nucleus, thus decreasing the activation of the inhibitory reticular neurons and decreasing the hyperpolarization of the thalamic relay neurons. T-type calcium channel inhibitors such as ethosuximide and valproic acid prevent the burst activity of thalamic relay neurons that is required for synchronous activation of cortical cells.

lacosamide over other effective sodium-modulating agents, and early data suggest that the therapeutic window may be limited by dose-dependent adverse effects. At the least, lacosamide provides another medication option for patients with drug-resistant epilepsy.

Drugs That Inhibit Calcium Channels Drugs used to treat epilepsy through inhibition of calcium channels fall into two main classes: those that inhibit the Ttype calcium channel and those that inhibit the high-voltageactivated (HVA) calcium channel. The T-type calcium channel is depolarized and inactive during the awake state (Fig. 15-5B). In absence (petit mal)

seizures, paroxysmal hyperpolarization is thought to activate the channel during the awake state, initiating the spike and wave discharges characteristic of this seizure type. Thus, drugs inhibiting the T-type calcium channel are specifically used to treat absence seizures. HVA calcium channels play an important role in controlling the entry of calcium into the presynaptic terminal and therefore help to regulate neurotransmitter release. The HVA calcium channel is formed by an ␣1 protein that assembles into the channel pore, and it has several auxiliary subunits. Drugs that inhibit HVA calcium channels tend to have pleiotropic effects; although they are used primarily for focal seizures with or without secondary generalization, they can also be used for generalized seizures other than absence seizures.

234 Principles of Central Nervous System Pharmacology

Ethosuximide In vitro, ethosuximide has a highly specific molecular profile. In experiments on thalamocortical preparations from rats and hamsters, ethosuximide has been shown to reduce lowthreshold T-type currents in a voltage-dependent manner. This inhibition occurs without altering the voltage dependence or recovery kinetics of the Na⫹ channel. Ethosuximide does not have any effect on GABA-mediated inhibition. Ethosuximide is often the first-line therapy for uncomplicated absence seizures. Consistent with its molecular profile as a specific T-type Ca2⫹ channel blocker, ethosuximide is not effective in the treatment of focal or secondary generalized seizures. Valproic Acid As is the case for many other AEDs, valproic acid acts pleiotropically in vitro. Similar to phenytoin and carbamazepine, valproic acid slows the rate of Na⫹ channel recovery from the inactivated state. At slightly higher concentrations than those necessary to limit repetitive firing, valproic acid has also been shown to limit the activity of the low-threshold T-type calcium channel. A third proposed mechanism of valproic acid action occurs at the level of GABA metabolism. In vitro, valproic acid increases the activity of glutamic acid decarboxylase, the enzyme responsible for GABA synthesis, while it inhibits the activity of enzymes that degrade GABA. Taken together, these effects are thought to increase the availability of GABA in the synapse and thereby to increase GABA-mediated inhibition. Perhaps because of its many potential sites of action, valproic acid is one of the most effective antiepileptic drugs for the treatment of patients with generalized epilepsy syndromes having mixed seizure types. Valproic acid is also the drug of choice for patients with idiopathic generalized seizures and is used for the treatment of absence seizures that do not respond to ethosuximide. Valproic acid is also commonly used as an alternative to phenytoin and carbamazepine for the treatment of focal seizures. Gabapentin Gabapentin was one of the first AEDs developed using the concept of “rational drug design.” That is, with the recognition that GABA receptors play an important role in the control of seizure spread, gabapentin was synthesized as a structural analogue of GABA and was predicted to enhance GABA-mediated inhibition. Consistent with this hypothesis, gabapentin has been shown to increase the content of GABA in neurons and glial cells in vitro. However, the main antiseizure effect of gabapentin appears to be through its inhibition of HVA calcium channels, which results in decreased neurotransmitter release. A main advantage of gabapentin is that, because its structure is similar to that of endogenous amino acids, it has few interactions with other drugs. On the other hand, gabapentin does not appear to be as effective as the other AEDs, and it is not generally used as a first-line agent. Pregabalin Like gabapentin, pregabalin is structurally related to GABA, but it exerts its main therapeutic effect through inhibition of HVA calcium channels, reducing the release of several neurotransmitters including glutamate and norepinephrine. It also has effects on substance P and calcitonin, which may contribute to its varied clinical uses. More potent than gabapentin, pregabalin is a reasonable adjunctive treatment for

focal seizures. It is particularly useful in patients with hepatic dysfunction, since it is metabolized in the kidney and has few drug–drug interactions.

Drugs That Enhance GABA-Mediated Inhibition In contrast to Na⫹ channel blockers and calcium channel inhibitors, whose mechanistic properties correlate well with their clinical activity, the enhancers of GABA-mediated inhibition have more varied effects and tend not to be as interchangeable. This is largely because of the diversity of GABAA receptors in the brain. The GABAA receptor channel has five subunits, with at least two alternative splice variants of several of the subunits (see Chapter 12). There are at least 10 known subtypes of the GABAA receptor, with varying distributions of these subtypes throughout the brain. Barbiturates and benzodiazepines both increase Cl⫺ influx through GABAA channels, but benzodiazepines act on a specific subset of GABAA channels, whereas barbiturates appear to act on all GABAA channels. The recently approved medication vigabatrin enhances GABA-mediated activity indirectly via inhibition of GABA metabolism. These different mechanisms of action result in distinct clinical profiles. Drugs that nonspecifically increase GABA content (e.g., through enhancement of synthetic pathways or reduced metabolism of GABA) tend to have a profile similar to the barbiturates. Benzodiazepines (Diazepam, Lorazepam, Midazolam, Clonazepam) Benzodiazepines increase the affinity of GABA for the GABAA receptor and enhance GABAA channel gating in the presence of GABA, and thereby increase Cl⫺ influx through the channel (see Chapter 12). This action has the dual effect of suppressing the seizure focus (by raising the threshold of the action potential) and strengthening surround inhibition. Thus, benzodiazepines such as diazepam, lorazepam, and midazolam are well suited for the treatment of focal and tonic–clonic seizures. The benzodiazepines cause prominent adverse effects, however, including dizziness, ataxia, and drowsiness. Thus, these drugs are typically used only to ablate seizures acutely. Clonazepam is unique among the benzodiazepines in its ability to inhibit T-type Ca2⫹ channel currents in in vitro preparations of thalamocortical circuits. In vivo, clonazepam acts specifically at GABAA receptors in the reticular nucleus (Fig. 15-5B), augmenting inhibition in these neurons and essentially “turning off” the nucleus. By this action, clonazepam prevents GABA-mediated hyperpolarization of the thalamus and thereby indirectly inactivates the T-type Ca2⫹ channel, which is the channel thought to be responsible for generating absence seizures (see above). However, as with diazepam, clonazepam use is limited because of its extensive adverse effects. Clonazepam is the fourth drug of choice in the treatment of absence seizures, after ethosuximide, valproic acid, and lamotrigine. Barbiturates (Phenobarbital) Phenobarbital binds to an allosteric site on the GABAA receptor and thereby potentiates the action of endogenous GABA by increasing the duration of Cl⫺ channel opening. In the presence of phenobarbital, there is a much greater influx of Cl⫺ ions for each activation of the channel (see Chapter 12). Barbiturates also display weak agonist activity at the GABAA channel, perhaps furthering the ability of this drug to increase Cl⫺ influx. This enhancement of GABAmediated inhibition, similar to that of the benzodiazepines,

CHAPTER 15 / Abnormal Electrical Neurotransmission in the Central Nervous System 235

may explain the effectiveness of phenobarbital in the treatment of focal seizures and tonic–clonic seizures. In contrast to the benzodiazepines, which are sometimes useful in treating the spike-and-wave discharges of the absence seizure, the barbiturates may actually exacerbate this type of seizure. This exacerbation may be caused by two factors. First, barbiturates act at all GABAA receptors. Unlike benzodiazepines, which selectively augment GABA inhibition in the reticular nucleus, barbiturates potentiate GABAA receptors in both the reticular nucleus and the thalamic relay cells. Importantly, the latter effect enhances the T-type calcium currents that are responsible for the absence seizure (Fig. 15-5B). Second, unlike benzodiazepines, which are purely allosteric enhancers of endogenous GABA activity, barbiturates can also act on the GABAA channel in the absence of the native ligand. The latter property may function to increase nonspecific activity of the barbiturates. Phenobarbital is used primarily as an alternative drug in the treatment of focal seizures and tonic–clonic seizures. Because of the pronounced sedative effects of this drug, its clinical use has been decreasing as more effective antiepileptic medications have become available. Vigabatrin Vigabatrin is a structural analogue of GABA that irreversibly inhibits the enzyme GABA transaminase, thereby increasing levels of GABA in the brain (see Fig. 12-2). Serious adverse effects, most notably peripheral visual field defects, limit the clinical utility of vigabatrin. The drug is generally used for infantile spasms and refractory focal epilepsy, and any patient treated with vigabatrin should undergo baseline and routine visual field testing.

Drugs That Inhibit Glutamate Receptors Glutamate is the principal excitatory neurotransmitter of the CNS (see Chapter 12). Not surprisingly, excessive activation of excitatory glutamatergic synapses is a key component of many forms of seizure activity. Numerous studies using animal models have shown that inhibition of the NMDA and AMPA subtypes of glutamate receptors can inhibit the generation of seizure activity and protect neurons from seizure-induced injury. However, none of the specific and potent glutamate receptor antagonists have been routinely used clinically for the treatment of seizures because of unacceptable behavioral adverse effects. Felbamate Felbamate has a variety of actions, including the inhibition of NMDA receptors. It appears to have some selectivity for NMDA receptors that include the NR2B subunit. Because this receptor subunit is not expressed ubiquitously throughout the brain, NMDA receptor antagonism by felbamate is not as widespread as that with other NMDA antagonists. This relative selectivity may explain why felbamate lacks the behavioral adverse effects observed with the other agents. Benefits of felbamate include its potency as an antiepileptic drug and its lack of the sedative effects common to many other antiepileptic drugs. However, felbamate has been associated with a number of cases of fatal aplastic anemia and liver failure, and its use is now restricted primarily to patients with refractory epilepsy.

Rufinamide Rufinamide is a recently approved drug for the treatment of focal seizures and drop attacks in Lennox-Gastaut syndrome (a syndrome characterized by childhood onset of frequent and sometimes refractory seizures). While rufinamide acts predominantly by prolonging sodium channel inactivation, it is structurally unrelated to the other antiepileptic agents with this mechanism of action. At higher doses, it may have an inhibitory effect on a subset of glutamate receptors (mGluR5 subtype), and it is included here based on that secondary mechanism and because its clinical profile is most similar to felbamate. Unlike felbamate, however, rufinamide has not been demonstrated to have any serious adverse effects, and it may provide an alternative option for patients with refractory epilepsy.

 CONCLUSION AND FUTURE DIRECTIONS In recent years, improved understanding of the physiology and pathophysiology of neuronal signaling in the CNS has led to a more thorough understanding of the current antiepileptic drugs (AEDs), as well as the design and discovery of novel agents. Under physiologic conditions, Na⫹ channel inactivation and GABA-mediated surround inhibition prevent uncontrolled, rapid spread of electrical activity. There are, however, numerous potential alterations in the brain that can weaken these inhibitory forces, such as damage and degeneration of GABAergic neurons, abnormal ion gradients induced by space-occupying lesions, and gene mutations that alter channel function. The AEDs described in this chapter restore the inherent inhibitory capacity of the brain. These include drugs such as phenytoin, which increases Na⫹ channel inactivation, and clonazepam, which enhances GABA-mediated inhibition. Newer classes of AEDs extend this repertoire by acting through modulation of the Ca2⫹ channel required for neurotransmitter release and modulation of excitatory receptors such as the NMDA receptor. Despite increased understanding of the mechanisms of certain seizure types, the efficacy of many of the antiepileptics is only partially explained by their known molecular profiles. Hence, current decisions about therapy are often driven by empirical example rather than by known molecular mechanisms. As a better understanding is gained of the role of genetics in not only simple inherited epilepsy but in complicated polygenic cases, the application of a more rational, mechanism-based pharmacology will become increasingly possible.

Suggested Reading Lowenstein DH. Seizures and epilepsy. In: Harrison’s principles of internal medicine. 17th ed. New York: McGraw Hill; 2008. (Discussion of seizure pathophysiology and extensive discussion of clinical uses of antiepileptic drugs.) Shorvon S. Drug treatment of epilepsy in the century of the ILAE: the second 50 years, 1959–2009. Epilepsia 2009;50(Suppl 3):93–130. (An historical perspective cataloging the introduction of each therapeutic agent over time.) Westbrook GL. Seizures and epilepsy. In: Kandel ER, Schwartz JH, Jessell TM, eds. Principles of neural science. 4th ed. New York: McGrawHill; 2000. (Detailed description of normal electrical signaling and seizure pathophysiology.)

16 General Anesthetic Pharmacology Jacob Wouden and Keith W. Miller

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 240-241 PHARMACODYNAMICS OF INHALED ANESTHETICS . . . . . . 240 The Minimum Alveolar Concentration (MAC). . . . . . . . . . . 241 Therapeutic and Analgesic Indices . . . . . . . . . . . . . . . . . . 241 The Meyer–Overton Rule . . . . . . . . . . . . . . . . . . . . . . . . . 242 PHARMACOKINETICS OF INHALED ANESTHETICS . . . . . . . 244 Concepts from Respiratory Physiology . . . . . . . . . . . . . . . 245 Local Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Global Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 The Uptake Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Equilibration of Alveolar with Inspired Partial Pressure . . . . . . . . . . . . . . . . . . . . . . . 246 Equilibration of Tissue with Alveolar Partial Pressure . . . . . . . . . . . . . . . . . . . . . . . 247 The Rate-Limiting Step . . . . . . . . . . . . . . . . . . . . . . . . 249 Applications of the Uptake Model . . . . . . . . . . . . . . . . . . . 250 Effects of Changes in Ventilation . . . . . . . . . . . . . . . . . 251 Effects of Changes in Cardiac Output . . . . . . . . . . . . . 251 Effects of Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Effects of Abnormal States . . . . . . . . . . . . . . . . . . . . . 252 Control of Induction. . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 PHARMACOLOGY OF GENERAL ANESTHETICS AND ADJUVANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Inhaled Anesthetic Agents . . . . . . . . . . . . . . . . . . . . . . . . 254 Intravenous Anesthetic Agents . . . . . . . . . . . . . . . . . . . . . 255 Adjuvant Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Balanced Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 MECHANISM OF ACTION OF GENERAL ANESTHETICS . . . . 256 The Meyer–Overton Rule and the Lipid Solubility Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Effects on Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . 257 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 258 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 APPENDIX B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

 INTRODUCTION

muscle relaxation, loss of autonomic reflexes, analgesia, and anxiolysis. All of these effects facilitate safe and painless completion of the procedure; some effects are more important in certain types of surgery than others. For example, abdominal surgery necessitates near complete relaxation of the abdominal muscles, whereas neurosurgery often requires light anesthesia that may be lifted rapidly when the neurosurgeon needs to judge the patient’s ability to respond to commands. This chapter provides a framework for understanding the pharmacodynamics and pharmacokinetics of general anesthetics in the context of physiologic and pathophysiologic variables. After discussing the pharmacology of specific agents and how a balanced anesthetic approach is achieved, the chapter considers what is currently known about the mechanism of action of general anesthetics.

Before the discovery of general anesthetics, pain and shock severely limited the possibilities for surgical intervention. Postoperative mortality dropped dramatically following the first public demonstration of diethyl ether at Massachusetts General Hospital in 1846. Since then, the administration of agents for the induction and maintenance of anesthesia has become a separate medical specialty. The modern anesthesiologist is responsible for all aspects of patient health during surgery. As part of this process, the anesthesiologist controls the depth of anesthesia and maintains homeostatic equilibrium with an arsenal of inhaled and intravenous anesthetics as well as many adjuvant drugs. General anesthetics induce a generalized, reversible depression of the central nervous system (CNS). Under general anesthesia, there is a lack of perception of all sensations. The anesthetic state includes loss of consciousness, amnesia, and immobility (a lack of response to noxious stimuli), but not necessarily complete analgesia. Other desirable effects provided by anesthetics or adjuvants during surgery may include 240

 PHARMACODYNAMICS OF INHALED ANESTHETICS General anesthetics distribute well to all parts of the body, becoming most concentrated in the fatty tissues. The CNS

Awake

Awake

Analgesia (depends on agent) Amnesia Euphoria Stage II: Excitement Excitement Delirium Combative behavior

Recovery from anesthesia

Deepening anesthesia

Stage I: Analgesia

Stage III: Surgical Anesthesia

Commence surgery

Unconsciousness Regular respiration Decreasing eye movement Stage IV: Medullary Depression Respiratory arrest Cardiac depression and arrest No eye movement

Surgery completed

MAC

Percentage of patients exhibiting each endpoint

100 Non-responsive to trapezius squeeze

80

LP 50

Non-responsive to skin incision

Non-responsive to intubation

60

40

Cardiac arrest (death)

20

0 0.01

0.02

0.03

0.04

Alveolar partial pressure of isoflurane (atm)

0.05

MAC 艐 1.3/(oil/gas)

Equation 16-1

CHAPTER 16 / General Anesthetic P harmacology 245

10,000 Thiomethoxyflurane

Potency (1/atm)

1,000

Methoxyflurane Halothane Isoflurane Diethylether Enflurane

100

Cyclopropane

10

Xenon

1

Nitrous oxide

0.1 Nitrogen 0.01 0.01

0.1

1

10

100

1,000 10,000

Oil/Gas partition coefficient

FIGURE 16-3. The Meyer–Overton rule. Molecules with a larger oil/gas partition coefficient [(oil/gas)] are more potent general anesthetics. This log–log plot shows the very tight correlation between lipid solubility [(oil/gas)] and anesthetic potency over five orders of magnitude. Note that even such gases as xenon and nitrogen can act as general anesthetics when breathed at high enough partial pressures. The equation describing the line is: Potency  (oil/gas) / 1.3. Recall that Potency  1/MAC.

As discussed below, the uptake model depends on calculations of the time required for the equilibration of anesthetic partial pressures in the tissues with the inspired anesthetic partial pressure.

Concepts from Respiratory Physiology Local Equilibration During general anesthesia, the patient breathes, either spontaneously or via a ventilator, an anesthetic or mixture of anesthetics together with oxygen and/or normal air. Once the anesthetic gas reaches the alveoli, it must diffuse across the respiratory epithelium into the alveolar capillary bed. According to Fick’s law, the rate of diffusion of gas through a sheet of tissue down its partial pressure gradient is proportional to the tissue area and the partial pressure difference between the two sides, and is inversely proportional to the thickness of the sheet: Diffusion rate  D  (A/ l)  P

Equation 16-2

where D  diffusion constant; A  surface area; l  thickness; and P  partial pressure difference. One principle evident from Fick’s law is that the equalization of the partial pressure of the gas, not its concentration, defines the approach to equilibrium across a boundary sheet. Thus, at equilibrium (i.e., when the net diffusion rate is zero), the partial pressure in the two compartments is the same, even though the concentration in the two compartments may be different. With its enormous alveolar surface area (⬃75 m2, or nearly half a tennis court) and thin epithelium (⬃0.3 m, which is less than 1/20th the diameter of a red blood cell),

the lung optimizes the rate of gas diffusion. Accordingly, the alveolar partial pressure Palv and the systemic arterial partial pressure Part are nearly the same at all times. (In normal individuals, small amounts of physiologic shunting keep Part slightly lower than Palv.) By using the lungs as an uptake system for inhaled anesthetics, anesthesiologists take advantage of the body’s system for absorbing oxygen. Similarly, the capillary beds in tissues have evolved to deliver oxygen rapidly to all cells in the body. The distances between arterioles are small, and diffusion pathways are on the order of one cell diameter. Consequently, the arterial partial pressure of a general anesthetic can equilibrate completely with tissues in the time required for blood to traverse the capillary bed. Likewise, the partial pressure in the postcapillary venules Pvenule equals the partial pressure in the tissue Ptissue. Another way of stating the above conclusion is that the transfer of anesthetic in both the lungs and the tissues is limited by perfusion rather than diffusion. Because perfusion is rate-limiting, increasing the rate of diffusion (e.g., by using a lower molecular weight anesthetic) will not, by itself, increase the rate of induction of anesthesia. Global Equilibration If an anesthetic is inspired for a long enough period of time, all compartments in the body will equilibrate to the same partial pressure (equal to PI). This global equilibration may be divided into a series of partial pressure equilibrations between each successive compartment and its incoming flow of anesthetic. In the case of the tissues, the incoming flow is the arterial blood flow, with partial pressure approximately equal to Palv. In the case of the alveoli, the incoming flow is the alveolar ventilation with partial pressure PI. The time constant  describes the rate of approach of a compartment’s partial pressure to that of its incoming flow. Specifically,  is the time required for equilibration to be 63% complete. This time constant is convenient because it can be calculated by dividing the compartment’s volume capacity (relative to the delivering medium; see below) by the flow rate. In other words, once a volume of flow equal to the capacity of a compartment has gone through that compartment, the partial pressure of anesthetic in the compartment (i.e., in the tissues or alveoli) will be 63% of the partial pressure in the incoming flow (i.e., in the arterial blood flow or alveolar ventilation, respectively). Equilibration is 95% complete after three time constants.   Volume Capacity/Flow Rate

Equation 16-3

Pcompartment  Pflow [ 1  e(t/)]

Equation 16-4

where t  elapsed time. These equations describe what should make intuitive sense: equilibration of the partial pressure of the compartment with the incoming flow takes place more quickly (i.e., the time constant is smaller) when the inflow is larger or the compartment capacity is smaller.

The Uptake Model For simplicity, the model of anesthetic uptake and distribution organizes the tissues of the body into groups based on similar characteristics. Each group can be modeled as a container with a particular capacity for anesthetic and a particular level

246 Principles of Central Nervous System Pharmacology PI

Palv Part

PMVR

Tissue group

% Cardiac output

% Body weight

Vol. cap. for N2O at Palv = 0.8atm

Vol. cap. for halo. at Palv = 0.8atm

VRG: brain, liver, kidneys

VRG

75%

9%

2.6 L

0.30 L

MG: muscle, skin

MG

18%

50%

16 L

3.0 L

FG: fat

FG

5.5%

19%

12 L

17 L

VPG

1.5%

22%

7.0 L

1.3 L

PVRG

PMG

PFG

VPG: bone, cartilage, ligaments PVPG

FIGURE 16-4. Distribution of cardiac output and volume capacity for general anesthetics among the major tissue compartments. The tissues of the body can be divided into four groups based on their level of perfusion and their capacity to take up anesthetic. These include the vessel-rich group (VRG), muscle group (MG), fat group (FG), and vessel-poor group (VPG). (The contribution of the VPG is generally ignored in most pharmacokinetic models of anesthesia.) The VRG, which contains the internal organs including the brain, constitutes a small percentage of the total body weight (9%), has the lowest capacity for anesthetic, and receives most of the cardiac output (75%). The high perfusion and low capacity allow PVRG to equilibrate rapidly with Part. Also, the VRG makes the largest contribution to the mixed venous return partial pressure PMVR, which is equal to (0.75 PVRG  0.18 PMG  0.055 PFG  0.015 PVPG). N2O, nitrous oxide; Halo., halothane; Vol. cap., volume capacity.

of blood flow delivering anesthetic. An adequate approximation groups the tissues into three main compartments that are perfused in parallel (Fig. 16-4). The vessel-rich group (VRG), which consists of the CNS and visceral organs, has a low capacity and high flow. The muscle group (MG), which consists of muscle and skin, has a high capacity and moderate flow. The fat group (FG) has a very high capacity and low flow. (A fourth group, the vessel-poor group [VPG], which consists of bone, cartilage, and ligaments, has a negligible flow and capacity, and its omission does not significantly affect the model.) The rate of increase of the partial pressure in the VRG (PVRG) is of the greatest interest because the VRG includes the CNS. The overall equilibration of PVRG with the inspired partial pressure occurs in two steps, either of which may be rate-limiting. First, the alveolar and inspired partial pressures equilibrate (Palv approaches PI, or Palv → PI). Second, PVRG

(and specifically PCNS) equilibrates with the arterial partial pressure (which is essentially equal to the alveolar partial pressure) (PVRG → Part). The discussion will now consider the time constant for each of these two steps and define conditions under which one or the other is rate-limiting. Equilibration of Alveolar with Inspired Partial Pressure The equilibration of Palv with PI is conceptually the first step of the equilibration of PVRG with PI. During induction of anesthesia, PVRG can never be higher than Palv; if Palv rises slowly, then PVRG must also rise slowly. To calculate the time constant for the approach of Palv to PI, {Palv→PI}, the flow rate and volume capacity must be defined. The delivering medium is free gas arriving through the airways, and the compartment is the lung and alveoli. The volume capacity is simply the volume of gas that remains in the lungs after

CHAPTER 16 / General Anesthetic P harmacology 247

normal exhalation, or the functional residual capacity (FRC, typically ⬃3 L for an average adult). Assume initially that the only component of the flow rate is the rate of alveolar ventilation, which delivers the anesthetic (Valv  {Tidal Volume Dead Space}  Respiratory Rate; for an average adult, Valv  {0.5 L 0.125 L}  16 min 1 ⬇ 6 L/min). Then, because {P alv Æ PI }  FRC/Valv

Equation 16-5

a typical value for {Palv→PI} is 3 L / 6 L/min, or 0.5 min— independent of the particular gas being inhaled. In children, the increased alveolar ventilation rate and decreased FRC (smaller lungs) both tend to shorten the time constant and to accelerate equilibration between the alveolar and inspired partial pressures. The assumption to this point has been that no uptake of anesthetic into the bloodstream occurs, as would be the case if the solubility of the anesthetic in blood were zero. In practice, at the same time that alveolar ventilation is delivering anesthetic to the alveoli, anesthetic is also being removed from the alveoli by diffusion into the bloodstream. The balance between delivery and removal is analogous to adding water into a leaky bucket (Fig. 16-5). The level of water in the bucket (which represents the alveolar partial pressure) is determined both by the rate at which the water is added (the minute ventilation) and the size of the leak (the rate of anesthetic uptake from the alveoli into the bloodstream). Increasing anesthetic delivery (for example, by using a higher ventilation rate or a higher inspired partial pressure) will increase the alveolar partial pressure of the gas, just as adding water faster will increase the level of water in the

bucket. Conversely, increasing anesthetic removal (for example, by increasing the perfusion rate or using a more blood-soluble anesthetic) will decrease the alveolar partial pressure of the gas; this is analogous to increasing the leakiness of the bucket. Thus, uptake of anesthetic from the alveoli into the bloodstream constitutes a negative component to the flow (i.e., a flow out of the lungs), which makes the time constant longer than the theoretical case where {Palv→PI} equals FRC divided by Valv. The magnitude of the increase in the time constant compared to the limiting case depends on the rate of uptake of anesthetic by the blood, with longer {Palv→PI} resulting from greater uptake. If one knows the cardiac output (i.e., the volume of blood pumped by the heart in 1 minute) and the value of the instantaneous difference between the pulmonary arterial partial pressure (which equals the systemic partial pressure of the mixed venous return, PMVR) and the pulmonary venous partial pressure (which equals the systemic arterial partial pressure, Part), then one can calculate the rate of uptake of a gas from the alveoli: Rate of uptake {in Lgas /min}  (blood/gas)  (Part  PMVR)  CO

Equation 16-6

where CO  cardiac output in liters of blood per minute. Equation 16-7 follows from Equation 16-6 because the anesthetic concentration [A]blood is equal to (blood/gas)  Pblood (see Box 16-2): Rate of uptake  ([ A]art  [ A]MVR )  CO Equation 16-7 If any of the terms in these equations approaches zero, the rate of uptake becomes small, and the delivery of anesthetic by ventilation drives the alveolar partial pressure toward the inspired partial pressure. In other words, equilibration of alveolar with inspired partial pressure is faster (i.e., {Palv→PI} is smaller) with lower blood solubility of the anesthetic [smaller (blood/gas]), lower cardiac output, or smaller arterial (⬇alveolar) to venous partial pressure difference.

Ventilation brings anesthetic into alveoli

Palv

The balance between input and output sets the level of Palv

Uptake into bloodstream removes anesthetic from alveoli

FIGURE 16-5. Determinants of the alveolar partial pressure of an inhaled anesthetic. The alveolar partial pressure, represented by the depth of fluid in the bucket, results from the balance between delivery by ventilation and removal by uptake into the bloodstream. Increased delivery of anesthetic, resulting from either increased ventilation or an increased inspired partial pressure of anesthetic, raises Palv. In contrast, increased uptake into the bloodstream, caused by a large (blood/gas) or increased cardiac output, lowers Palv.

Equilibration of Tissue with Alveolar Partial Pressure In addition to the equilibration between Palv and PI, equilibration between Ptissue and Part (which is nearly equal to Palv) must occur for Ptissue to equilibrate with PI. Changes in Palv are transmitted rapidly to systemic arterioles, because equilibration across the pulmonary epithelium is fast and the circulation time from pulmonary veins to tissue capillaries is generally less than 10 seconds. Thus, the time constant for equilibration between Ptissue and Palv can be approximated as the time constant for equilibration between Ptissue and Part. To calculate the time constant {Ptissue→Part}, one must define the capacity of the compartment and the flow rate of the delivering medium. The flow rate is simply the rate at which blood perfuses the tissue. Recall that capacity is a volume capacity relative to the delivering medium. Specifically, the capacity is the volume that the tissue would need to contain all of its gas if the solubility of the gas in the tissue were the same as that in the blood. (This definition is similar to that of the volume of distribution of a drug; see Chapter 3, Pharmacokinetics): Relative Volume Capacity of Tissue  ([A]tissue  Voltissue)/[A]blood

Equation 16-8

Relative Volume Capacity of Tissue   (tissue/blood)  Voltissue

Equation 16-9

{Ptissue Æ Part } 艐 {Ptissue Æ Palv }  Relative Vol. Cap. of Tissue /Qtissue Equation 16-10 {Ptissue Æ Part}  (tissue/blood)  Voltissue /Qtissue

Equation 16-11

Nitrous oxide (PI = 0.75)

Halothane (PI = 0.01) Alveolar VRG

MG FG

80 63% equilibration 60

40

20

0 1

10

Time (min)

100

1

10

100

Time (min)

Alveolar partial pressure as fraction of inspired partial pressure (Palv/PI)

% of inspired partial pressure

100

1.0 Nitrous oxide, λ = 0.47 Desflurane, λ = 0.45

0.8

Isoflurane, λ = 1.4 63% equilibration

0.6 Halothane, λ = 2.3

0.4

0.2 Ether, λ = 12.0

0.0 0

10

20

Minutes of administration

30

250 Principles of Central Nervous System Pharmacology A Initial Palv = 0.1 atm λ (blood/gas) = 0.5 Final Palv = Part = 0.067 atm

B Initial Palv = 0.1 atm λ (blood/gas) = 11 Final Palv = Part = 0.0083 atm

Anesthetic A to achieve the anesthetic partial pressure in the CNS (Fig. 16-8).a

Applications of the Uptake Model

Anesthetic Alveolus Capillary

Why do anesthetics with smaller (blood/gas) have shorter induction times? Consider two equally potent anesthetics inspired at the same partial pressure, PI. Before any anesthetic molecules have been taken up from the alveolus into the blood, the alveolar partial pressure, Palv, of each anesthetic is 0.1 atm. This partial pressure would be represented in the diagram by 12 anesthetic “spheres” in each alveolus. For each anesthetic, equilibration of the partial pressures in the alveolus and the capillary then takes place. For a relatively blood-insoluble agent with (blood/gas)  0.5 (Anesthetic A, which closely resembles nitrous oxide, desflurane, sevoflurane, and cyclopropane), the transfer of a small amount of anesthetic from the alveolus significantly raises the partial pressure in the capillary. To illustrate, consider a time, tv, when the volume of blood that has flowed past the alveolar wall is equal to the volume of the alveolus. At that time, the concentration in the alveolus will be twice that in the capillary (because  (blood/gas)  0.5; see Box 16-2) when four of the “spheres” have been transferred from the alveolus to the capillary and eight “spheres” remain in the alveolus. The partial pressure in the alveolus will now have dropped to (8/12)  0.1  0.067 atm. This is also the partial pressure in the capillary. In contrast, for a very blood-soluble agent with (blood/gas)  11 (Anesthetic B, which closely resembles diethyl ether), much larger amounts of anesthetic must dissolve in the blood to raise the partial pressure in the capillary. Using the same illustration as above, at tv, 11 of the 12 “spheres” will have been transferred from the alveolus to the capillary, and the remaining Palv will be given by (1/12)  0.1  0.0083 atm. Thus, although the inspired partial pressure of the two anesthetics is the same, at time tv, the Palv and Part of Anesthetic A will be eight times higher than that of Anesthetic B. Within approximately 2 minutes (Table 16-3), Pbrain will also reach these values. Thus, the brain partial pressure rises toward the inspired partial pressure much more rapidly for Anesthetic A than for Anesthetic B (i.e., the induction time for Anesthetic A is much shorter than that for Anesthetic B). If the reader is confused by the fact that more molecules of Anesthetic B are being carried to the brain, recall that (brain/blood) is ⬃1 for all of the commonly used anesthetics [that is, for each agent, (blood/gas) is approximately equal to (brain/gas); see Table 16-2]. Thus, proportionally many more molecules of Anesthetic B than Anesthetic A must be delivered to the brain in order to raise the partial pressure of each anesthetic by an equivalent amount. See Boxes 16-1 and 16-2 and the Appendix for definitions.

FIGURE 16-8.

has a small (blood/gas), while Anesthetic B has a large (blood/gas). Because Anesthetics A and B are identical in (oil/gas), they have the same MAC. They also have identical (brain/blood), so their {Pbrain→Palv} is the same (see Equations 16-12 and 16-13). To cause anesthesia, both must achieve the same partial pressure in the CNS. At any particular partial pressure, however, the blood and CNS contain more moles of Anesthetic B than Anesthetic A because Anesthetic B is more soluble than Anesthetic A in the blood and CNS. The transfer of a larger number of moles of Anesthetic B out of the lungs slows the rate of rise of Palv, so a longer period is necessary for Anesthetic B than for

Throughout the following discussion, it is critical to remember that the primary responsibility of the anesthesiologist is to keep the patient well oxygenated and the vital signs stable while manipulating the inspired partial pressure of anesthetic to maintain the desired depth of anesthesia. Armed with the uptake model, the anesthesiologist can predict the effects of cardiopulmonary changes and pathologic states on the depth of anesthesia. Changes in ventilation and cardiac output may be caused by the general anesthetic itself, by the trauma of surgery, or by some other physiologic or pathophysiologic process. The effects of changes in both ventilation and cardiac output on PCNS are greatest when the difference between PI and Palv is greatest; that is, early in the course of anesthesia (Fig. 16-6). To understand this, consider the partial pressure in the mixed venous return (MVR), PMVR, which is a weighted average of the partial pressures in each of the tissue groups, with PVRG making the largest contribution because the VRG receives the majority of the cardiac output (Fig. 16-4). When Palv (and thus PVRG) is much less than PI, PMVR is low, and the bloodstream is capable of carrying large amounts of anesthetic away from the alveoli to the tissues. Under these conditions, the rate of uptake of anesthetic from the alveoli into the bloodstream can be greatly modified by cardiopulmonary changes, and PCNS can be greatly affected by changes in ventilation and cardiac output. As each successive tissue group approaches saturation with anesthetic, PMVR approaches PI. When PMVR is nearly equal to PI, the bloodstream cannot remove much anesthetic from the lungs under any circumstances, and changes in ventilation or cardiac output have little effect on PCNS. Upon commencement of anesthetic administration, the length of time during which there is a significant difference between PI and Palv increases with (blood/gas). With ventilation-limited anesthetics, such as diethyl ether and halothane, the prolonged time during which Palv lags behind PI allows cardiopulmonary changes to modulate Palv significantly, potentially leading to unexpected CNS partial pressures. With perfusion-limited anesthetics, such as nitrous oxide, the alveolar partial pressure rises so rapidly that Palv is significantly less than PI for only a short time, minimizing the time during which cardiopulmonary changes could have a significant effect on PCNS (Fig. 16-6). a

In this hypothetical model, one may correctly note that the concentration of Anesthetic B in the CNS as a whole will be higher than that of Anesthetic A at any particular time point. One may, therefore, wonder how Anesthetic B can have a slower induction, if anesthesia results when a particular concentration (0.05 M) is reached at the site of action (see Pharmacodynamics, above). At this point, one must recognize that the brain is primarily aqueous, but that anesthetics are likely to have a hydrophobic site of action, and that both Anesthetic A and Anesthetic B must have the same concentration (0.05 M) in the key hydrophobic portions of the brain at their anesthetic partial pressures. However, Anesthetic B, with its larger aqueous solubility [(blood/gas)], will partition relatively more than Anesthetic A into the aqueous portions of the brain. To provide the higher aqueous concentrations, many more moles of Anesthetic B than Anesthetic A must be transferred from the lungs. The overall conclusion still holds if (oil/gas) and thus MAC differ for the two hypothetical anesthetics. Palv for a less blood-soluble agent will rise proportionally faster toward its PI than a more blood-soluble agent, independent of what that PI is (note that PI will be larger for the less oil-soluble anesthetic). A larger (oil/gas) allows the anesthetic to cause anesthesia at a lower partial pressure, but does not affect the proportional rate at which the partial pressure rises.

CHAPTER 16 / General Anesthetic P harmacology 251

Effects of Changes in Ventilation Hypoventilation decreases the delivery of anesthetic to the alveoli. Meanwhile, removal of anesthetic from the alveoli continues provided that cardiac output is maintained. Consequently, the alveolar partial pressure rises more slowly, and {Palv→PI} is prolonged. In other words, hypoventilation slows induction. This effect is greater with ventilation-limited than with perfusion-limited anesthetics (Fig. 16-9A). General anesthetics themselves can cause hypoventilation by depressing the medullary respiratory center. In this manner, anesthetic-induced hypoventilation sets up a beneficial

A Ventilation Effects 1.0 Nitrous oxide

Palv/PI

63% equilibration Halothane

0.5

Diethyl ether

0.0 0

20

40

Minutes 2 L/min ventilation

8 L/min ventilation

B Cardiac Output Effects 1.0

Palv/PI

Nitrous oxide 63% equilibration

0.5

Halothane

Diethyl ether

0.0 0

20

40

Minutes 2 L/min cardiac output

FIGURE 16-9.

negative feedback loop on the depth of anesthesia. Increasing anesthetic depth leads to medullary depression, which, in turn, depresses respiration. The beneficial effect of this physiologic response is that the depressed ventilation slows the rate of rise of the alveolar partial pressure, while perfusion continues to remove anesthetic from the lung at the same rate (Fig. 16-5). Thus, Palv falls, and shortly thereafter, the partial pressure of anesthetic in the medulla falls as well. This decrease in PCNS relieves the respiratory depression. In the extreme example of a full respiratory arrest, there is no ventilation to deliver anesthetic to the alveoli, but cardiac output continues to distribute anesthetic from the alveoli and VRG to the MG and FG. In the case of diethyl ether, the decrease in PCNS can be of a sufficient magnitude that spontaneous ventilation resumes. Hyperventilation delivers anesthetic more quickly to the alveoli. This decreases the time constant for equilibration of the alveolar with the inspired partial pressure (recall that {Palv→PI}  FRC / Valv, in the limiting case). However, the hyperventilation-induced hypocapnia may concomitantly decrease cerebral blood flow, increasing {PCNS→Part}. Thus, while the partial pressure in the alveoli rises faster, the rate of equilibration between the CNS and the alveoli could be slower. The net effect depends on which of these two steps is rate-limiting. For perfusion-limited anesthetics such as nitrous oxide, the decrease in cerebral blood flow results in a slower induction. For the most soluble ventilation-limited anesthetics such as diethyl ether, the faster delivery of anesthetic to the alveoli hastens induction. For less soluble ventilation-limited anesthetics such as isoflurane, the effects roughly balance, and induction is not significantly affected.

18 L/min cardiac output

Effects of changes in ventilation and cardiac output on the rate at which alveolar partial pressure rises toward inspired partial pressure. The rate of equilibration of the alveolar partial pressure with the inspired partial pressure can be affected by changes in ventilation (A) and cardiac output (B). Increasing ventilation from 2 L/min (dashed lines) to 8 L/min (solid lines) accelerates equilibration. On the other hand, increasing cardiac output from 2 L/min (dashed lines) to 18 L/min (solid lines) slows equilibration. Both effects are much larger for more blood-soluble gases, such as halothane and diethyl ether, which have rather slow induction times. For nitrous oxide, the rate of equilibration is so fast that any changes caused by hyperventilation or decreased cardiac output are small. The dashed horizontal line represents 63% equilibration of Palv with PI ; the time required for each curve to cross this line represents {Palv→PI }.

Effects of Changes in Cardiac Output At anesthetic partial pressures higher than those required to depress the respiratory center, cardiac output falls. When cardiac output falls, the bloodstream removes anesthetic from the alveoli at a slower rate. Consequently, the alveolar partial pressure rises faster (Fig. 16-9B). Because the alveolar partial pressure equilibrates relatively quickly with the VRG (even at the lower cardiac output), the partial pressure in the CNS also rises more rapidly. In other words, decreased cardiac output speeds induction. This effect is more marked with ventilationlimited than with perfusion-limited anesthetics. Moreover, cardiac depression by anesthetics sets up a harmful positive feedback loop on the depth of anesthesia. Increasing PCNS depresses cardiac function, which further increases Palv, which further increases PCNS, which further depresses cardiac function. If cardiac arrest occurs, then positive measures must be taken to restore the circulation (e.g., CPR) while reducing the alveolar partial pressure through controlled breathing with oxygen. Increased cardiac output increases perfusion to the lungs and accelerates equilibration between the alveoli and the tissues. However, because the increased blood flow to the lungs removes anesthetic from the alveoli at a faster rate, the rate of rise of the alveolar partial pressure is slowed. Thus, increased cardiac output slows induction. This effect is greater with ventilation-limited than with perfusion-limited agents. Effects of Age Relative to their body weight, young children such as Matthew have higher ventilation than do adults. This effect tends to speed induction. However, young children also have

0.9 Children (1–5 years) 0.8

Palv/PI

0.7 Adults 0.6

0.5

0.4 0

10

20

30

40

Minutes of anesthesia

50

60

Clinical Status of Patient

Alveolar partial pressure (Atm)

0.03

Respiratory depression (toxic range)

Continuing at PI = 0.04 Atm 0.02 Varying PI between 0.015 and 0.02 Atm

0.01

Desired Pbrain for anesthesia PI = 0.04 Atm

PI = 0.01 Atm

0.00 0

5

10

Time (min)

15

Anesthesia (therapeutic range)

Awake (subtherapeutic range)

Nitrous oxide λ (blood/gas) = 0.47

Halothane λ (blood/gas) = 2.3

Methoxyflurane λ (blood/gas) = 13.0

1.0 Minutes of Anesthesia

PE/PE0

∞

240 120 60 30 15 0.5

0 0

40

80

Time (min)

120

0

40

80

Time (min)

120

0

40

80

Time (min)

120

CHAPTER 16 / General Anesthetic P harmacology 255

Intravenous Anesthetic Agents Intravenous anesthetics, such as barbiturates (see also Chapter 12), allow for rapid induction. Ultrashort-acting barbiturates, such as thiopental, are capable of inducing surgical anesthesia within seconds. As nonvolatile compounds, intravenous agents differ from inhaled anesthetics in that they cannot be removed from the body by ventilation. Accordingly, one must take great care during their administration to avoid severe medullary depression that is not easily reversible. The primary method of removal of these agents from the CNS is by redistribution from the VRG to the MG and finally to the FG. Metabolism and/or excretion then slowly decrease the overall body levels of drug (Fig. 16-13). Propofol is an important intravenous anesthetic prepared in an intralipid formulation. This agent produces anesthesia at a rate similar to the ultrashort-acting barbiturates. Propofol is both rapidly redistributed and rapidly metabolized, resulting in a faster recovery than for barbiturates. Propofol is used both for induction and for maintenance, especially in short day-surgery procedures where its fast elimination favors prompt recovery and early discharge. The intralipid preparation of propofol can rarely be a source of infection,

100 Blood MG

80

Percent of dose

Isoflurane and enflurane are somewhat less potent than halothane [they have a smaller (oil/gas)], but they equilibrate faster because they have a smaller (blood/gas). Enflurane is metabolically defluorinated to a greater extent than isoflurane, and may thus have a greater risk of causing renal toxicity. It also induces seizure-like activity in the EEG of some patients. Isoflurane is probably the most widely used general anesthetic today. Although less potent than isoflurane and enflurane, diethyl ether is still quite potent with a rather high (oil/gas). However, because of its flammability and very slow induction attributable to its extremely high (blood/gas), this agent is no longer in common use in the United States and Europe. In developing countries, however, its low price and simplicity of application favor its continued use. Nitrous oxide has a very low (blood/gas) and thus equilibrates extremely rapidly. However, its low (oil/gas) results in a very high MAC, close to one atmosphere. Thus, the need to maintain an acceptable partial pressure of oxygen (normally greater than 0.21 atm) prevents the attainment of full anesthesia using nitrous oxide alone, and this agent is commonly employed in combination with other agents (see Balanced Anesthesia below). Desflurane and sevoflurane are newer anesthetics that, by design, have low (blood/gas); times of equilibration between their alveolar and inspired partial pressures are nearly as short as that of nitrous oxide. Furthermore, they are much more potent than nitrous oxide because their oil/gas partition coefficients are higher. Thus, these agents offer great improvements over earlier agents. However, desflurane is a poor induction agent because its pungency irritates the airway, potentially causing cough or laryngospasm. Sevoflurane is sweet-tasting, but can be chemically unstable when exposed to some carbon dioxide adsorbents in anesthetic machinery, degrading to an olefinic compound that is potentially nephrotoxic. These disadvantages have been overcome with improved machinery, and sevoflurane is gaining in popularity.

VRG

60

40 FG 20

0 0.1

1

10

100

Time (min)

FIGURE 16-13.

Distribution of a bolus of intravenous anesthetic. When a bolus of intravenous anesthetic is administered, it is initially transported through the vascular system to the heart and then distributed to the tissues. The vesselrich group (VRG) receives the highest percentage of the cardiac output; its anesthetic concentration rises rapidly, reaching a peak within 1 minute. Redistribution of anesthetic to the muscle group (MG) then quickly decreases the anesthetic level in the VRG. Because of very low fat group (FG) perfusion, redistribution from the MG to the FG does not occur until much later. Note that rapid redistribution from the VRG to the MG does not occur if the MG has previously approached saturation through prolonged administration of anesthetic (not shown); this can lead to significant toxicity if intravenous barbiturates are administered continuously for long periods of time. Newer agents, such as propofol, are designed to be eliminated by rapid metabolism and, therefore, can be used safely for longer periods of time.

and the lipid preparation provides a large caloric source; these considerations can be important in critically ill patients who may receive prolonged propofol infusions. Etomidate is an imidazole that is used for induction of anesthesia because its kinetics are similar to those of propofol. This agent causes minimal cardiopulmonary depression, perhaps because of its unique lack of effect on the sympathetic nervous system. Unlike the above agents, ketamine produces dissociative anesthesia, in which the patient seems to be awake but is actually in an analgesic and amnesic state. Ketamine has the unusual property that it increases cardiac output by increasing sympathetic outflow; for this reason, it is occasionally useful in emergency trauma situations. However, it can also produce unpleasant hallucinations. This agent is rarely used today.

Adjuvant Drugs Adjuvant drugs provide additional effects that are desirable during surgery but are not necessarily provided by the general anesthetics. Benzodiazepines (see Chapter 12), such as diazepam, lorazepam, and midazolam, are often given for their anxiolytic and anterograde amnesic properties. These agents are administered 15 to 60 minutes before the induction of anesthesia to calm the patient and obliterate memory of the induction, although they may also be used for intraoperative sedation. If necessary, benzodiazepine effects can be reversed with the antagonist flumazenil.

CHAPTER 16 / General Anesthetic P harmacology 257

A Inhaled anesthetics

B Intravenous anesthetics

O N

N

O

HN

Nitrous oxide F

O

F

F3C

O

* HN O Pentobarbital

F

If interfacial solubilities (i.e., the solubility of a compound at an aqueous–lipid interface) are considered instead of simple lipid solubilities, then the Meyer–Overton Rule is much more successful at explaining the activity of transitional and nonanesthetic compounds. This finding is likely to mean that anesthetics act at a hydrophobic–hydrophilic interface. Examples of such an interface could include a water–membrane interface, a protein–membrane interface, or an interface between a hydrophobic protein pocket and the hydrophilic lumen of an ion-conducting pore.

Desflurane

O CF3 F3C

Effects on Ion Channels

HN

O

F

S

* HN

Sevoflurane

O Thiopental

O

Diethyl ether (Ether)

OH

Br F3C

* Cl

Propofol

Halothane

O Cl Cl

F

*

* F3C

O

F

NH

Isoflurane Ketamine F

F

F F O

* Cl

Enflurane

O

F

O

* N N Etomidate

FIGURE 16-14.

Structures of general anesthetics. A. Structures of some inhaled anesthetics. B. Structures of some intravenous anesthetics. The extreme variability in the structures of these molecules, all of which are capable of causing general anesthesia, suggests that not all general anesthetics interact with a single receptor site. * Indicates carbons where asymmetry results in enantiomeric structures.

and anesthetic steroids) do exhibit substantial stereoselectivity. That is, one enantiomer is more potent than the other. Second, many so-called nonanesthetics or nonimmobilizers are chemically similar to known anesthetics but do not cause anesthesia. For example, straight-chain alcohols with more than 12 carbons lack anesthetic activity, even though their (oil/gas) is larger than the shorter alcohols. Other compounds, called transitional anesthetics, have a much higher MAC than that predicted by the Meyer–Overton Rule. Refinements to the Meyer–Overton Rule have been proposed to account for some of the weaknesses listed above.

Current research has focused on proteins that may alter neuronal excitability when acted on by anesthetics, either directly or indirectly. Anesthetics affect both axonal conduction and synaptic transmission, but modulation of synaptic transmission occurs at lower anesthetic concentrations and is, therefore, likely to be the pharmacologically relevant action. Accordingly, anesthetics are thought to act on ligand-gated ion channels at lower concentrations than on voltage-gated ion channels. Both presynaptic and postsynaptic modulation occur, although the postsynaptic actions seem to dominate. A superfamily of genetically and structurally related ligand-gated channels is sensitive to modulation by anesthetics at clinically relevant concentrations. Members of this superfamily have five homologous subunits, each with four transmembrane regions. The sensitivity of these ligand-gated ion channels to anesthetics can vary with their subunit composition. The superfamily includes the excitatory nicotinic acetylcholine and 5-HT3 receptors, as well as the inhibitory GABAA and glycine receptors (see Fig. 9-2 and Fig. 12-3). Although receptors for glutamate, the major excitatory neurotransmitter in the brain, do not belong to this superfamily, NMDA glutamate receptors are also modulated by some anesthetics (e.g., ketamine and nitrous oxide). Excitatory receptors (nicotinic acetylcholine, 5-HT3, and NMDA) are inhibited by anesthetics. The binding of anesthetic to these receptors lowers their maximum activation, without changing the concentration of agonist required to achieve a half-maximal effect (EC50) (Fig. 16-15). This action is consistent with noncompetitive inhibition and an allosteric site of action (see also Chapter 2). In contrast, inhibitory receptors (GABAA and glycine) are potentiated by anesthetics. The binding of anesthetic to these receptors decreases the concentration of agonist required to achieve a maximum response, and thereby prolongs the synaptic current. The activation curves for these receptors are shifted to the left (lower EC50), and the maximal response often increases as well because the anesthetics stabilize the open state of the receptor (Fig. 16-15). Until recently, GABAA receptors seemed to be the most relevant receptors for general anesthetic action, based on the sensitivity of GABAA receptors to clinical concentrations of anesthetics and the wide range of agents that act on these receptors. However, it now seems that glycine receptors and some neuronal acetylcholine receptors are equally sensitive to many anesthetics, and that nonpolar anesthetics such as xenon and cyclopropane (both of which have been used in clinical practice), as well as nitrous oxide and ketamine, act by inhibiting nicotinic acetylcholine and NMDA glutamate receptors rather than by potentiating GABAA receptors. Thus, it currently appears that an agent must cause either

258 Principles of Central Nervous System Pharmacology 150

Inhibitory synapse with general anesthetic

Relative response (%)

125

100 Control EC50

75

EC50

50

EC50

25

Excitatory synapse with general anesthetic

0 0.01

0.1

1.0

10

100

Relative agonist concentration

FIGURE 16-15.

Actions of anesthetics on ligand-gated ion channels. Anesthetics potentiate the action of endogenous agonists at inhibitory receptors, such as GABAA and glycine receptors, and inhibit the action of endogenous agonists at excitatory receptors, such as nicotinic acetylcholine, 5-HT3, and NMDA glutamate receptors. At GABAA receptors, anesthetics both decrease the EC50 of GABA (i.e., GABA becomes more potent) and increase the maximum response (i.e., GABA becomes more efficacious). The latter effect is thought to be due to the ability of anesthetics to stabilize the open state of the receptor channel. At excitatory receptors, anesthetics decrease the maximum response while leaving the EC50 unchanged; these are the pharmacologic hallmarks of noncompetitive inhibition.

sufficient enhancement of inhibition (e.g., etomidate) or inhibition of excitation (e.g., ketamine), or a mixture of the two (e.g., volatile anesthetics), to cause anesthesia. This hypothesis also suggests that surgical anesthesia may represent more than one neurological state. Direct anesthetic–protein interactions are probably responsible for the effects of anesthetics on ligand-gated ion channels. Anesthetics could bind in the pore of excitatory channels and thereby directly plug the channel. Alternatively, anesthetics could bind elsewhere on the protein and thereby affect the channel’s conformation (and, thus, the equilibrium among its open, closed, and desensitized states). Site-directed mutagenesis, photolabeling, and kinetic studies suggest that inhibition of excitatory acetylcholine receptors probably occurs at a site in the pore of the ion channel that is on the central axis of symmetry and in contact with all five subunits. However, the site of anesthetic binding to inhibitory GABAA receptors cannot be in the ion pore because potentiation, not inhibition, is observed at therapeutic concentrations. Indeed, GABAA receptors lack a stretch of hydrophobic amino acids that is present in the pore of excitatory receptors. Instead, site-directed mutagenesis studies suggest that an anesthetic binding site is located on the “outside” of one of several alpha helices that line the GABAA ion channel. This evidence places the volatile anesthetic site within the four transmembrane helices of a single receptor subunit. In contrast, recent photolabeling evidence, using an analog of the highly potent intravenous anesthetic etomidate, places the binding site for that anesthetic at the interface between

the and  subunits of the GABAA receptor; the site is located within the membrane region and about 50 Å below the agonist (GABA) site in the same subunit interface. Although current research is focused on protein sites of anesthetic action, no single site has been found that accounts for the Meyer–Overton Rule or the pharmacology of all general anesthetics. Consequently, adoption of such protein-site theories may have to be accompanied by an abandonment of the unitary hypothesis. Some new unifying principles are emerging, however. For example, a single mutation in the alpha helix of the GABAA receptor 2 or 3 subunit that lines the ion channel (see paragraph above) attenuates the action of etomidate on this receptor, although this mutation has no effect on the potency of volatile anesthetics. Mice genetically engineered to contain the mutation are normally sensitive to volatile anesthetics but much less sensitive to anesthesia with etomidate. In contrast, the equivalent mutation in the subunit attenuates the channel’s response to volatile anesthetics but not to etomidate. Thus, although different classes of anesthetics act on different GABAA receptor subunits, and by implication different binding sites, all these sites are within the membrane region where the conformation of the receptor changes upon gating. It is possible that each class acts in a similar manner on the subunit to which it binds and that selectivity is a function of the detailed architecture of each subunit at that site.

 CONCLUSION AND FUTURE DIRECTIONS Inhaled and intravenous anesthetics are used to produce the clinical features of general anesthesia, including unconsciousness, immobility, and amnesia. The pharmacodynamics of general anesthetics are unique. Anesthetics have steep dose–response curves and small therapeutic indices, and they lack a pharmacologic antagonist. According to the Meyer–Overton Rule, the potency of a general anesthetic can be predicted simply from its oil/gas partition coefficient. The pharmacokinetics of inhaled anesthetics can be modeled assuming three principal tissue compartments that are perfused in parallel. Equilibration of the partial pressure of anesthetic in the CNS with the inspired partial pressure proceeds in two steps: (1) equilibration between the alveolar partial pressure and the inspired partial pressure; and (2) equilibration between the CNS partial pressure and the alveolar partial pressure. With ventilation-limited anesthetics, which have a large blood/gas partition coefficient, the first of these steps is slow and rate-limiting. With perfusion-limited anesthetics, which have a small blood/gas partition coefficient, both steps are rapid and neither is clearly rate-limiting; changes in either can affect induction time. Recovery from anesthesia occurs roughly as the reverse of induction, except that redistribution of anesthetic from the vessel-rich group to the muscle group and fat group can also occur. The “ideal” inhaled anesthetic has not yet been found. Future researchers may attempt to identify a nonflammable anesthetic with high (oil/gas), low (blood/gas), high therapeutic index, good vapor pressure, and few or no significant adverse effects. Currently, the combined use of adjuvants and balanced anesthesia with multiple inhaled and/or intravenous anesthetics achieves all of the goals of general anesthesia, including fast induction and a state of analgesia, amnesia, and muscle relaxation.

CHAPTER 16 / General Anesthetic P harmacology 259

The exact mechanism of action of general anesthetics remains a mystery. Although the site of action was formerly thought to reside in lipid bilayers, direct interactions with several ligand-gated ion channels—members of the four-transmembrane–helices, Cys-loop superfamily and the glutamate receptor family—now seem to be more likely. More research is required to elucidate the mechanisms of action of general anesthetics. Once discovered, these mechanisms could shed light on such far-reaching issues as the generation of consciousness itself.

Suggested Reading Campagna JA, Miller KW, Forman SA. The mechanisms of volatile anesthetic actions. N Engl J Med 2003;348:2110–2124. (Reviews how general anesthetics act.)

Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia. Philadelphia: Churchill Livingstone; 2000:74–95. (Pharmacokinetics and uptake of inhaled anesthetics.) Rudolph U, Antkowiak B. Molecular and neuronal substrates for general anesthetics. Nat Rev Neurosci 2004;5:709–720. (A short review with good diagrams.) Various authors. Molecular and cellular mechanisms of anaesthesia. In: Can J Anesth 2011; Feb issue. (This special issue is a compilation of detailed reviews relating to all major current theories on the mechanism of action of general anesthetics.) Wiklund RA, Rosenbaum SH. Anesthesiology. N Engl J Med 1997;337: 1132–1151, 1215–1219. (Two-part review covers many aspects of the modern practice of anesthesiology.) Winter PM, Miller JN. Anesthesiology. Sci Am 1985;252:124–131. (A good account of the clinical approach of the anesthesiologist.)

Appendix A Abbreviations and Symbols PI  inspired partial pressure PE  exhaled partial pressure Palv  alveolar partial pressure Part  arterial partial pressure Ptissue  partial pressure in a tissue Pvenule  partial pressure in a venule PMVR  mixed venous partial pressure Psolvent  partial pressure in a solvent PCNS  partial pressure in the central nervous system PVRG  partial pressure in the vessel-rich group (oil/gas)  partition coefficient defining solubility of a gas in a lipophilic solvent such as oil (blood/gas)  partition coefficient defining solubility of a gas in blood (tissue/gas)  partition coefficient defining solubility of a gas in a tissue (tissue/blood)  partition coefficient describing ratio of solubility in tissue to solubility in blood  (tissue/gas) / (blood/gas)  time constant for 63% equilibration {Palv→PI}  time constant for 63% equilibration of Palv with PI {Ptissue→Palv}  time constant for 63% equilibration of Ptissue with Palv

262

[A]  concentration of gas A, in terms of either Lgas/Lsolvent or mol/Lsolvent CNS  central nervous system VRG  vessel-rich group (includes CNS, liver, kidney) MG  muscle group (includes muscle, skin) FG  fat group (includes adipose tissue) VPG  vessel-poor group (includes bone, cartilage, ligaments, tendons) FRC  functional residual capacity of lung Valv  alveolar ventilation CO  cardiac output Q  perfusion rate Voltissue  volume of tissue MAC  minimum (or median) alveolar concentration P50  alveolar partial pressure sufficient for immobility in 50% of patients ⬅ MAC AP50  alveolar partial pressure sufficient to cause analgesia in 50% of patients LP50  alveolar partial pressure sufficient to cause death in 50% of subjects EC50  concentration of agonist required to activate 50% of channels

Appendix B Equations  GAS CONCENTRATIONS

 EQUILIBRATION TIME CONSTANTS (FOR 63% EQUILIBRATION)

In an ideal gas mixture: [A]mixture  nA / V  PA / RT {in terms of mol/L} In solution (Henry’s law): [A]solution  Psolvent  (solvent/gas) {in terms of Lgas/Lsolvent} [A]solution  Psolvent  (solvent/gas) / 24.5 {in terms of mol/ Lsolvent} {where nA  moles of gas A, V  total volume, PA  partial pressure of A, R  universal gas constant, T  temperature in degrees Kelvin}

  Volume Capacity / Flow Rate {Ptissue→Palv}⬇ {Ptissue→Part}  Volume Capacity of Tissue / Tissue Blood Flow  (tissue/blood)  Volume of Tissue / Tissue Blood Flow {Pbrain→Part}  (brain/blood)  Volume of Brain / Blood Flow to Brain Pcontainer  Pflow [1 e (t/)]

 MEYER–OVERTON RULE

 VOLUME CAPACITY

MAC ⬇ 1.3 / (oil/gas)

 FICK’S LAW FOR DIFFUSION ACROSS A BOUNDARY Rate of diffusion  D  (A / l)  P {where D  Diffusion constant; A  Surface area; l  Thickness; P  Partial pressure difference}

Volume Capacity  ([A]compartment  Volume of compartment) / [A]medium {at equilibrium}  (compartment/medium)  Volume of Compartment

 MIXED VENOUS PARTIAL PRESSURE PMVR  0.75 PVRG  0.18 PMG  0.055 PFG  0.015 PVPG

 ALVEOLAR CAPILLARY RATE OF UPTAKE Rate of uptake  ([A]art [A]MVR)  CO {in Lgas/min} Rate of uptake  (blood/gas)  (Part PMVR)  CO {where CO  cardiac output}

263

17 Pharmacology of Analgesia Robert S. Griffin and Clifford J. Woolf

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 264-265 PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 Sensory Transduction: Excitation of Primary Afferent Neurons . . . . . . . . . . . . . . . . . . . . . . . . . 265 Conduction from the Periphery to the Spinal Cord . . . . . . 267 Transmission in the Dorsal Horn of the Spinal Cord . . . . . 267 Descending and Local Inhibitory Regulation in the Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Clinical Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Peripheral Sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Central Sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Neuropathic Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 Migraine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 273 Opioid Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . 273 Mechanism of Action and Major Adverse Effects . . . . . 273

Morphine, Codeine, and Derivatives . . . . . . . . . . . . . . 274 Synthetic Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Partial and Mixed Agonists . . . . . . . . . . . . . . . . . . . . . 275 Opioid Receptor Antagonists . . . . . . . . . . . . . . . . . . . . 275 Nonsteroidal Anti-Inflammatory Drugs and Nonopioid Analgesics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 General Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Specific Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Antiepileptic Drugs and Antiarrhythmics . . . . . . . . . . . . . . 277 NMDA Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . 277 Adrenergic Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Migraine Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 278 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

 INTRODUCTION

syndrome, results from an abnormal function of the nervous system. These categories of pain—physiologic, inflammatory, neuropathic, and dysfunctional—are produced by a number of different mechanisms. Ideally, treatment should be targeted at specific mechanisms rather than at suppressing the symptom of pain. A number of pharmacologic agents are currently available to relieve pain. These drugs have mechanisms of action that interfere with the response of primary sensory neurons to somatic or visceral sensory stimuli, with the relaying of information to the brain, and with the perceptual response to a painful stimulus. The following discussion of pain and analgesic pharmacology begins by describing the mechanisms by which noxious stimuli lead to the perception of pain. The chapter continues by considering the processes responsible for the heightened pain sensitivity that occurs in response to inflammation and lesions of the nervous system. The chapter concludes by describing the mechanisms of action of the major drug classes used for clinical pain relief.

Everyone has experienced pain in response to an intense or noxious stimulus. This physiologic “ouch” pain helps us to avoid potential damage by acting as an early warning or protective signal. Pain can, however, also be incapacitating, as after trauma, during recovery from surgery, or in association with medical conditions that are characterized by inflammation, such as rheumatoid arthritis. Under circumstances where tissue injury and inflammation are present, noxious stimuli elicit more severe pain than normal because of increases in the excitability of the somatosensory system, and stimuli that would not normally cause pain become painful. In addition, nerve injury produced by disease or trauma, as in amputation, HIV infection, varicella zoster (VZV) infection, cytotoxic treatment, and diabetes mellitus, evokes pain that persists long after the initiating cause has disappeared. In these conditions, pathologic and sometimes irreversible alterations in the structure and function of the nervous system lead to severe and intractable pain. For such patients, the pain is the pathology rather than a physiologic defense mechanism. Finally, there are patients with no noxious stimuli and no inflammation or lesions to the nervous system, but who experience considerable pain. This dysfunctional pain, as in tension-type headache, fibromyalgia, or irritable bowel 264

 PHYSIOLOGY Pain is the end perceptual consequence of the neural processing of particular sensory information. The initial stimulus usually arises in the periphery and is transferred

Central perception

Thalamus

Relay and descending modulation

Brainstem

Transmission Spinal cord

Conduction Peripheral stimulus

Signal transduction

Cortex

Chemical

ASIC, P2X, P2Y, B1, B2 receptors

Mechanical Mec Mechanosensitive ion channels

Thermal TRPV1, TRPV2 T receptors

Na+/Ca2+ influx

Generator potential (membrane depolarization)

Reach voltage-gated sodium channel threshold

Action potential

268 Principles of Central Nervous System Pharmacology

that descend from the brainstem to the dorsal horn. Because these systems can limit transfer of incoming sensory information to the brain, they represent an important site for pharmacologic intervention. The major inhibitory neurotransmitters in the dorsal horn of the spinal cord are opioid peptides, norepinephrine, serotonin (5-HT), glycine, and GABA (Fig. 17-4). The physiology of GABA receptors is discussed in Chapter 12, Pharmacology of GABAergic and Glutamatergic Neurotransmission. The opioid peptides inhibit synaptic transmission and are released at several CNS sites in response to noxious

Primary sensory neuron central terminal Action potential

Glu Ca2+

Calcium influx Synaptic vesicle release

Glu

Neuropeptides CGRP Substance P

Na+ 2+ Ca Na

Glu

Primary sensory neuron central terminal

Glu

Glu

+

α2

Action potential

NMDA-R

Glu

Voltage-dependent Na+ and Ca2+ AMPA-R influx + Rapid Na influx

mGluR

Ca2+

GABA

NK1 CGRP-R Calcium influx

Slow modulatory response

μ Endorphins Enkephalins Neuropeptides CGRP Substance P

Reach voltage-gated Na+ channel threshold

Na+ 2+ Ca Na

+

neuropeptide-containing synaptic vesicles requires higherfrequency and longer-lasting action potential trains than release of glutamate-containing vesicles.

mGluR

Synaptic transmission in the spinal cord is regulated by the actions of both local inhibitory interneurons and projections

NK1

CGRP-R

GABAB

Voltage-dependent AMPA-R + 2+ Rapid Na+ Na and Ca influx influx

Cl- conductance GABAA

Cl-

μ K+

K+ conductance K+

Cl-

Secondary relay neuron (postsynaptic membrane)

Postsynaptic hyperpolarization Voltage-gated Na+ channels reaching threshold

Action potential generation

FIGURE 17-4.

Descending and Local Inhibitory Regulation in the Spinal Cord

α2

NMDA-R

Glu

Neurotransmission in the spinal cord dorsal horn. An incoming action potential from the periphery activates presynaptic voltage-sensitive calcium channels, leading to calcium influx and subsequent synaptic vesicle release. The released neurotransmitters (i.e., glutamate and neuropeptides, such as calcitonin gene-related peptide [CGRP] and substance P) then act on postsynaptic receptors. Stimulation of ionotropic glutamate receptors leads to fast postsynaptic depolarization, while activation of other modulatory receptors mediates slower depolarization. Postsynaptic depolarization, if sufficient, leads to action potential production (signal generation) in the secondary relay neuron.

Glu

Glu

Action potential

FIGURE 17-3.

GABAB

Synaptic vesicle Glu release

Postsynaptic depolarization Secondary relay neuron (postsynaptic membrane)

Norepinephrine

Inhibitory regulation of neurotransmission. Norepinephrine, GABA, and opioids, released by descending and/or local-circuit inhibitory neurons, act both presynaptically and postsynaptically to inhibit neurotransmission. Presynaptic inhibition is mediated through reduced activity of voltage-sensitive calcium channels, whereas postsynaptic inhibition is mediated primarily by enhanced chloride influx and potassium efflux.

CHAPTER 17 / Pharmacology of Analgesia 269

stimuli. All endogenous opioid peptides, which include ␤endorphin, the enkephalins, and the dynorphins, share the N-terminal sequence Tyr-Gly-Gly-Phe-Met/Leu. The opioids are proteolytically released from the larger precursor proteins pro-opiomelanocortin, proenkephalin, and prodynorphin. Opioid receptors traditionally fall into three classes, designated ␮, ␦, and ␬, all of which are seventransmembrane G protein-coupled receptors. The ␮-opioid receptors mediate morphine-induced analgesia. This conclusion is based on the observation that the ␮-opioid receptor knockout mouse exhibits neither analgesia nor adverse effects in response to morphine administration. The endogenous opioid peptides are receptor-selective: the dynorphins act primarily on ␬ receptors, while both enkephalins and ␤endorphin act on ␮ and ␦ receptors. The related ORL receptor for the peptide nociceptin has recently been identified. The physiologic role of these endogenous opioid peptides remains poorly understood. The effects of opioid receptor signaling include reduced presynaptic calcium conductance, enhanced postsynaptic potassium conductance, and reduced adenylyl cyclase activity. The first function impedes presynaptic neurotransmitter release; the second reduces postsynaptic neuronal responses to excitatory neurotransmitters; the physiologic role of the last remains unknown. Opioids produce analgesia because of their action in the brain, brainstem, spinal cord, and peripheral terminals of primary afferent neurons. In the brain, opioids alter mood, produce sedation, and reduce the emotional reaction to pain. In the brainstem, opioids increase the activity of cells that provide descending inhibitory innervation to the spinal cord; here, opioids also produce nausea and respiratory depression. Spinal opioids inhibit synaptic vesicle release from primary afferents and hyperpolarize postsynaptic neurons (see above). There is also evidence that peripheral opioid receptor stimulation reduces the activation of primary afferents and modulates immune cell activity. Action of opioids at these serially located sites is thought to have a synergistic effect to inhibit information flow from the periphery to the brain. Norepinephrine is released by projections that descend from the brainstem to the spinal cord. The ␣2-adrenergic receptor, a seven-transmembrane G protein-coupled receptor (see Chapter 10, Adrenergic Pharmacology), is the primary receptor for norepinephrine in the spinal cord. As with opioid receptor activation, ␣2-adrenergic receptor activation inhibits presynaptic voltage-gated calcium channels, opens postsynaptic potassium channels, and inhibits adenylyl cyclase. Because ␣2-adrenergic receptors are expressed both presynaptically and postsynaptically, spinal norepinephrine release can both reduce presynaptic vesicle release and decrease postsynaptic excitation. The ␣2-adrenergic receptor agonist clonidine is sometimes used to treat pain, although this application is limited by adverse effects that include sedation and postural hypotension. Serotonin is also released in the spinal cord by projections that descend from the brainstem. This neurotransmitter acts on several receptor subtypes that mediate both excitatory and inhibitory effects on nociception. The 5-HT3 ligand-gated channel may be responsible for the excitatory actions of serotonin in the spinal cord; several of the 5-HT G protein-coupled receptors may mediate the inhibitory actions of 5-HT. Given this complexity, the mechanism of the analgesic effect of serotonin is not fully understood. Selective serotonin reuptake inhibitors have been tested in the treatment of pain but generally have had little beneficial

effect. Selective norepinephrine (NE) reuptake inhibitors do have an analgesic action, as do dual NE/5-HT reuptake inhibitors such as duloxetine. Tramadol, a weak centrally acting opioid, also has monoaminergic actions and is widely used to treat mild pain. Its relatively weak efficacy as a single agent is increased when combined with acetaminophen, and its lack of abuse potential makes the drug attractive to prescribers. Other compounds also have regulatory roles in the spinal cord. The cannabinoid receptors and the endogenous cannabinoids have recently become a focus for research on pain regulation. There are two cannabinoid receptors, both of which are G protein-coupled: CB1, expressed in the brain, spinal cord, and sensory neurons; and CB2, largely expressed in nonneural tissues, especially immune cells including microglia. Several endogenous cannabinoids have been identified, including members of the anandamide and 2-arachidonylglycerol (2AG) families. Anandamide and 2-AG are synthesized via separate pathways for immediate use without storage. Anandamide has relatively low efficacy at CB1 and CB2, whereas 2-AG has high efficacy at both receptors. Clearance of anandamide is mediated by fatty acid amino hydrolase (FAAH); 2-AG is cleared via monacylglycerol lipase. A combination of anecdotal evidence and clinical trial data suggests that marijuana has an analgesic effect in patients with AIDS neuropathy or multiple sclerosis. Selective cannabinoid receptor agonists and FAAH inhibitors under development may prove useful for pain management. Preclinical data have specifically implicated the CB1 receptor as a mediator of analgesia following a stressor, whereas CB2 receptors are up-regulated in spinal cord microglia after peripheral nerve injury. Endogenous cannabinoids could modulate pain via cannabinoid receptors that are located peripherally or in the spinal cord and that affect nociceptive transmission or via receptors in the periaqueductal gray that affect descending inhibitory projections. Centrally acting CB1 agonists could have psychotropic effects and may have abuse potential. The CB1 receptor antagonist rimonabant was approved in 2006 in Europe for use in the treatment of obesity but was later withdrawn because of concerns about adverse effects including severe depression and suicidality. This agent was never approved for use in the United States.

 PATHOPHYSIOLOGY The pain processing circuit described above is responsible for producing acute nociceptive pain, a physiologic, adaptive sensation elicited only by noxious stimuli that acts as a warning or protective signal. There are some clinical situations, such as acute trauma, labor, or surgery, in which it is necessary to control nociceptive pain. In these cases, the pain pathway can be interrupted either by blocking transmission with local anesthetics (see Chapter 11) or by administering high-dose opioids. The opioids may be rapidly acting, such as remifentanil for intraoperative use, or more slowly acting, such as morphine; administered perioperatively, morphine retains activity for postoperative pain control. Both peripheral inflammation and nervous system damage produce pain that is characterized by hypersensitivity to noxious and innocuous stimuli and by spontaneous pain that arises in the absence of any obvious stimulus. Understanding the mechanisms responsible for these types of clinical pain will facilitate both the appropriate use of currently available drugs and the development of novel therapeutic agents.

Mechanical Chemical

P

Na+/Ca2+ influx

Thermal

Generator potential (membrane depolarization)

Reach voltage-gated sodium channel threshold

PKC activation Sensitizing agents

PKA activation P Tyr

TrkA

Na+

Action potential

Primary sensory neuron central terminal Action potential

Glu Ca2+

Calcium influx Glu

Synaptic vesicle S release

BDNF Substance P Ca2+ Mg2+

Glu

Glu

Na+

Glu

NMDA-R P

mGluR

AMPA-R Ca2+

P

Na

NK1

P Tyr

Tyr

P

+

TrkB

Initial depolarization

Kinase activation (PKC, CAMK II, ERK)

Phosphorylation of postsynaptic proteins

Phosphorylation of gene regulatory proteins

Secondary relay neuron (postsynaptic membrane)

Altered gene expression

Short term sensitization

Long term sensitization

CHAPTER 17 / Pharmacology of Analgesia 273

To brain Dorsal root ganglion

Altered gene expression and sensitivity

Dorsal horn

Schwann cell reaction, inflammatory cell infiltration, cytokine and growth factor secretion Free nerve endings

Neurotrophic support

Loss of neurotrophic support

Site of axonal injury Spinal cord

FIGURE 17-7.

Schematization of neuropathic pain. Nerve injury results in a combination of negative signals and positive signals that alter the physiology of the nociceptive system. The loss of neurotrophic support alters gene expression in the injured nerve fiber, whereas the release of inflammatory cytokines alters gene expression in both the injured and adjacent uninjured nerve fibers. These changes in gene expression can lead to altered sensitivity and activity of nociceptive fibers and, thus, to the continued perception of injury that is characteristic of neuropathic pain.

Evidence from a rare autosomal dominant disorder, familial hemiplegic migraine (FHM), may shed light on the mechanisms of migraine in general. This disorder consists of migraine attacks with a particular aura characterized by one-sided motor paralysis. Three genes have been associated with FHM: CACNA1A, ATP1A2, and SCNA1. CACNA1A encodes a Cav2.1 voltage-sensitive calcium channel subunit. In animal models, Cav2.1 gain-of-function mutations cause increased presynaptic calcium and increased glutamate release, which may help explain the trigger for cortical spreading depression. ATP1A2 encodes a subunit of the Na⫹/K⫹ ATPase, which is critical for the maintenance of neuronal membrane potential and which produces the Na⫹ gradient needed for glutamate transport. SCNA1 encodes a voltagesensitive sodium channel subunit that is involved in action potential conduction. Whether the more common forms of migraine are associated with similar changes in these genes remains unknown.

 PHARMACOLOGIC CLASSES AND AGENTS Several drug classes are widely used for pain relief. These include opioid receptor agonists, NSAIDs (see Chapter 42), tricyclic antidepressants (see Chapter 14, Pharmacology of Serotonergic and Central Adrenergic Neurotransmission), antiepileptic drugs (sodium channel blockers) (see Chapter 15, Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System), NMDA receptor antagonists (see Chapter 12, Pharmacology of GABAergic and Glutamatergic Neurotransmission), and adrenergic agonists. In addition, 5-HT1 receptor agonists have specific applications in the acute treatment of migraine.

Opioid Receptor Agonists Opioid receptor agonists are the primary drug class used in the acute management of moderate to severe pain. The

naturally occurring opioid receptor agonist morphine has the greatest historical importance and remains in wide use, but synthetic and semisynthetic opioids add pharmacokinetic versatility. Historically, opioids have been most widely used to treat acute and cancer-related pain, but in recent years, they have become one component of the management of chronic noncancer pain as well. Mechanism of Action and Major Adverse Effects Opioid receptor agonists produce both analgesia and other effects by acting on ␮-opioid receptors (Fig. 17-8). Sites of analgesic action include the brain, brainstem, spinal cord, and primary afferent peripheral terminals, as described previously. Opioids produce a wide array of adverse effects. These effects are qualitatively similar across opioids, but may vary in intensity. In the cardiovascular system, opioids can reduce sympathetic tone and lead to orthostatic hypotension; a subgroup of opioids, most notably morphine, causes histamine release that can also contribute to orthostatic hypotension via vasodilation. Opioids can also cause bradycardia. Respiratory effects are often the major, doselimiting adverse effect of opioids. By acting on the medullary respiratory control center, opioids blunt the respiratory response to carbon dioxide and can cause periods of apnea. Importantly, the respiratory effects of opioids interact with other stimuli; painful or other arousing stimuli can promote ventilation, while natural sleep synergizes with opioids to suppress ventilation. Acting through receptors in the medullary chemoreceptor zone and the gastrointestinal tract, opioids also produce nausea, vomiting, and constipation. Acting through receptors in the genitourinary system, opioids can cause urinary urgency and urinary retention. In the central nervous system, opioids can cause sedation, confusion, dizziness, euphoria, and myoclonus. It has recently become apparent that excessive use of opioids can lead to a paradoxical opioid-induced hyperalgesia. Opioid use is often associated with the development of tolerance, in which repeated use of a constant dose of a drug

Primary sensory neuron central terminal Action potential

μ-agonist Glu

μ

Ca2+

Calcium influx

Glu

Synaptic vesicle release

Neuropeptides CGRP Substance P K+ Na+

Descending modulation or exogenous drug

μ-agonist (opioid, enkephalin, endorphin)

K+ channel

μ-opioid receptor

Glu

AMPA-R K+ Na+

K+ conductance

Postsynaptic hyperpolarization

Secondary relay neuron (postsynaptic membrane)

Voltage-gated Na+ channels reaching threshold

Action potential generation

276 Principles of Central Nervous System Pharmacology Peripheral inflammation

Central cytokine release

COX-2 upregulation in inflammatory cells

COX-2 upregulation in dorsal horn neurons and supporting cells

Acetaminophen Celecoxib NSAIDs Prostaglandin production

Prostaglandin production

Constitutive COX-1 Action on peripheral terminal PGE2 receptors

Action on PGE2 receptors on dorsal horn neurons

Peripheral sensitization

Enhanced depolarization of secondary sensory neurons

FIGURE 17-9. Mechanism of analgesic action of cyclooxygenase inhibitors. Inflammatory states are often associated with the production of prostaglandins, which are important mediators of both peripheral (left) and central (right) pain sensitization. In the periphery, prostaglandins produced by inflammatory cells sensitize peripheral nerve terminal prostaglandin (EP ) receptors, making them more responsive to a painful stimulus. In central pain pathways, cytokines released in response to inflammation induce prostaglandin production in the dorsal horn of the spinal cord. These prostaglandins sensitize secondary nociceptive neurons and thereby increase the perception of pain. Nonsteroidal anti-inflammatory drugs (NSAIDs) block peripheral and central sensitization mediated by prostanoids that are released in inflammation; NSAIDs also reduce the extent of inflammation.

effects are primarily attributable to inhibition of COX-1, a constitutive enzyme responsible for the production of prostanoids involved in physiologic tissue maintenance and vascular regulation. However, this view may be an oversimplification because COX-2 may be induced to support COX-1 activity in the setting of gastric mucosal injury, and COX-1 may produce prostaglandins in tandem with COX-2 in inflammatory states. There is also concern that COX-2 inhibition may promote thrombosis and reduce or delay wound healing. Specific Agents There are several major classes of NSAIDs, including the salicylates (aspirin or acetylsalicylate), indole acetic acid derivatives (indomethacin), pyrrole acetic acid derivatives (diclofenac), propionic acid derivatives (ibuprofen), and benzothiazines (piroxicam). The para-aminophenols (acetaminophen) are a related class of compounds with analgesic and antipyretic activity, but not anti-inflammatory activity. The COX-2 selective inhibitors celecoxib, rofecoxib, and valdecoxib were designed to produce analgesia equivalent to that of the NSAIDs while decreasing the adverse effects associated with chronic NSAID use. This has turned out to be a disappointment, and both rofecoxib and valdecoxib have been withdrawn from the market because of an increased risk of cardiovascular effects and skin reactions. Representative agents are discussed here; further information on their

anti-inflammatory uses and adverse effects is discussed in Chapter 42. • Acetylsalicylic acid (aspirin) acts by covalently acetylating the cyclooxygenase active site in both COX-1 and COX-2. Aspirin is rapidly absorbed and distributed throughout the body. Chronic aspirin use can produce gastric irritation and erosion, hemorrhage, vomiting, and renal tubular necrosis. Aspirin is of great value in the treatment of mild or moderate pain. • The coxibs are COX-2 selective enzyme inhibitors. Currently, only celecoxib remains in clinical use in the United States. This class of drugs was originally reserved for patients who required NSAIDs but were at high risk for developing gastrointestinal (GI), renal, or hematologic adverse effects, although there is no clinical evidence that celecoxib reduces the risk of GI adverse effects. • The widely used compound ibuprofen is a derivative of propionic acid. Used primarily for analgesia and antiinflammatory action, it also is an antipyretic, and it has a lower incidence of adverse effects than aspirin. Another common propionic acid derivative is naproxen. Compared to ibuprofen, naproxen is more potent and has a longer half-life; therefore, it can be administered less frequently with equivalent analgesic efficacy. Its adverse effect profile is similar to ibuprofen, and it is generally well tolerated. As with all NSAIDs, ibuprofen and naproxen can cause GI complications ranging from dyspepsia to gastric bleeding. • The pyrrole acetic acid derivatives diclofenac and ketorolac are used to treat moderate to severe pain. Ketorolac can be administered orally or parenterally, while diclofenac is available in oral formulations. Both agents carry a risk of severe adverse effects, including anaphylaxis, acute renal failure, Stevens–Johnson syndrome (a diffuse life-threatening rash involving the skin and mucous membranes), and gastrointestinal bleeding. Ketorolac is valuable for shortterm pain control when avoidance of opioid adverse effects is desirable, for example, in day-surgery patients. Topical formulations of these drugs may have some utility. • Acetaminophen (paracetamol) preferentially reduces central prostaglandin synthesis by an uncertain mechanism; as a result, the drug produces analgesia and antipyresis but has little anti-inflammatory efficacy. Acetaminophen is frequently combined with weak opioids for the treatment of moderate pain, and preparations featuring acetaminophen combined with codeine, hydrocodone, oxycodone, pentazocine, or propoxyphene are available. Following deacetylation to its primary amine, acetaminophen is conjugated to arachidonic acid by fatty acid amide hydrolase in the brain and spinal cord; the product of this reaction, N-arachidonoylphenolamine, may inhibit COX-1 and COX-2 in the CNS. N-arachidonoylphenolamine is an endogenous cannabinoid and an agonist at TRPV1 receptors, suggesting that direct or indirect activation of TRPV1 and/ or cannabinoid CB1 receptors could also be involved in the mechanism of acetaminophen action. A major concern for acetaminophen is its low therapeutic index, as the drug is hepatotoxic and overdose can result in liver failure. Tramadol is a centrally acting analgesic. Analgesia apparently results from a monoaminergic effect within the CNS, as well as an opioid effect mediated by a metabolite that is formed by O-demethylation of the parent drug by CYP2D6. Tramadol has minimal abuse liability, but it does

278 Principles of Central Nervous System Pharmacology

Clonidine does, however, cause postural hypotension; this effect limits its usefulness in pain control.

Migraine Therapy The treatment of pain associated with migraine has features distinct from the treatment of other pain conditions. In many but not all patients, an effective treatment for migraine is the triptan class of serotonin receptor agonists; the most well-studied example is sumatriptan. The triptans are selective for the 5-HT1B and 5-HT1D receptor subtypes of the 5-HT1 family, one of the seven families of serotonin receptor. 5-HT1B receptors are located on vascular endothelial cells, smooth muscle cells, and neurons, including trigeminal nerves. 5-HT1D receptors are present in the trigeminal nerves that innervate the meningeal blood vessels. Triptrans reduce both sensory activation in the periphery and nociceptive transmission in the brainstem trigeminal nucleus, where they diminish central sensitization. The triptans also cause vasoconstriction, opposing the vasodilation thought to be involved in the pathophysiology of migraine attacks. It remains unclear whether the vasoconstriction is helpful in producing the antimigraine actions of these drugs, however. Furthermore, as a result of this vasoconstrictive effect, the triptans can be dangerous in patients with coronary heart disease. The triptans can reduce the pain and other symptoms associated with acute migraine attack and have largely replaced the vasoconstrictive agent ergotamine in the treatment of migraine. Sumatriptan can be administered subcutaneously, orally, or by nasal inhalation; the nasal formulation may have an improved therapeutic index. Several other orally administered agents in the triptan class are also available, including zolmitriptan, naratriptan, and rizatriptan (see Drug Summary Table). NSAIDs, opioids, caffeine, and antiemetics also have activity and some utility for treatment of acute migraine headaches. For example, a combination of indomethacin, prochlorperazine, and caffeine may have effectiveness similar to triptans in the treatment of migraine attacks. During an attack, migraine patients often experience gastric stasis that can reduce the bioavailability of oral medications. CGRP receptor antagonists are promising candidates for migraine therapy. Although the triptans are relatively effective in ameliorating the acute symptoms of migraine, other classes of drugs are used to reduce the frequency of attacks. Numerous drugs are used for migraine prophylaxis, including ␤-adrenergic blockers, valproic acid, serotonin antagonists, and calcium channel blockers. These agents are generally chosen based on the severity and frequency of the migraine attacks, the cost of the drug, and the adverse effects of the drug in the context of the individual patient. None has been shown to have a high level of efficacy, and new drugs need to be developed for more effective migraine prophylaxis.

 CONCLUSION AND FUTURE DIRECTIONS Because of the limited efficacy of any single drug, it is common in clinical practice to use a polypharmacy approach to manage pain. In combination, several drugs that are only moderately effective as single agents can have additive or supra-additive effects. This is largely a consequence of the multiple processing events and mechanisms responsible for

producing pain; intervention at several steps may be required to achieve adequate analgesia (Fig. 17-10). Because many drugs used to treat pain are also active systemically and/or in parts of the nervous system that are not related to somatic sensation, analgesics can produce deleterious adverse effects. One approach to limiting toxicity is to use localized (nonsystemic) forms of drug delivery. In particular, epidural and topical delivery limit the drug to a local site of action.

Central perception (opioids)

Cortex

Thalamus

Relay and descending modulation

Brainstem

Transmission (opioids, antidepressants, NSAIDs, antiepileptics, α2 adrenergic agonists, celecoxib, α2δ binding agents)

Spinal cord

Conduction (sodium channel blockers)

Peripheral stimulus

Signal transduction (NSAIDs)

FIGURE 17-10. Summary of the sites of action of the major drug classes used for pain management. Analgesics target various steps in pain perception, from the initiation of a pain stimulus to the central perception of that pain. NSAIDs modulate the initial membrane depolarization (signal transduction) in response to a peripheral stimulus. Sodium channel blockers decrease action potential conduction in nociceptive fibers. Opioids, antidepressants, NSAIDs, antiepileptic drugs (anticonvulsants), and ␣2-adrenergic agonists all modulate transmission of pain sensation in the spinal cord by decreasing the signal relayed from peripheral to central pain pathways. Opioids also modulate the central perception of painful stimuli. The multiple sites of action of analgesics allow a combination drug approach to be used in pain management. For example, moderate pain is often treated with combinations of opioids and NSAIDs. Because these drugs have different mechanisms and sites of action, the combination of the drugs is more effective than one drug alone.

CHAPTER 17 / Pharmacology of Analgesia 279

Many of the opioids are short-acting and must be administered frequently to patients in severe pain. Modes of drug delivery have also been developed to optimize the pharmacokinetics of the short-acting opioids; these methods include transdermal and buccal dosage forms, patient-controlled analgesia devices, and controlled-release oral preparations. Patient-controlled devices ensure that patients do not suffer pain because of waning drug effects, and instrumental controls can effectively prevent overdose. At the present time, however, patient-controlled technologies are suitable only for inpatient treatment. Most of the currently available analgesics have been identified by empirical observation (opioids, NSAIDs, and local anesthetics) or serendipity (antiepileptic drugs). Now that the mechanisms responsible for pain are being explored at a molecular level, many new targets are being revealed that are likely to lead to new and different classes of analgesics. It is hoped that drugs active at these targets will achieve greater efficacy and have fewer adverse effects than current therapies. Effective pain management approaches must not only rely on pharmacologic intervention; physical therapy and rehabilitation and, in some very limited situations, surgical approaches may also have a role. The placebo reaction produces analgesia and may explain the limited success produced by treatments such as acupuncture and homeopathy. These effects are usually unpredictable, modest, and of short duration. The growing complexity of pain management has spawned specialized pain services for inpatient pain control, as well as pain clinics and centers for the outpatient management of chronic pain.

Acknowledgments The authors thank Salahadin Abdi, MD, Rami Burstein, PhD, Carl Rosow, MD, PhD, and Joachim Scholz, MD, for their valuable comments.

Suggested Reading Costigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 2009;32:1–32. (Overview of mechanisms of neuropathic pain.) Drenth JP, Waxman SG. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders. J Clin Invest 2007;117:3603– 3609. (Reviews channelopathies that produce pain and their treatment.) Eisenberg E, McNicol ED, Carr DB. Efficacy and safety of opioid agonists in the treatment of neuropathic pain of nonmalignant origin. JAMA 2005;293:3043–3052. (A systematic review of published randomized controlled trials using opioids for nonmalignant neuropathic pain.) Finnerup NB, Otto M, McQuay HJ, et al. Algorithm for neuropathic pain treatment: an evidence-based proposal. Pain 2005;118:289–305. (Clinical approach to management of neuropathic pain.) Patapoutian A, Tate S, Woolf CJ. Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov 2009;8:55–68. (Review of the status of TRP channels as analgesic targets.) Rosow CE, Dershwitz M. Pharmacology of opioid analgesics. In: Longnecker D, Brown DL, Newman MF, Zapol WM, eds. Anesthesiology. New York: McGraw Hill; 2008. (Detailed review of opioid pharmacology.) Taylor CP. Mechanisms of analgesia by gabapentin and pregabalin–calcium channel alpha2-delta [Cavalpha2-delta] ligands. Pain 2009;142:13–16. (Review on the mechanisms of action of alpha 2 delta ligands as analgesics.) Woolf CJ. Pain: moving from symptom control toward mechanism-specific pharmacologic management. Ann Intern Med 2004;140:441–451. (Advances in molecular understanding of pain pathways.)

18 Pharmacology of Drugs of Abuse Peter R. Martin, Sachin Patel, and Robert M. Swift

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 284-285 DEFINITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 MECHANISMS OF TOLERANCE, DEPENDENCE, AND WITHDRAWAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Dependence and Withdrawal . . . . . . . . . . . . . . . . . . . . . . 289 MECHANISMS OF ADDICTION . . . . . . . . . . . . . . . . . . . . . . . 291 Learning and Development of Addiction . . . . . . . . . . . . . . 291 Variables Affecting the Development of Addiction . . . . . . . 293 Role of Personality Characteristics and Co-Occurring Disorders in Addiction. . . . . . . . . . . . . . . . . 295 DRUGS OF ABUSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Opioids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Benzodiazepines and Barbiturates . . . . . . . . . . . . . . . . . . 296

Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Nicotine and Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Cocaine and Amphetamine . . . . . . . . . . . . . . . . . . . . . . . 299 Marijuana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Other Abused Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 MEDICAL COMPLICATIONS OF DRUG ABUSE AND DEPENDENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 TREATMENTS FOR ADDICTION . . . . . . . . . . . . . . . . . . . . . . 302 Detoxification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Self-Help and Mutual Support Programs . . . . . . . . . . . . . 303 Pharmacologic Treatment of Addiction . . . . . . . . . . . . . . . 304 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 306 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

 INTRODUCTION

development of addiction. For example, societal attitudes toward drug use often influence the likelihood that a drug will be taken in the first place. The availability and cost of a drug are also affected by its legal and tax status. The availability of other, nondrug alternatives may be a key factor in determining the likelihood of drug use and addiction. This chapter describes the mechanisms of action of selected representative drugs of abuse, and the mechanisms of other important drugs of abuse are summarized in Table 18-1. Since addiction is a disorder of brain reward pathways, learning, and motivated behavior, the use of medications in an integrated pharmacopsychosocial approach to treat addiction is also discussed.

This chapter considers pharmacologic agents implicated in drug abuse and dependence and the brain processes relevant to understanding drug addiction. While the pharmacology of these agents is important to understanding their effects on behavior and their abuse liability, personality characteristics, as well as the presence of co-occurring psychiatric and medical conditions, may also contribute to the risk of developing drug abuse and addiction. Understanding abuse and addiction as a biopsychosocial disorder, rather than simply the pharmacologic consequences of chronic drug use, has led to recognition of the central role that learning plays in drug addiction and the potential for an integrated pharmacopsychosocial approach to treatment. Most individuals with drug use disorders also have a second diagnosable psychiatric condition, but it is not easy to determine whether psychiatric symptoms are a cause or consequence of drug use. For example, although alcohol is widely used to self-medicate depression and/or anxiety, it may be difficult to determine whether such psychiatric symptoms in alcoholics are the cause of drinking or its effect, because the actions of alcohol per se, as well as alcohol withdrawal and dependence, can also result in significant anxiety or depression. Genetic determinants of the psychopharmacologic actions of abused drugs are increasingly recognized. Nonetheless, environmental variables have a significant influence on the 284

 DEFINITIONS The empirically based nomenclature promulgated by the American Psychiatric Association (APA) in the Diagnostic and Statistical Manual (DSM, Box 18-1) defines substance dependence (used interchangeably in this chapter with addiction) as “maladaptive use” leading to “significant impairment or distress.” This definition avoids value judgment and is generalizable across cultures. Psychosocial features of dependence are similar for diverse psychopharmacologic agents with abuse liability and are likely more important in the development and maintenance of pathologic drug use than the unique pharmacologic profile of any given drug. Diagnostic features are conceptualized as clinical clusters of symptoms: loss of control,

288 Principles of Central Nervous System Pharmacology Acute use

Withdrawal

A

GABAA-R(α4) GABAA-R

Cl-

Cl-

Cl-

Cl-

Ca2+

Cl-

P Ca2+

Ca2+

Ca2+

Ca2+ Cl-

Cl-

Alcohol

P

Ca2+ Ca2+

Ca2+

NMDA-R(NR2B) P

NMDA-R

Decreased excitability

Increased excitability

B

Anxiolysis/ sedation

Anxiety

Coma

Seizures

Acute use

Withdrawal

GABA Morphine αi β

Neuroadaptation

γ

GDP

γ

K+

Time x Dose

K+channel

Dependence

AC AC

PKA PKA

CREB

Gene transcription

C

Arousal/pleasure

Fatigue/dysphoria

Dopamine receptor Dopamine

Acute use

Withdrawal

Cocaine Amphetamine

Decreased excitability

Increased excitability

Decreased neurotransmitter release

Increased neurotransmitter release

Euphoria

Withdrawal symptoms (dysphoria, lacrimation, diarrhea)

Neuroadaptation Time x Dose DA transporter Dopamine D2 receptor DA

DA

L-DOPA

Tyrosine hydroxylase

L-DOPA

Tyrosine hydroxylase

Tyrosine

Neuroadaptation Time x Dose

Dependence

Tyrosine

αi GDP

AC

μ-opioid receptor

β

Dependence

P

Receptor degradation K+

K+ channel (closed)

CHAPTER 18 / Pharmacology of Drugs of Abuse 289

FIGURE 18-1.

Mechanisms of acute drug action for depressants, opioids, and psychostimulants, and development of neuroadaptation and dependence in response to chronic drug use. (A) Alcohol modulates the major inhibitory and excitatory neurotransmitter systems of the brain via effects on GABAA and NMDA receptors, respectively. Alcohol is a positive allosteric modulator of GABAA receptors. Alcohol increases chloride conductance through GABAA receptors, resulting in cellular hyperpolarization. Alcohol also decreases calcium conductance through NMDA receptors, further decreasing cellular excitation. These dual actions on GABAA and NMDA receptors contribute to alcohol’s anxiolytic, sedative, and CNS-depressant effects. Molecular adaptations to chronic alcohol exposure include: (1) internalization and decreased surface expression of “normal” ␣1 subunit-containing GABAA receptors; (2) increased surface expression of “low alcohol sensitivity” ␣4 subunit-containing GABAA receptors; and (3) increased phosphorylation of NMDA receptors containing “high conductance” NR2B subunits. Thus, neuroadaptation results in tolerance to the acute depressant effects of alcohol and occurs concomitantly with dependence. During withdrawal (i.e., in the dependent state but in the absence of alcohol), these adaptations result in generalized hyperexcitability of neurons. CNS excitation is expressed as anxiety, insomnia, delirium, and potentially seizures. (B) Opioids activate ␮-opioid receptors located on synaptic nerve terminals. Acute activation of ␮-opioid receptors results in G protein-dependent activation of potassium channels and inhibition of adenylyl cyclase activity. These effects result in cellular hyperpolarization and decreased GABA release from the nerve terminal; the decreased GABA release results in disinhibition of ventral tegmental area (VTA) dopamine neurons. Molecular adaptations to chronic ␮-opioid receptor stimulation include: (1) increased ␮-opioid receptor phosphorylation, resulting in receptor internalization and degradation; (2) decreased efficacy of ␮-opioid signal transduction; and (3) hyperactivation of adenylyl cyclase signaling, leading to enhanced GABA release and to increased gene transcription via activation of transcription factors including cyclic AMP response element binding protein (CREB). Thus, neuroadaptation results in tolerance to the euphoric effects of opioids. During withdrawal (i.e., in the dependent state but in the absence of opioid), the enhanced GABA release from inhibitory interneurons results in inhibition of VTA dopamine neurons, dysphoria, and anhedonia. (C) Acute cocaine exposure inhibits dopamine reuptake transporters (DAT), resulting in increased synaptic dopamine levels and increased postsynaptic dopamine receptor activation at synapses in the nucleus accumbens; in turn, these effects cause feelings of euphoria and increased energy. Increased extrasynaptic dopamine also results in D2 autoreceptor activation, which decreases dopamine synthesis. Amphetamine both releases vesicular transmitter stores into the cytoplasm and inhibits neurotransmitter reuptake into vesicles; these combined actions cause neurotransmitter concentrations to increase in the synaptic cleft. During chronic psychostimulant exposure, DAT expression increases, the number of postsynaptic dopamine receptors decreases, and presynaptic dopamine is depleted. Thus, neuroadaptation results in tolerance to the euphoric effects of psychostimulants. During withdrawal (i.e., in the dependent state but in the absence of psychostimulant), the decreased synaptic levels of dopamine that result from reduced dopamine synthesis and increased clearance through DAT cause decreased activation of postsynaptic dopamine receptors and feelings of dysphoria, fatigue, and anhedonia.

Dependence and Withdrawal Dependence is typically associated with tolerance, and it results from mechanisms closely related to those that produce pharmacodynamic and learned tolerance. The dependence syndrome results from the need for the drug to be present in the brain to maintain “near-normal” functioning. If the drug is eliminated from the body so that it no longer occupies its site of action, the adaptations that produced dependence are unmasked and manifested as an acute withdrawal syndrome that lasts until the system re-equilibrates to the absence of drug (days). Subsequently, a protracted withdrawal syndrome, characterized by craving for the drug (i.e., an intense preoccupation with obtaining the drug), may emerge and continue indefinitely (years). Protracted withdrawal is also associated with subtle dysregulation of learning, drives/motivations, and reward. This syndrome should be distinguished from premorbid risk factors for addiction that do not resolve with abstinence and from brain injury that is sustained as a result of drug use. Like tolerance, dependence is associated with changes in cellular signaling pathways (Fig. 18-1). For example, upregulation of the cAMP pathway by a drug contributes to acute withdrawal upon discontinuation of the drug because up-regulated adenylyl cyclase effects a “supranormal” response in neurons when physiologic amounts of neurotransmitter stimulate the cAMP-coupled receptor. Conversely, a drug that produces dependence by decreasing receptor number or receptor sensitivity renders the down-regulated receptors understimulated after drug discontinuation. The effects of alcohol illustrate that excitatory and inhibitory mechanisms can act in a synergistic fashion on opposing neurotransmitter systems. Acute alcohol intake causes sedation by facilitating the inhibitory activity of GABA at its receptors and inhibiting the excitatory activity of glutamate at its receptors. Over time, the GABA receptors are downregulated and their subunit structure is modified through a variety of molecular mechanisms, thus decreasing the

level of inhibition to counter the sedative effects of alcohol. Simultaneously, the NMDA receptors are up-regulated, also decreasing the level of inhibition due to alcohol. If the alcohol is abruptly removed, the decreased GABAergic inhibition and enhanced glutamatergic excitation result in a state of central nervous system hyperactivity, which causes the signs and symptoms of alcohol withdrawal. The balance between these inhibitory (GABAergic) and excitatory (glutamatergic) pathways may explain the alternating sedation and hyperactivity characteristic of alcohol withdrawal. Because dependence can occur without tolerance and vice versa, it is clear that learning-related changes, not necessarily due to the pharmacologic actions of a drug, are also involved. In the 1950s, Olds and Milner implanted electrodes in various regions of the rat brain to systematically determine which neuroanatomic areas could reinforce self-stimulation. (Self-stimulation consisted of a short pulse of nondestructive electric current that was delivered in the brain at the site of the electrode upon the animal’s pressing of a lever.) The medial forebrain bundle and ventral tegmental area (VTA) in the midbrain were found to be particularly effective sites. These sites have been termed “pleasure centers,” or the foci of reward in the brain. A subset of dopaminergic neurons projects directly from the VTA to the nucleus accumbens (NAc) via the medial forebrain bundle. It is believed that these neurons are crucial for the brain reward pathway, which reinforces motivated behavior and facilitates learning and memory via links to the hippocampus, amygdala, and prefrontal cortex. Severing this pathway, or blocking dopamine receptors in the NAc with a dopamine receptor antagonist (such as haloperidol; see Chapter 13, Pharmacology of Dopaminergic Neurotransmission), decreases electrical self-stimulation of the VTA. Moreover, release of dopamine in the NAc can be detected in vivo using the technique of microdialysis, whereby a cannula is inserted into a specific brain region in order to determine the concentrations of neurotransmitters. These measurements show that increases in

290 Principles of Central Nervous System Pharmacology From cortex

A

B

Sensory cues (action potential)

Sensory cues (action potential)

C

Glutamate NMDA-R Dopamine receptor

Neuroadaptation Na+

Ca2+

non-NMDA-R Ca2+ Na+

Natural reward

Na+

Ca2+

Time x Dose

Increase in glutamate receptors

Dependence Ca2+ Na+

Drug of abuse

Increase in structural proteins

From VTA

Dopamine

CaMKII

NAc

CaMKII

NAc Gene transcription

Reward learning

NAc Gene transcription

Out of control drug use

Dependence

Period of abstinence

D Relapse mechanisms Sensory cues

1

2

Ca2+ Na+

3

Ca2+ Na+

Stress

Ca2+ Na+

Drug re-exposure

NAc

Cellular excitation

Relapse

FIGURE 18-2.

NAc

Cellular excitation

Relapse

NAc

Cellular excitation

Relapse

Synaptic changes linking environmental stimuli, drug effects, and reward learning in drug dependence and mechanisms of relapse after abstinence. (A) Natural rewards such as food or sex increase dopamine release in the nucleus accumbens (NAc) and give rise to reward learning that links relevant environmental stimuli (sensory cues) with concurrent rewarding elements by altering neural circuitry in associative areas of the brain. Spiny neurons within the NAc receive glutamatergic inputs from the cortex that relay sensory cue information and dopaminergic inputs from the ventral tegmental area (VTA). The glutamatergic inputs act via NMDA receptors (permeable to calcium) and non-NMDA receptors (permeable to sodium). Coincident release of dopamine and glutamate results in potentiation of NMDA signaling, activation of calcium-calmodulin dependent kinase (CaMKII), and ultimately alterations in transcription of structural protein genes and glutamate receptor genes. These synaptic changes are thought to underlie reward learning. (B) Drugs of abuse induce amplified dopamine release and activate the same synaptic adaptations as natural reinforcers. Thus, drugs of abuse are thought to “hijack” evolutionary brain reward learning systems in a manner that leads to out-of-control drug use. (C) After chronic drug use, synaptic adaptations result in “potentiated synapses.” This potentiation is mediated via increased dendritic spine size, increased structural protein expression, and increased glutamate receptor surface expression; all of these adaptations occur in response to long-term transcriptional changes. (D) After a period of abstinence from drug use, multiple mechanisms can induce relapse to drug-taking behavior. (1) Stress can trigger relapse by increased dopamine release. In this potentiated state, dopamine can trigger cellular excitation and trigger relapse behaviors. (2) Exposure to drug-related sensory cues can trigger relapse via increased glutamate release, and the increased surface expression of glutamate receptors can lead to cellular excitation and relapse. (3) Exposure to small amounts of drug can reactivate relapse to drug self-administration in this potentiated state, since the amplified dopamine release can trigger cellular excitation.

CHAPTER 18 / Pharmacology of Drugs of Abuse 291

Prefrontal cortex Executive function Cognitive control Hippocampus Context / Memory NAc Orbitofrontal cortex Judgment Decision making

Amygdala Stress / Anxiety

Locus ceruleus

FIGURE 18-3. Integration of brain behavioral systems via connections to the mesolimbic dopamine pathway. Noradrenergic neurons originating in the locus ceruleus (black) relay information regarding novelty and arousal to dopaminergic neurons in the ventral tegmental area (VTA). The VTA projects to the nucleus accumbens (NAc) and cortex (red). Multiple inputs from the brain modify VTA output: glutamatergic input from the prefrontal cortex relays executive function and cognitive control; excitatory input from the amygdala signals stress and anxiety; and glutamatergic input from the hippocampus conveys contextual information and past experiences (blue). Together, these multiple inputs modify signaling in the mesolimbic dopamine pathway and modulate the perception of pleasure.

Arousal / Novelty

Ventral tegmental area

concentrations of dopamine are associated with drug selfadministration by laboratory animals and that dopaminergic synapses in the NAc are active during electrical stimulation of the brain reward pathway, supporting the hypothesis that NAc dopamine is necessary for reward. Drugs capable of causing dependence are readily self-administered by animals directly into the VTA, NAc, or the cortical or subcortical areas that innervate these two areas, often at the cost even of eating food (Fig. 18-3). Although the dopaminergic pathway mediates reward, dopamine may also increase the salience of stimuli, alert the organism to the importance of stimuli, and guide motor activity to seek rewarding stimuli. As discussed above, the dopamine pathway is activated by all drugs of abuse. Importantly, behaviors that are necessary for survival of the species (e.g., feeding, reproduction, and exploration) also result in dopamine release in the NAc but to a much smaller degree, suggesting that drugs of abuse may pharmacologically “hijack” the normal evolutionary functions of reward pathways. With repeated experiences via conditioning (i.e., the association of an element of the environment with the reward through rewiring of brain circuits), this dopamine pathway is also activated during anticipation of the reward, as can be demonstrated in humans using functional neuroimaging techniques such as positron emission tomography (PET) when addicts are exposed to drug-related sensory cues. Although the dopaminergic neurons that link the VTA and the NAc serve as the final common pathway of reward, these neurons receive inputs from a number of brain regions (cortex, hippocampus, thalamus, amygdala, and raphe nuclei) that modify reward and thereby mediate reward-associated learning (Fig. 18-4). Since withdrawal from certain drugs of abuse can be aversive, avoiding acute withdrawal was for many years thought to be the primary motivation for continued abuse. However, this explanation is not consistent with the observations that: the effects of addiction are felt long after the physical symptoms of withdrawal have abated; withdrawal can occur without concomitant drug-seeking, as is often the case after treatment for acute pain; and drugs

such as stimulants, hallucinogens, and cannabinoids cause significant dependence without a striking acute withdrawal syndrome. Years after an addict has discontinued use of a substance, s/he can experience intense cravings and, thus, is prone to relapse. The likelihood of relapse is especially strong in situations in which individuals simultaneously encounter both stress and the context in which the drug was previously used. In part, this is due to the interplay between reward and memory circuitry in the brain that, under normal circumstances, assigns emotional value to certain memories. Hence, the motivational underpinnings of drug-seeking are tied to both socioenvironmental stimuli and subjective effects of the drug, each of which can have both rewarding and aversive linkages with previous experiences via learning. This is a more complex explanation than the “simple” avoidance of acute withdrawal.

 MECHANISMS OF ADDICTION The drug-seeking activity characteristic of addiction results from the interplay of learning, reward mechanisms, and individual propensity toward the development of addiction.

Learning and Development of Addiction Recognition that chronic drug self-administration results in long-lasting changes in the experience of reward has led to our understanding that the relevant neural circuits can never return to their predrug state. The term allostasis describes this enduring, progressively evolving adaptive process in brain reward pathways upon repeated exposure to abused drugs. Allostasis means that the baseline to which the brain returns upon discontinuing drug use can change even after acute withdrawal has abated. (This is in contrast to homeostasis, which is defined as the process whereby a system repeatedly equilibrates to the same baseline.) Accordingly, even when the drug is no longer present in the brain, the addict cannot experience positive emotions in the way he did prior to beginning drug use (termed anhedonia); the unsuccessful attempt to recapture the previous “near-normal”

292 Principles of Central Nervous System Pharmacology

Prefrontal cortex

Nicotine

DA ACh

Ventral tegmental area

Nicotinic ACh receptor

NAc

Cannabinoids Opioids μ-opioid receptor

CB1 receptor C

GABA

Cortex

NMDA-R μ-opioid receptor Opioids NAc Alcohol PCP

Cocaine Amphetamine

Cannabinoids DAT CB1 receptor

DA

FIGURE 18-4.

The mesolimbic dopamine pathway: a final common substrate for the rewarding actions of drugs. All drugs of abuse activate the mesolimbic dopamine pathway, which comprises ventral tegmental area (VTA) dopamine neurons that project to the nucleus accumbens (NAc). Different interneurons interact with VTA neurons and NAc neurons to modulate mesolimbic neurotransmission. Nicotine interacts with excitatory nicotinic cholinergic receptors located on VTA dopamine neuron cell bodies to enhance dopamine release in the nucleus accumbens (NAc). Cocaine acts predominantly at the dopamine nerve terminal to inhibit reuptake of dopamine via the dopamine transporter (DAT), thus increasing synaptic levels of dopamine that can impinge on NAc. Amphetamine also acts at the dopamine nerve terminal to facilitate release of dopamine-containing vesicles, and possibly to enhance reverse transport of dopamine through DAT (not shown). Both cannabinoids and opioids decrease GABA release from local inhibitory interneurons in the VTA, resulting in disinhibition of dopamine neuron activity and increased dopaminergic neurotransmission. Cannabinoids and opioids can also act within the NAc. Alcohol and other CNS depressants act on NMDA receptors (NMDA-R) to reduce glutamatergic neurotransmission in the NAc. The effects of alcohol on dopaminergic neurons in the VTA appear to be both excitatory and inhibitory, and are the subject of active investigation (not shown).

state fuels drug-seeking. Human and animal studies have found evidence for long-term neuroadaptation in altered neurotransmitter levels (e.g., dopamine and serotonin depletion after chronic alcohol or stimulant use), changes in neurotransmitter receptors, altered signal transduction pathways, changes in gene expression, and altered synaptic configuration and function. Clinically, abstinent patients report not only craving but also dysphoria, sleep disturbances, and increased stress reactivity (e.g., panic attacks), which can last for weeks, months, or years after detoxification. A common misconception is that addicts are pleasure seekers and that their focus on drugs represents withdrawal from life into irresponsible hedonism. Current thinking

about addiction recognizes the heterogeneity of the addictive process. For some individuals, reward factors (positive reinforcement) may predominate, and getting high or feeling euphoric motivates drug use. For others, relief factors (negative reinforcement) predominate, such as drinking to reduce stress or to reduce the dysphoria of protracted withdrawal. A large proportion of addicts self-medicate to reduce distress associated with co-occurring psychiatric and medical disorders. Furthermore, the motivations to use early in the course of addiction may differ substantially from motivations as the illness progresses (Fig. 18-5). As a result of allostasis, positive reinforcement is rare in the later stages of the illness. For example, drinking in one’s teens to relieve

CHAPTER 18 / Pharmacology of Drugs of Abuse 293 Socio-environmental stimuli Peer group Drug paraphernalia

Discriminative stimuli Subjective effects of drug Drug taste, smell, appearance

Initiation of drug use

Chronic drug dependence Reinforcers

Reinforcers Euphoria Behavioral activation Novelty Anxiolysis Analgesia

Continued drug seeking

Social interaction Prevention of withdrawal

Aversive effects

Aversive effects

Sedation Acute withdrawal (hangover) Nausea Legal problems

Organic disease Societal stigma Legal problems

Cessation of drug

FIGURE 18-5.

Clinical determinants of drug-seeking change throughout the life course of addiction. The motivational underpinnings of drug-seeking are determined by socioenvironmental stimuli paired with subjective effects of the drug. Reinforcers of drug self-administration result in continued drug use, whereas aversive drug effects contribute to cessation of drug self-administration: whether an individual continues to use is a function of whether reinforcing or aversive effects predominate under the circumstances. Brain reward pathways are modified during the course of repeated drug self-administration, such that reinforcing and aversive effects are often different when drug use first begins compared to later in the course when drug self-administration may have become repetitive and out-ofcontrol. Ultimately, whether addiction progresses or the addictive disorder can be successfully arrested is determined by learning-related modification of reinforcing and aversive effects of drugs using pharmacopsychosocial interventions.

shyness may progress to drinking for euphoria and disinhibition. Ultimately, after years of drinking to intoxication, the middle-aged person may drink to prevent withdrawalassociated depression and anxiety, or perhaps to alleviate chronic pain. Drug use in each of these situations is linked via learning to elements of the environment associated with drug use or to memories and emotions, each of which can trigger craving and drug-seeking. The essence of addiction is drug-seeking behavior, whereby an individual cannot control the urge to obtain and use a psychoactive substance despite recognized negative consequences and at the exclusion of other needs that typically constitute a balanced life. Studies in laboratory animals suggest that drug-seeking behavior is the result of dysfunctional “reward learning,” i.e., the processes that guide the organism to fulfill needs or goals have gone awry. Thus, if the organism initiates an action that results in a goal or “reward” (e.g., self-administration of a psychoactive agent), and if the organism “learns” that its action resulted in the reward, the likelihood of engaging in that behavior is enhanced. For example, if a person uses cocaine for the first time and finds it pleasurable or that it alleviates depressive symptoms from which the individual is suffering, obtaining and using cocaine are reinforced. The intense experience of cocaine, relative to natural rewards such as food and sex, results in a preferential expenditure of energy to obtain

cocaine over other rewards. Thus, cocaine has effectively “hijacked” reward-learning systems, biasing future behavior in favor of obtaining cocaine over natural rewards. Reexposure to environmental or affective states that are associated with cocaine use serve as cues to increase drug-seeking behavior. For example, reexposure to drug paraphernalia can induce intense craving, drug-seeking behavior, and relapse in cocaine addicts.

Variables Affecting the Development of Addiction The development of addiction is dependent on the nature of the drug; genetic, acquired, psychological, and social traits of the drug user; and environmental factors. The ability of a drug to activate reward mechanisms is strongly correlated with its ability to cause addiction. Pharmacokinetic properties of the drug can significantly influence its effects on the brain. In general, the more rapid the rise in drug concentrations at the target neurons, the greater the activation of reward pathways. For example, many drugs of abuse are highly lipophilic and can easily permeate the blood–brain barrier. In addition, direct injection or rapid absorption of drug through a large surface area (e.g., through the lungs via smoking) is more highly reinforcing than slower absorption through the intestinal or nasal mucosa. Furthermore, rapidly eliminated drugs are more addictive than slowly eliminated drugs, since slow clearance of a drug maintains the drug concentration at the site of action for a longer duration, diminishing the severity of acute withdrawal. The importance of pharmacokinetic effects is demonstrated by the potential for abuse of various forms of cocaine (Fig. 18-6), and these principles are readily applicable to other drugs of abuse. The use of coca leaves as a chew or in teas is widely practiced among people living in the Andean mountains: this has a relatively low potential for addiction, because of the slow rate of rise and low peak concentration of drug attained by absorption through the buccal or intestinal mucosa. The rapid absorption of extracted cocaine through the nasal mucosa is substantially more reinforcing. The most reinforcing and addictive forms of cocaine are intravenous injections and inhalation of smoked freebase (crack cocaine), both of which result in a very rapid rise in plasma concentration and a high peak concentration of drug. Different people react differently to drugs. Some individuals use a drug once and never use it again; others use a drug repeatedly in moderate amounts without developing addiction; in others, the first use of a drug produces such an intense effect that the likelihood of addiction is high. The factors that make individuals more or less vulnerable to addiction upon exposure to a given drug are of continued research interest. A variety of predisposing or protective genetic, acquired, psychosocial, and environmental factors have been identified, but—as expected in a complex, multifactorial illness—individually each can explain only a relatively small component of the risk for addiction. Individual factors include: (1) resistance or sensitivity to the acute effects of a given drug; (2) differences in drug metabolism; (3) the potential for neuroadaptive changes with chronic drug exposure; (4) personality traits and co-occurring psychiatric and medical disorders that incline an individual to drug use;

294 Principles of Central Nervous System Pharmacology

Plasma concentration of cocaine (ng/ml)

A 500 400 300 200 100 0 0

60

120

180

240

300

360

Time (minutes after dose)

B

Intoxication level (0 –100 scale)

50

Route and Dose 40

IV–0.6 mg/kg Smoked – 100 mg base

30

Nasal – 2 mg/kg Oral – 2 mg/kg Placebo

20

10

0 0

60

120

180

240

300

360

Time (minutes after dose)

FIGURE 18-6.

Plasma cocaine concentrations and levels of intoxication as a function of route of administration of the drug. The pharmacokinetics (A) and pharmacodynamics (B) of cocaine are highly dependent on the route of administration of the drug. Intravenous (IV) cocaine and smoked free-base cocaine are associated with very rapid attainment of peak plasma drug concentrations (A) and high levels of intoxication (B). In contrast, the nasal and oral routes of administration are associated with a slower rise in plasma drug concentrations (A) and lower levels of intoxication (B). Because of the very rapid rise in plasma drug concentration and very high intoxication levels, intravenous and smoked cocaine carry a higher risk of addiction than cocaine taken nasally or orally.

and (5) susceptibility of the individual to brain injury associated with drug use that may modify drug effects. Genetic influences have been best studied in individuals with alcohol dependence. Heritability estimates suggest that genetic factors account for 50–60% of the variance associated with alcohol abuse, but the specific determinant(s) that lead to alcoholism in an individual are not known. In fact, many individuals whose family history highly predisposes them to alcohol dependence do not develop the disorder. Alcohol abuse and dependence are complex phenotypes determined by multiple genes, environmental exposures throughout the lifespan, gene– environment interactions, gene–behavior interactions, and gene–gene interactions. The best known examples of candidate genes that alter risk for alcohol dependence are the alcohol metabolism genes, including those encoding the alcohol dehydrogenases ADH1B*2, ADH2, and ADH3 that metabolize alcohol more rapidly and those encoding certain aldehyde dehydrogenases (particularly

ALDH2*2). Polymorphisms in these genes alter enzymatic activity and increase the levels of acetaldehyde, which causes aversive symptoms that act as a deterrent to drinking alcohol and to the development of alcohol dependence. Sensitivity to alcohol is also a physiologically based trait influenced by genetic inheritance. Low sensitivity to alcohol (high innate tolerance) is associated with an increased risk for developing alcoholism. Schuckit and colleagues have found evidence for genetic linkage of the “low level of response” phenotype to the same region on chromosome 1 that is linked to the “alcohol dependence” phenotype. However, subjective response to alcohol is a complex trait affected by several neurotransmitter systems. For example, individuals with the alcohol dependence-associated GABRA2 allele have a blunted subjective response to alcohol, and individuals carrying the ASP40 variant of the ␮-opioid receptor or those with a certain single nucleotide polymorphism of the cannabinoid receptor appear to have an enhanced euphoric response to alcohol.

CHAPTER 18 / Pharmacology of Drugs of Abuse 295

Role of Personality Characteristics and Co-Occurring Disorders in Addiction The clinical characterization of individuals who develop a drug-use disorder has been most extensively studied for alcohol dependence. The Cloninger classification of alcoholic subtypes relates genetic and neurobiological differences to the age of alcoholism onset and to personality traits. Type 1 (“late” onset) alcoholism is characterized by alcohol-related problems beginning after 25 years of age, less antisocial behavior, infrequent spontaneous drinking or loss of control, and guilt and concern about one’s alcoholism. Type 1 alcoholics are low in thrill-seeking, are harm-avoidant, and are dependent on approval from others. In contrast, type 2 alcoholism is characterized by early onset of alcoholrelated problems (before age 25), antisocial behavior, frequent spontaneous alcohol-seeking and loss of control, and little concern about the consequences of one’s drinking or its effects on others. Genetic predispositions to late-onset alcohol dependence are significantly influenced by precipitating environmental factors, whereas genetic predispositions to early-onset alcohol dependence are less influenced by the environment. The Lesch classification envisions four alcoholism subtypes: type 1 exhibits withdrawal symptoms, including alcohol-related delirium and seizures, relatively early in the drinking history; type 2 exhibits anxiety related to premorbid conflicts; type 3 is characterized by associated mood disorders; and type 4 has premorbid cerebral injuries and associated social problems. Alcohol subtypes are now being examined as predictors of response to medications used for the treatment of alcoholism. For example, earlyonset alcoholics may worsen their drinking and impulsive behavior in response to a selective serotonin reuptake inhibitor (SSRI), whereas late-onset alcoholics may improve with an SSRI. According to a major epidemiologic survey in the United States, the odds of having a mental disorder are three times greater if an individual also has a drug-use disorder than if the individual has no drug-use disorder. In decreasing order of association, these psychiatric diagnoses include bipolar affective disorder, antisocial personality disorder, schizophrenia, major depressive disorder, and anxiety disorders. Drug-use disorders occur at higher rates in those with alcoholism, and alcoholism is more prevalent among individuals with addiction to other drugs. The association between psychiatric disorders and drug-use disorders has led to theories of common pathogenesis and treatment strategies. For example, individuals with major depressive disorder are two to three times more likely to have a drug-use disorder throughout their lifetime than those without depression, and exacerbations of mood symptoms are prime precipitants of relapse to drug use (and vice versa). Of note, these associations seem to be generic with respect to which drugs are abused, suggesting that such abuse is related more to availability than to a specific pharmacologic mechanism of action. Physical disability and pain associated with medical illness or traumatic injury can greatly enhance the risk of a co-occurring drug-use disorder. Moreover, drug use not only complicates certain medical conditions, but for many of these illnesses (e.g., cirrhosis or traumatic brain injury due to motor vehicle accidents), alcohol and drug use should also be considered a significant causal factor. Similarly, increased pain perception is now understood to be a frequent

complication of chronic opioid administration (opioid hyperalgesia). Thus, many pain physicians no longer advise long-term use of opioid analgesics for treatment of chronic (nonterminal) pain, recognizing that detoxifying a patient from chronic opioid use can often result in a preferable outcome to continuing to increase the opioid dose. In conclusion, drug-use disorders are not only illnesses in their own right, but also common consequences of many psychiatric and medical conditions that, in turn, are further exacerbated by continued drug use.

 DRUGS OF ABUSE Many psychoactive substances have abuse potential through their activation of inputs in brain reward pathways. It is vital to understand the unique pharmacology of each agent to appropriately address overdose complications, metabolic consequences, and organ toxicity associated with abuse of these drugs. Several drugs with the potential to cause dependence are readily available and widely used, and they exact an enormous toll on public health (e.g., alcohol, nicotine). Other drugs are commonly prescribed for accepted medical purposes, and their mechanisms of action have been discussed in detail in previous chapters (e.g., opioids, barbiturates, benzodiazepines, stimulants). These drugs represent a significant cause of iatrogenic dependence in patients, and prescription drug abuse represents perhaps the fastest growing U.S. drug problem. Other commonly abused drugs are not generally prescribed in medical practice and are typically available only from illicit sources (e.g., cocaine, heroin). Finally, some drugs affect receptors that are actively being pursued as potential targets for therapeutic intervention, and it is controversial whether or how they should be regulated (e.g., cannabis, nicotine).

Opioids Opioid alkaloids have been used medically for centuries for analgesia, treatment of diarrhea and cough, and sleep induction. Central effects of opioids are biphasic, with behavioral activation at low doses and sedation at higher doses. These drugs depress respiration, and death from opioid overdose is invariably due to respiratory arrest. The ␮-opioid receptor appears to be the most important subtype for the reinforcing actions of opioids. Addicts describe an intense euphoric feeling (“rush”) that lasts for less than a minute upon the intravenous injection of heroin and that seems to be the reason for abuse. There appear to be two pathways by which opioids interact with the brain reward system. One site of action lies in the ventral tegmental area, where GABAergic interneurons tonically inhibit the dopaminergic neurons responsible for activating the brain reward pathway in the nucleus accumbens. These GABAergic interneurons can be inhibited by endogenous enkephalins, which bind to ␮-opioid receptors on the GABAergic terminals. Because exogenous opioids such as morphine also bind to and activate ␮-opioid receptors (see Chapter 17), exogenously administered opioids can activate the brain reward pathway by disinhibiting dopaminergic neurons in the ventral tegmental area (Fig. 18-4, Fig. 18-7). The second pathway is localized in the nucleus accumbens. Opioids acting in this region may inhibit GABAergic neurons that project back to the ventral tegmental area, perhaps as

Inhibitory neuron

A

Endogenous enkephalins

GABA

Dopaminergic neuron

Tonic dopamine release REWARD

Ventral tegmental area

Nucleus accumbens

Inhibitory neuron

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Exogenous opioids or Endogenous enkephalins

GABA

Dopaminergic neuron

Increased dopamine release

REWARD

Ventral tegmental area

Nucleus accumbens

CHAPTER 18 / Pharmacology of Drugs of Abuse 297

A

B 100 Full agonist

% Maximal binding

% Maximal signal transduction

100

Partial agonist

50

Antagonist

Buprenorphine Naloxone

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cAMP

cAMP

cAMP

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nAChR (closed)

nAChR (open)

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γ

ATP

Nicotine

D

αi β GDP

Drug withdrawal and craving

Treatment with partial agonist reduced withdrawal symptoms

FIGURE 18-8. Partial agonists in the treatment of addiction. (A) Full agonists at ␮-opioid receptors, such as morphine, produce maximal signal transduction (100%). Partial agonists, such as buprenorphine, produce reduced signal transduction (⬃50% of a full agonist). Antagonists, such as naloxone, do not stimulate signal transduction. (B) Both buprenorphine and naloxone have very high binding affinities for ␮-opioid receptors compared to morphine. Consequently, when ␮-opioid receptors are fully occupied by an agonist like morphine, both naloxone and buprenorphine displace morphine from the receptor and lead to withdrawal. (C) Upon morphine binding to ␮-opioid receptors, intracellular signaling leads to inhibition of adenylyl cyclase activity and a decrease in cyclic AMP (cAMP) production. Upon removal of morphine from ␮-opioid receptors, either by discontinuing morphine or by administration of an antagonist or partial agonist (withdrawal), the inhibition of adenylyl cyclase is released. The resulting large increase in cAMP production causes withdrawal symptoms, such as diarrhea, hyperalgesia, tachypnea, and photophobia. The use of a partial agonist, buprenorphine, can alleviate these withdrawal symptoms by “partial” activation of ␮-opioid receptors. In addition, binding of the high-affinity buprenorphine molecule to ␮-opioid receptors prevents lower affinity full agonists, such as morphine, from binding to and activating the receptor. Thus, the physiologic antagonist property of buprenorphine prevents the “high” associated with morphine use, but also alleviates craving and drug-seeking behavior. (D) Nicotine activates nicotinic acetylcholine receptors (nAChR), causing neuronal excitation. Nicotine withdrawal causes a rapid decrease in nAChR activity and a withdrawal syndrome associated with intense craving. Treatment with the partial nAChR agonist varenicline results in partial activation of nAChR and alleviation of withdrawal symptoms, but this activation is insufficient to cause dependence or a “high.” Importantly, binding of the high-affinity varenicline molecule to nAChRs prevents the lower affinity nicotine molecule from binding to and activating the receptor. Thus, varenicline can prevent the subjective “high” associated with nicotine use.

298 Principles of Central Nervous System Pharmacology

window than benzodiazepines and are used less frequently. For both of these drug classes, euphoric feelings are often reported in the early stage of intoxication and typically are the expressed reason for drug self-administration. Anxiolytic and tension-reducing properties may also contribute to the reinforcing actions and abuse potential of these drugs. All sedative-hypnotics can cause dependence, but the risk of abuse can be limited if they are used judiciously in a timelimited fashion. Benzodiazepines and barbiturates increase the efficiency of GABAergic pathways, and chronic use can induce down-regulation of these pathways by neuroadaptation. One possible mechanism of down-regulation is uncoupling of the benzodiazepine site from the GABA site on GABAA receptors (see Chapter 12). Thus, the binding of benzodiazepines to GABAA receptors would remain unchanged, but the drug would have little or no potentiating effect on the binding of GABA to the receptor. Downregulation of inhibitory GABAergic pathways would be expected to leave the brain “underinhibited,” increasing the possibility of seizures and delirium upon abrupt withdrawal of the benzodiazepine or barbiturate (see Chapter 15). Associated central sympathetic hyperactivity can lead to physical symptoms such as anxiety, sleep disturbance, and dizziness and to emotional concomitants such as fear and panic. Because the central nervous system depressant actions of barbiturates are more widespread than those of the GABAA-specific benzodiazepines (Fig. 18-9), barbiturate dependence is associated with a more severe and potentially dangerous withdrawal syndrome than benzodiazepine dependence. Within a given class of sedative-hypnotics, the onset, amplitude, and duration of the withdrawal syndrome are determined by the rate of elimination of the drug and its active metabolites. For example, among the barbiturates and

Plasma drug concentration

A Alcohol Alprazolam Diazepam

0

1

2

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Withdrawal severity

Alcohol Alprazolam Diazepam

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Withdrawal severity

Pharmacokinetic determinants of CNS depressant-induced withdrawal severity. (A) Due to rapid elimination of alcohol and alprazolam, plasma levels fall rapidly after cessation of drug use. Plasma levels of diazepam, which has a long elimination half-life, decline at a slower rate. Furthermore, the effective biological half-life of diazepam is longer still because the active metabolites desmethyldiazepam (which has an even longer elimination half-life) and oxazepam are formed via the metabolism of diazepam. (B) The onset, severity, and duration of the CNS-depressant withdrawal syndrome are directly related to the rate of elimination of the drug, and thus the rate of removal of the drug from its target receptor. Alprazolam and alcohol withdrawal are more rapid in onset, of greater severity, and of relatively limited duration compared to diazepam withdrawal. (C) Treatment of CNS-depressant withdrawal is aimed at maintaining occupancy of the target receptor for a sufficiently long period of time that the system can re-equilibrate and thereby minimize the risk of severe withdrawal symptoms. This is accomplished by using a cross-tolerant drug (i.e., another CNS depressant) with a relatively slower rate of removal from the target receptor than the abused drug. Administration of diazepam to treat alcohol withdrawal illustrates this point. Although plasma levels of alcohol drop rapidly, administration of diazepam results in continued occupancy and activation of receptor sites (such as the GABAA receptor) for a much longer period of time and throughout the period of highest risk for withdrawal-related seizures. (D) The more gradual reduction in receptor occupancy after diazepam administration reduces the severity of alcohol withdrawal symptoms, prevents seizures, and reduces the morbidity and mortality from alcohol withdrawal. (E) In addition to its slower elimination compared to alcohol, diazepam has higher efficacy at GABAA receptors than alcohol, resulting in enhanced GABAA receptor activation. This property holds even when the receptor is in a desensitized state due to chronic alcohol consumption. Thus, the combination of the slower elimination and the higher efficacy of diazepam compared to alcohol makes it the medication of choice for the treatment of alcohol withdrawal.

Alcohol alone Alcohol + diazepam treatment

0

1

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3

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Days after last dose E

Efficacy at GABAA receptor (arbitrary units)

FIGURE 18-9.

Diazepam Alcohol

Time after drug administration (arbitrary units)

7

CHAPTER 18 / Pharmacology of Drugs of Abuse 299

benzodiazepines, withdrawal usually begins within 12 hours after drug discontinuation and is most severe for rapidly eliminated compounds (e.g., amobarbital and triazolam); withdrawal may be delayed for several days and is less severe for slowly eliminated compounds (e.g., phenobarbital, diazepam, and clonazepam) (Fig. 18-9). Co-occurring dependence on benzodiazepines or barbiturates and alcohol is particularly prevalent due to the similarity of these drugs’ effects on GABAergic neurotransmission (Fig. 18-9). Benzodiazepines (not barbiturates) are the accepted treatment for alcohol withdrawal; these drugs are efficacious in alleviating “rough spots” when alcoholics cannot drink, and the effects of alcohol are greatly accentuated by benzodiazepines (or barbiturates). Benzodiazepines are almost never associated with mortality due to overdose when used alone; combined with alcohol, however, they can be fatal because of synergistic depression of cardiorespiratory centers. Benzodiazepines and opioids can sometimes be coprescribed under conditions when pain is associated with significant anxiety. This combination can also be fatal due to synergistic effects on respiration; in fact, even the relatively safe partial agonist buprenorphine can cause respiratory arrest when combined with benzodiazepines. Physicians may try to limit use of these dangerous combinations, but some drug-seeking patients may resort to obtaining prescriptions from multiple physicians or even forging prescriptions, especially after suboptimal management of the underlying condition. Nonetheless, undermedication of pain must be avoided, and benzodiazepines should be used for treatment of alcohol withdrawal or significant anxiety. Another serious concern is the misuse of prescription opioids (or, less commonly, benzodiazepines or barbiturates) by health professionals. For at least two reasons, health professionals who misuse prescription medication are at greater risk for developing addiction. First, they have more ready access to prescription medication. Second, they may mistakenly believe that, because they understand a drug’s effects, they will be able to control its use more easily.

Alcohol Alcoholic beverages are readily available at affordable cost with minimal legal restriction. Alcohol abuse stands as the most prevalent drug problem in the United States. Early in intoxication, CNS stimulation and euphoria result from depression of inhibitory control, and aspects of discrimination, memory, and insight are impaired. As blood levels rise, judgment, emotional control, and motor coordination suffer. Traumatic injuries sustained while intoxicated are likely the most common public health problem associated with alcohol abuse. Respiratory depression and death can result from overdose, and the most serious consequences occur when alcohol is combined with other psychoactive agents. Ethanol affects GABAA receptors, NMDA glutamate receptors, and cannabinoid receptors. Although the specific sites of action are unknown, GABAA channels are believed to mediate the anxiolytic and sedative effects of alcohol, as well as the effects of alcohol on motor coordination, tolerance, dependence, and self-administration. Alcohol increases GABA-mediated chloride conductance and enhances hyperpolarization of the neuron. Its mechanisms of dependence are likely similar to those of other sedative-hypnotic drugs affecting GABA

neurotransmission. In severity and time course, the symptoms of alcohol withdrawal lie between those of short-acting barbiturates and intermediate-acting benzodiazepines. Evidence also points to a role for NMDA receptors in the development of tolerance and dependence to alcohol, and NMDA receptors also have a role in the alcohol withdrawal syndrome. Specifically, alcohol inhibits subtypes of NMDA receptors that seem to be capable of long-term potentiation. The rewarding effects of alcohol may also be mediated in part by indirect activation of cannabinoid receptors. Endogenous cannabinoids are “retrograde” neuromodulators that act as a feedback mechanism to enhance dopaminergic activity in the mesolimbic reward pathway (Fig. 18-10; see also Fig. 18-4). Endocannabinoid signaling has been implicated in reward learning, appetite regulation, mood regulation, pain modulation, and cognition. Thus, although GABA receptors have a vital role in mediating the effects of alcohol, the ability of alcohol to interact with a number of different receptor types suggests that our understanding of its mechanisms of action remains incomplete.

Nicotine and Tobacco Smoking, or the combustion of tobacco for the purpose of nicotine self-administration, represents a major source of preventable medical morbidity and mortality. Nicotine activates nicotinic acetylcholine receptors that are located centrally, peripherally, and at the neuromuscular junction. Cholinergic neurons arising from the laterodorsal tegmental area (near the border of the midbrain and pons) activate nicotinic and muscarinic acetylcholine receptors on dopaminergic neurons in the ventral tegmental area; stimulation of these nicotinic receptors by nicotine activates the dopaminergic brain reward pathway (Fig. 18-4). In addition, activation of presynaptic nicotinic receptors on dopaminergic axon terminals facilitates the release of dopamine. These strong and direct effects on the mesolimbic reward pathway, combined with the inhalational route of administration and short half-life of nicotine, explain the high addiction potential of nicotine, and hence of cigarettes and other forms of tobacco. Activation of central nicotinic receptors also produces anxiolytic effects, increases arousal, and suppresses appetite, while activation of peripheral nicotinic receptors increases blood pressure and stimulates smooth muscle contraction. A strong and spontaneous withdrawal syndrome is associated with the decreases in plasma levels of nicotine that occur upon cessation of smoking. The major symptoms include irritability, anxiety, autonomic arousal, and intense craving and associated drug-seeking behavior. These symptoms are readily relieved by smoking, and given the widespread availability of tobacco products, it is easy to see why smoking is so recalcitrant to treatment. Because smoking can alleviate a number of symptoms associated with depression and anxiety, it is commonly associated with the use of other drugs and with mental disorders.

Cocaine and Amphetamine Cocaine is isolated from the South American shrub Erythroxylon coca and has been used as a local anesthetic since 1884. Amphetamine and congeners are used clinically as nasal decongestants, analeptics, antidepressants, and diet pills, and for treatment of attention-deficit hyperactivity disorder (ADHD). Cocaine and many amphetaminerelated drugs have substantial abuse liability; hence, other

300 Principles of Central Nervous System Pharmacology

A

B

GABA terminal

GABA terminal

Rimonabant 2-AG

CB1 receptor

GABA Action potential

DAGL

Cl-

GABAA-R

Enhanced excitation of VTA dopaminergic neuron

Action potential

DAGL

Cl-

Increased inhibition of VTA dopaminergic neuron

FIGURE 18-10. Endogenous cannabinoid neurotransmission in the mesolimbic dopamine pathway. (A) Endogenous cannabinoids are a class of lipid neurotransmitters that act as “retrograde signals” to inhibit release of other neurotransmitters. Here, activation of dopaminergic neurons in the ventral tegmental area (VTA) results in rapid synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via the activity of diacylglycerol lipase (DAGL). 2-AG then activates CB1 cannabinoid receptors located on presynaptic GABAergic terminals. Activation of CB1 receptors causes a transient decrease in vesicular release of GABA on a time scale of seconds to minutes. This results in “feed-forward” enhancement of VTA dopaminergic neuron activity and could contribute to drug-seeking behavior. Thus, endocannabinoids can modulate VTA dopaminergic neuronal activity by inhibiting GABAergic (inhibitory) inputs to the VTA. Activation of VTA dopaminergic neurons in response to environmental cues associated with drug use can often trigger relapse (see Fig. 18-2). (B) The CB1 receptor antagonist rimonabant has been shown to inhibit cue-induced relapse in preclinical studies. A putative mechanism of action of rimonabant involves blockade of CB1 receptors on presynaptic GABAergic terminals in the VTA, which would sustain high levels of GABA and thus inhibit VTA dopaminergic neuron activity in response to drug-associated cues, and possibly reduce relapse.

medications with lower risk profiles have taken their place for many of their uses. Nevertheless, these drugs are widely available by prescription and through illicit sources. They are highly reinforcing because of the profound sense of well-being, energy, and optimism associated with stimulant intoxication; however, this state can rapidly progress to psychomotor agitation, severe paranoia, and even psychosis due to augmented dopamine neurotransmission. The initial euphoric effects of cocaine appear to be more pronounced than those of amphetamine, while amphetamine intoxication far outlasts that of cocaine. Elevated mood is often followed by listlessness, drowsiness, and depressed mood upon the withdrawal of stimulants. Appetite suppression can be followed by ravenous hunger. Stimulants are almost always taken with another drug of abuse, most commonly alcohol, since the other drug accentuates the “high” and alleviates the sleeplessness and sense of being “wired” (Fig. 18-4). By blocking or reversing the direction of the neurotransmitter transporters that mediate reuptake of the monoamines dopamine, norepinephrine, and serotonin into presynaptic terminals, cocaine and amphetamine potentiate dopaminergic, adrenergic, and serotonergic neurotransmission. Cocaine is most potent at blocking the dopamine transporter (DAT), although higher concentrations block the serotonin and norepinephrine transporters (SERT and NET, respectively). Recall that the tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs) function in a similar manner, blocking reuptake of norepinephrine and serotonin

(TCAs) or serotonin alone (SSRIs) into presynaptic neurons. Amphetamine reverses the direction of all three monoamine transporters, although this drug is more effective at the norepinephrine transporter. Amphetamine also releases vesicular transmitter stores into the cytoplasm; these combined actions cause the catecholamine neurotransmitter to be transported into, rather than out of, the extracellular space. By these actions, cocaine and amphetamine increase the concentration of monoamine neurotransmitters in the extracellular space, potentiating neurotransmission (Fig. 18-1). Although cocaine and amphetamine act on monoaminergic neurons throughout the body, it is the action of these drugs on neurons in two major centers in the brain that likely governs their potential for abuse. The first set of neurons, in the locus ceruleus in the pons, sends ascending adrenergic projections throughout the hypothalamus, thalamus, cerebral cortex, and cerebellum and descending projections to the medulla and spinal cord. These projections maintain alertness and responsiveness to unexpected stimuli (see Chapter 10, Adrenergic Pharmacology). Thus, drugs such as cocaine and amphetamine, which potentiate the actions of norepinephrine by inhibiting neurotransmitter reuptake, produce enhanced arousal and vigilance and are called psychostimulants. The second major site at which cocaine and amphetamine act is on midbrain dopaminergic neurons, the axons of which terminate in the nucleus accumbens, striatum, and cortex (Fig. 18-4). As discussed above, these dopaminergic terminals in the nucleus accumbens are a critical component of the brain’s reward pathway.

CHAPTER 18 / Pharmacology of Drugs of Abuse 301

It was long believed that the psychostimulants do not cause significant withdrawal and that behaviors to seek these drugs rarely attain levels that are out of control. However, cocaine use can be associated with withdrawal symptoms such as bradycardia, sleepiness, and fatigue. Withdrawal from cocaine or amphetamine also produces psychological symptoms, such as dysphoria and anhedonia (an inability to experience pleasure), that are opposite to the euphoria experienced immediately following administration of the drug. Many of these symptoms are not strictly attributable to withdrawal because they cannot be alleviated by the administration of more cocaine or amphetamine. In fact, symptoms of withdrawal can appear even when psychostimulant levels in the plasma are high. This phenomenon occurs both because of allostasis of reward pathways (discussed earlier) and because these drugs cause tachyphylaxis, an acute process in which the target tissue becomes less and less responsive to constant concentrations of a drug. In the case of cocaine and amphetamine, tachyphylaxis may be caused by depletion of the neurotransmitter. Because the drugs block presynaptic neurotransmitter reuptake, the elevated levels of neurotransmitter in the extracellular space feed back to inhibit its synthesis, and neurotransmitter stores in the presynaptic terminal are progressively depleted. The combination of tachyphylaxis and allostasis makes discontinuation of stimulants particularly difficult for addicts, both in the short and long term.

Marijuana Cannabinoids are compounds derived from Cannabis sativa (marijuana). The primary psychoactive component of marijuana is ⌬9-tetrahydrocannabinol (THC), which is a partial agonist at the G protein-coupled type-1 cannabinoid receptor (CB1). The endogenous ligand of the CB1 receptor is the arachidonic acid derivative anandamide, which is representative of a class of endocannabinoid “retrograde” neuromodulators that act as a feedback mechanism to reduce neuronal excitation (Fig. 18-10). Since blockade of CB1 receptors by the antagonist rimonabant eliminates the effects of smoked marijuana in humans, the subjective effects of marijuana are thought to be mediated by the CB1 receptor. The CB1 receptor is widely distributed within the prefrontal cortex, hippocampus, amygdala, basal ganglia, and cerebellum. Endogenous cannabinoids appear to modulate a variety of appetitive (reinforcing and consumptive) behaviors including eating, smoking, and alcohol drinking. Cannabinoid use causes a prompt and generalized “high” characterized by euphoria, laughter, giddiness, and depersonalization. After 1–2 hours, cognitive functions such as memory, reaction time, coordination, and alertness are compromised, and the user has difficulty concentrating. This effect corresponds to a “mellowing” phase, which results in relaxation and even sleep. In rats, the administration of natural and synthetic cannabinoids causes dopamine release in the nucleus accumbens of the brain reward pathway. High doses of marijuana can cause anxiety, overt panic reactions, perceptual distortions, impairments in reality testing, and, rarely, overt psychosis in susceptible individuals. Overt panic reactions are the most common reason cited for stopping marijuana use. Tolerance to marijuana occurs via down-regulation of CB1 receptor expression and post-translational modifications that reduce signal transduction efficiency. Withdrawal from marijuana is

generally mild due to its high volume of distribution and long elimination half-life. Withdrawal symptoms can include insomnia, loss of appetite, irritability, and anxiety, perhaps due to activation of central corticotropin-releasing factor (CRF) systems, particularly in the amygdala.

Other Abused Drugs Phencyclidine (PCP) was developed initially as a dissociative anesthetic but is no longer used because of behavioral toxicity. PCP blocks NMDA glutamate receptors, which mediate excitatory synaptic transmission and are involved in synaptic plasticity and memory. By interfering with these processes, PCP produces complex effects such as anesthesia, delirium, hallucinations, intense paranoia, and amnesia. Methylenedioxymethamphetamine (MDMA), known colloquially as ecstasy, is one in the class of phenylethylamine hallucinogenics that unfortunately has been falsely advertised by some as a “safe” drug. Although it is chemically related to methamphetamine and has similar dopaminergic effects, the primary effect of MDMA is on serotonergic neurotransmission. MDMA causes serotonin release into the extracellular space, inhibition of serotonin synthesis, and block of serotonin reuptake. Together, these complex actions of MDMA increase serotonin in the extracellular space while depleting presynaptic stores of the neurotransmitter. The drug causes a central stimulant effect like cocaine and amphetamine but, unlike those drugs, it also has hallucinogenic properties. Like cocaine and amphetamine, MDMA affects the brain reward pathway through dopaminergic stimulation. MDMA may be neurotoxic to a subpopulation of serotonergic neurons when the drug is administered repeatedly or in large amounts. Caffeine and the related methylxanthines theophylline and theobromine are ubiquitous drugs found in coffee, tea, cola, “energy” drinks, chocolate, and many prescribed and over-the-counter medications. Methylxanthines act by blocking adenosine receptors that are expressed presynaptically on many neurons, including dopaminergic and adrenergic neurons. Because activation of adenosine receptors inhibits dopamine and norepinephrine release, competitive antagonism of the receptors by caffeine increases dopamine and norepinephrine release and, thus, acts as a stimulant. Caffeine may also block adenosine receptors on cortical neurons, and thereby disinhibit these neurons. Because CNS adenosine is a natural promoter of sleep and drowsiness, caffeine’s blocking of adenosine receptors has alerting effects and improves performance in a variety of circumstances, but can also produce insomnia. Symptoms of withdrawal from caffeine can include lethargy, irritability, and a characteristic headache, but addiction, although documented, is rare. Caffeine withdrawal symptoms are commonly observed in even low to moderate users of caffeine, but these typically resolve without treatment. Inhalants are volatile organic compounds that are inhaled (sometimes called huffing) for their psychoactive effects. The typical user of inhalants is a male teenager. Inhalants include organic solvents such as gasoline, toluene, ethyl ether, fluorocarbons, and volatile nitrates, including nitrous oxide and butyl nitrate. Inhalants are readily available in many households and workplaces. At low doses, inhalants produce mood changes and ataxia; at high doses, they may produce dissociative states and hallucinations. Dangers of organic

302 Principles of Central Nervous System Pharmacology

solvent use include suffocation and organ damage, especially hepatotoxicity and neurotoxicity in the central and peripheral nervous systems. Cardiac arrhythmias and sudden death can occur. Inhaled nitrates can produce hypotension and methemoglobinemia. Hydrocarbon inhalants do not appear to act at a specific receptor, but rather to disrupt cell functions by binding nonspecifically to hydrophobic sites on receptors, signal transduction proteins, and other macromolecules. Nitrates, however, act at specific receptors for nitric oxide, a small-molecule neuromodulator (see Chapter 21, Pharmacology of Vascular Tone).

 MEDICAL COMPLICATIONS OF DRUG ABUSE AND DEPENDENCE Individuals with drug-use disorders typically present to a physician complaining of the indirect effects of drug selfadministration. These can include family disruptions and emotional trauma, legal problems and physical injury, selfneglect (e.g., malnutrition, harm from adulterants mixed with drugs, infection from needle administration), inappropriate use of prescribed medication (e.g., analgesics, anxiolytics), and lack of adherence with medical regimens for coexisting illnesses. These effects are clearly not specific to the pharmacologic actions of any given drug but are the consequence of out-of-control, often self-destructive behaviors that interfere with a balanced life because the reward and salience of drug use supersede that of other elements of the environment. Less commonly, patients seek medical care for acute and chronic direct pharmacologic and toxic actions of substance(s) of abuse. Given the multiplicity of drugs, the means by which they are obtained, and the variety of routes of administration, complications may also be secondary to tissue toxicity and induced metabolic changes. Adequate treatment of the medical complications related to drug abuse requires knowledge of a given drug’s pharmacologic actions. Many patients who abuse drugs use more than one substance. Pharmacodynamic and pharmacokinetic effects of polysubstance abuse are often difficult to predict from the actions of each individual agent. For example, research has revealed a potentially dangerous interaction between cocaine and alcohol. When taken together, the two drugs are converted to cocaethylene. Cocaethylene has a longer duration of action in the brain and is more toxic than either drug alone. The vast majority of individuals with drug-use disorders also smoke cigarettes and, despite attaining abstinence from their “drug of choice,” the eventual cause of death is often related to complications of cigarettes (e.g., cancer, cardiovascular disease). Alcohol abuse is associated with widespread toxicity. Alcoholic cardiomyopathy can result in a life-threatening decrease in left ventricular function. Ethanol is directly toxic to heart muscle cells, affecting contractility of the myocytes and inhibiting the repair of injury to these cells. The mechanism of myocyte damage may relate to the overproduction of oxygen-containing molecules secondary to alcohol metabolism, with damage to the plasma membrane of the myocyte. Nutritional deficiencies of water-soluble vitamins such as thiamine may also be involved. With moderate drinking, there is typically an increase in systolic blood pressure. Alcohol withdrawal also plays a role in hypertension because sympathetic activity is increased during withdrawal. Stress appears to cause a greater rise in blood pressure in drinkers than in

nondrinkers. There appears to be a protective effect of drinking on coronary artery disease, at least in older individuals and those otherwise at risk for coronary disease. The so-called J-shaped mortality curve shows that these populations have decreased mortality with low to moderate drinking (generally 0.5–2 drinks/day) and increased mortality with heavy drinking. The mechanism of this protection involves beneficial effects of ethanol on lipoprotein metabolism and thrombosis: ethanol increases high-density lipoprotein (HDL) levels in a dose-dependent manner, and ethanol inhibits platelet aggregation and lowers plasma fibrinogen levels. Chronic alcoholism has other significant medical complications. Metabolic consequences of alcohol abuse include gout, hyperlipidemia and fatty liver, and hypoglycemia. Chronic alcoholics can develop obesity when the high caloric content of alcohol is added to normal food intake; when food intake is limited and/or malabsorption is present, weight loss with mineral and electrolyte imbalances and vitamin deficiencies can result. Alcohol toxicity can lead to pancreatic insufficiency and diabetes. The gastrointestinal system is frequently affected by chronic alcohol consumption, resulting in esophagitis, gastritis or ulcer, pancreatitis, and alcoholic hepatitis and cirrhosis. Effects of alcohol on the cytochrome P450 system alter drug and carcinogen metabolism, accounting for significant drug interactions and increased cancer incidence in chronic alcoholics. Alcohol increases the release of ACTH, glucocorticoids, and catecholamines and inhibits testosterone synthesis and the release of ADH and oxytocin. Neurologic complications of chronic alcoholism include dementia, amnestic disorder, cerebellar degeneration, and neuropathy, due to both direct neurotoxicity and thiamine deficiency. Finally, alcohol consumption during pregnancy has widespread teratogenic consequences, termed fetal alcohol spectrum disorder. Pharmacologic consequences of psychostimulant abuse relate to specific effects of these drugs on the nervous and cardiovascular systems. Potentiation of norepinephrine neurotransmission increases heart rate and blood pressure. Cocaine, in particular, can cause vasospasm leading to stroke, cerebrovasculitis, myocardial infarction, and aortic dissection. The inhibition of cardiac and CNS sodium channels by cocaine can cause arrhythmias and seizures. Psychostimulants can reset temperature regulation, causing hyperpyrexia and associated rhabdomyolysis. Cocaine and amphetamine can also cause involuntary movements through their action on the basal ganglia.

 TREATMENTS FOR ADDICTION Despite the high prevalence of alcohol and drug problems in medical practice (10–15% in ambulatory care, 30–50% in emergency departments, and 30–60% in general hospital settings), the diagnosis is often overlooked. As is the case with other stigmatized diseases, specialized services are often inaccessible. Recent health legislation in the United States promises parity for medical and mental disorders (including alcohol and drug problems) and more widespread availability of addiction treatment. Treatments for addiction can be divided into two broad approaches, pharmacologic and psychosocial. Traditionally, pharmacologic treatments for addiction have focused on acute detoxification to relieve the withdrawal symptoms that

304 Principles of Central Nervous System Pharmacology

Pharmacologic Treatment of Addiction The recognition that addiction is caused by fundamental changes in brain reward pathways indicates that pharmacotherapy could have an important role in the management of addiction. To date, several pharmacologic strategies have been employed. The first of these strategies is the chronic administration of an agent that causes aversive effects when the drug of abuse is used. For example, disulfiram inhibits aldehyde dehydrogenase, a critical enzyme in the alcohol metabolism pathway. In an individual who ingests ethanol while taking disulfiram, alcohol dehydrogenase oxidizes the ethanol to acetaldehyde, but disulfiram prevents aldehyde dehydrogenase from metabolizing the acetaldehyde. Therefore, this toxic metabolite accumulates in the blood. Acetaldehyde causes a number of aversive symptoms, including facial flushing, headache, nausea, vomiting, weakness, orthostatic hypotension, and respiratory difficulty. These symptoms can last from 30 minutes to several hours and are followed by exhaustion and fatigue. The aversive effects of alcohol consumption in the presence of disulfiram are intended as a deterrent to further drinking. Unfortunately, the effectiveness of disulfiram is limited by failures in adherence and by substantial toxicity. A second strategy used to treat addiction is to block the effects of the drug of abuse. Naltrexone is an opioid antagonist that competitively blocks the binding of opioids to the opioid receptor. Thus, a patient who injects an opioid, such as heroin, while taking naltrexone will not experience the “high” that normally accompanies drug use. Studies have shown that naltrexone also acts as an opioid inhibitor in the brain reward pathway. Thus, the effects of a drug such as ethanol, which releases endogenous opioids resulting in a disinhibition (or stimulation) of mesolimbic dopamine, share a final common reward pathway

involving the opioid receptor and dopamine and are therefore also inhibited by naltrexone. For this reason, naltrexone has been used to treat alcohol addiction. Placebo-controlled clinical trials have generally shown efficacy of naltrexone compared to placebo, particularly in reducing relapse to heavy drinking. Naltrexone should not be administered when there are traces of exogenous opioids in the system, because antagonism of remaining drug by naltrexone can lead to the development or exacerbation of opioid withdrawal symptoms. Although naltrexone can effectively prevent the “high” associated with opioid abuse, it does not alleviate cravings or withdrawal effects, and there is a relatively high likelihood of non-adherence. Therefore, naltrexone has been effective only in individuals addicted to opioids or alcohol who have a high motivation to stay drug-free or who have supervised administration. An injectable long-acting naltrexone preparation has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of alcohol dependence. This sustained-release naltrexone is injected intramuscularly once a month; it has been demonstrated to reduce heavy alcohol consumption and increase alcohol abstinence, and it may also be beneficial in opioid dependence, especially in those with low adherence to treatment. A third pharmacologic approach is the use of a longacting agonist for medication maintenance. Methadone, as discussed above, is a long-acting opioid agonist. Because it is taken orally, it is less likely to produce the sharp increases in plasma levels required to elicit a “high” such as that accompanying the injection of heroin or other opioids. Methadone also has a long half-life compared to heroin or morphine. Thus, once-daily administration of methadone produces plasma opioid levels that remain relatively constant over time and, therefore, mitigate cravings and prevent the emergence of withdrawal signs and symptoms (Fig. 18-11).

“High”

Plasma concentration of drug

Heroin Asymptomatic

Methadone

Withdrawal symptoms

0

6

12

18

24

30

36

42

48

54

60

Time (hours)

FIGURE 18-11. Pharmacokinetics and pharmacodynamics of a fast-acting opioid (heroin) compared to a slow-acting opioid (methadone). The plasma concentration of a fast-acting opioid such as heroin rises rapidly after intravenous administration, generating a “high,” but also falls quickly, producing withdrawal symptoms. In contrast, the plasma concentration of a slow-acting, long half-life drug such as methadone remains in the asymptomatic range for a period of over 24 hours, so that the patient does not experience either the “high” or the withdrawal symptoms. Moreover, because of its long plasma half-life, methadone needs to be administered only once daily.

306 Principles of Central Nervous System Pharmacology

dopamine ␤-hydroxylase and can increase brain dopamine levels, possibly counteracting the dopamine-depleting effects of chronic cocaine use. Because cocaine sensitization involves glutamate, antiepileptics such as topiramate are also being studied for efficacy in the treatment of cocaine dependence.

 CONCLUSION AND FUTURE DIRECTIONS This chapter has discussed the major causes of drug dependence and drug addiction. Dependence is defined as a maladaptive pattern of drug use associated with context-induced craving and drug-seeking, especially under situations of stress, that leads to clinically significant impairment or distress. Drug addiction is caused by an allostatic adaptation to the presence of the drug in brain reward pathways. Although each drug has its own molecular and cellular mechanism of action that may account for drug toxicity, all abused drugs specifically affect the mesolimbic dopamine brain reward pathway. This chapter has also discussed the major treatments for addiction, including the pharmacologic prevention and treatment of withdrawal symptoms, the long-term psychosocial management of addiction, and newer pharmacologic treatments that, when integrated with psychosocial approaches, promote long-lasting sobriety. Together, these addiction treatments achieve outcomes approximating those of other long-term chronic medical disorders, such as atherosclerosis, hypertension, and diabetes. New directions in addiction research are focused on the pharmacologic modulation of brain reward, stress responses, and learning-related neural processes. In addition, these approaches are complemented by basic and clinical studies of the neurobiology of learning and memory and the modification of these processes through psychosocial treatments. Current approaches to cocaine addiction provide two specific examples. First, drugs that specifically interact with different dopamine receptor subtypes have been explored, investigating the hypotheses that a D1-specific agonist or D4-specific antagonist could suppress drug cravings, and that a D2-specific antagonist could prevent the reinforcing effects of cocaine. Second, researchers have recently completed clinical trials of a cocaine vaccine, under the theory that cocaine will be less reinforcing in vaccinated persons who are exposed to the drug. If successful, this approach could be extended to other drugs of abuse. (Trials of an analogous anti-nicotine vaccine are also forthcoming.) However, vaccinated individuals may switch to other drugs of abuse for which they have not produced antibodies, and hence, this is not likely to be a totally satisfactory approach. Broader and more promising are efforts to develop pharmacologic treatments for addiction aimed at: (1) modulating the chemical mediators of synaptic plasticity that underlie reward learning and memory; and (2) modifying the negative affective states and stress responses, the previously mentioned “allostatic load,” associated with chronic drug abuse. These approaches address shared brain mechanisms of addiction to all drugs of abuse. For example, a failure of the prefrontal cortex to control drug-seeking behaviors has been linked to glutamatergic dysfunction in reward pathways, which may

be amenable to new glutamate- and neuroplasticity-based pharmacotherapies. Another approach targets neural systems mediating behavioral stress responses; for example, an antagonist of the neurokinin-1 receptor, which is expressed in brain areas involved in stress responses and drug reward, has been shown in preliminary studies to suppress alcohol cravings, improve well-being, and attenuate the cortisol stress response in abstinent alcoholics. Preclinical studies have shown that CRF antagonists may block stress-induced reinstatement of drug use in animal models of addiction. Endocannabinoid signaling has also been implicated in a variety of physiologic functions including reward learning, appetite, mood, pain, and cognition. Elucidation of endocannabinoid signaling as a pro-hedonic system involving CB1 receptor activation led to findings that the CB1 cannabinoid receptor antagonist rimonabant was effective in obesity treatment, and this drug is now being investigated as a treatment for drug addiction. Rimonabant has not received FDA approval because it is associated with significant psychiatric adverse effects, but this approach remains a promising direction for future research.

Acknowledgment We thank David C. Lewis, Joshua M. Galanter, and Alan A. Wartenberg for their valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Camí J, Farré M. Mechanisms of disease: drug addiction. N Engl J Med 2003;349:975–986. (Current understanding of neural mechanisms leading to addiction.) Dani JA, Harris RA. Nicotine addiction and comorbidity with alcohol abuse and mental illness. Nat Neurosci 2005;8:1465–1470. (Examines the interface between the neuropharmacologic underpinnings of nicotine addiction and psychiatric disorders, especially alcoholism.) Goldstein RZ, Craig AD, Bechara A, Garavan H, Childress AR, Paulus MP, Volkow ND. The neurocircuitry of impaired insight in drug addiction. Trends Cog Sci 2009;13:372–380. (Discusses current understanding of lack of insight and awareness in addiction.) Kalivas PW, O’Brien C. Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacol 2007;33:166–180. (Review that links learning mechanisms to reward through the glutamatergic system.) Koob GF, Le Moal M. Neurobiological mechanisms for opponent motivational processes in addiction. Philos Trans R Soc B Biol Sci 2008;363: 3113–3123. (Reviews relationships between stress and reward pathways.) McLellan AT, Lewis DC, O’Brien CP, Kleber HD. Drug dependence, a chronic medical illness: implications for treatment, insurance, and outcomes evaluation. JAMA 2000;284:1689–1695. (Seminal analysis of the status of drug use disorders in the health care system.) Nestler EJ. Transcriptional mechanisms of addiction: role of delta-FosB. Philos Trans R Soc B Biol Sci 2008;363:3245–3255. (Reviews the role of gene regulation as a unitary neurobiological mechanism in reward and stress responses.) Alcoholics Anonymous. www.aa.org. (Excellent information on Alcoholics Anonymous.) Substance Abuse and Mental Health Services Administration. www.samhsa .gov. (Contains a wealth of information about prevention and treatment and co-occurring diagnoses; also access to listings of evidence-based treatment practices.)

III Principles of Cardiovascular Pharmacology

19 Pharmacology of Cholesterol and Lipoprotein Metabolism David E. Cohen and Ehrin J. Armstrong

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 311-312 BIOCHEMISTRY AND PHYSIOLOGY OF CHOLESTEROL AND LIPOPROTEIN METABOLISM . . . . . . . . . . . . . . . . . . . . 311 Metabolism of ApoB-Containing Lipoproteins . . . . . . . . . . 313 Assembly of ApoB-Containing Lipoproteins . . . . . . . . . 313 Intravascular Metabolism of ApoB-Containing Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Receptor-Mediated Clearance of ApoB-Containing Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 Formation and Clearance of LDL Particles . . . . . . . . . . 317 HDL Metabolism and Reverse Cholesterol Transport . . . . . 318 HDL Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 Intravascular Maturation of HDL . . . . . . . . . . . . . . . . . 320 HDL-Mediated Cholesterol Efflux from Cells . . . . . . . . 320 Delivery of HDL Cholesterol to the Liver . . . . . . . . . . . 320 Biliary Lipid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Cholesterol Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Hypercholesterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Mixed Hyperlipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Disorders of HDL Metabolism. . . . . . . . . . . . . . . . . . . . . . 323 Secondary Hyperlipidemia . . . . . . . . . . . . . . . . . . . . . . . . 323 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 324 Inhibitors of Cholesterol Synthesis . . . . . . . . . . . . . . . . . . 324 Inhibitors of Bile Acid Absorption . . . . . . . . . . . . . . . . . . . 326 Inhibitors of Cholesterol Absorption . . . . . . . . . . . . . . . . . 326 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 Omega-3 Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 329 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

 INTRODUCTION

cutting-edge genomic approaches. It is apparent that genes modify both the sensitivity of individuals to adverse dietary habits and lifestyles and the response of individuals to lipidlowering therapies. This chapter highlights the biochemistry and physiology of cholesterol and lipoproteins, with an emphasis on the role of lipoproteins in atherogenesis, and the pharmacologic interventions that can ameliorate hyperlipidemia. Abundant clinical outcomes data have proven that morbidity and mortality from cardiovascular disease can be reduced by the use of lipid-lowering drugs.

Lipids are insoluble or sparingly soluble molecules that are essential for membrane biogenesis and maintenance of membrane integrity. They also serve as energy sources, hormone precursors, and signaling molecules. In order to facilitate transport through the relatively aqueous environment of the blood, nonpolar lipids, such as cholesteryl esters and triglycerides, are packaged within lipoproteins. Increased concentrations of certain lipoproteins in the circulation are associated strongly with atherosclerosis. Much of the prevalence of cardiovascular disease (CVD), the leading cause of death in the United States and most Western countries, can be attributed to elevated blood concentrations of cholesterol-rich low-density lipoprotein (LDL) particles as well as lipoproteins that are rich in triglycerides. Epidemiologically, decreased concentrations of high-density lipoproteins (HDL) also predispose to atherosclerotic disease. The major contributors to lipoprotein abnormalities appear to be Western diets combined with sedentary lifestyles, but a limited number of genetic causes of hyperlipidemia have also been identified. The role of genetics in the common forms of hyperlipidemia is the subject of intense study utilizing

 BIOCHEMISTRY AND PHYSIOLOGY OF CHOLESTEROL AND LIPOPROTEIN METABOLISM Lipoproteins are macromolecular aggregates that transport triglycerides and cholesterol in the blood. Circulating lipoproteins can be differentiated on the basis of density, size, and protein content (Table 19-1). As a general rule, larger, less dense lipoproteins have a greater percentage composition of lipids; chylomicrons are the largest and least dense 311

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 313

Core: Triglyceride and cholesteryl esters

Apolipoprotein B100 Apolipoprotein C

Free cholesterol Apolipoprotein E

Phospholipid monolayer

apolipoprotein B [apoB]-containing lipoproteins, chylomicrons, and VLDL) and lipoproteins that are involved primarily in cholesterol transport (HDL and the remnants of apoB-containing lipoproteins). HDL also serves as a reservoir for exchangeable apolipoproteins in the plasma, including apoAI, apoCII, and apoE. The following discussion presents each lipoprotein class in the context of its function.

Metabolism of ApoB-Containing Lipoproteins The primary function of apoB-containing lipoproteins is to deliver fatty acids in the form of triglycerides to muscle tissue for use in ATP biogenesis and to adipose tissue for storage. Chylomicrons are formed in the intestine and transport dietary triglycerides, whereas VLDL particles are formed in the liver and transport triglycerides that are synthesized endogenously. The metabolic lifespan of apoB-containing lipoproteins can be divided into three phases: assembly, intravascular metabolism, and receptor-mediated clearance. This is a convenient categorization because pharmacologic agents are available that influence each phase. FIGURE 19-1.

Structure of lipoprotein particles. Lipoproteins are spherical particles (7–100 nm in diameter) that transport hydrophobic molecules, principally cholesterol and triglycerides, as well as fat-soluble vitamins. The surface of the particle is composed of a monolayer of phospholipid and unesterified cholesterol molecules. These polar lipids form a coating that shields a hydrophobic core of nonpolar triglyceride and cholesteryl esters from interacting with the aqueous environment of plasma. Lipoproteins contain amphipathic apolipoproteins (also called apoproteins ) that associate with the surface lipids and hydrophobic core. Apolipoproteins provide structural stability to the lipoprotein particle and act as ligands for specific cell-surface receptors or as cofactors for enzymatic reactions. In the example shown, a very-low-density lipoprotein (VLDL) particle contains apolipoprotein E, apolipoprotein B100, and apolipoproteins CI, CII, and CIII (shown here as apolipoprotein C ).

lipoprotein subclass, whereas HDLs are the smallest lipoproteins, containing the lowest lipid content and the highest proportion of protein. Structurally, lipoproteins are microscopic spherical particles ranging from 7 to 100 nm in diameter. Each lipoprotein particle consists of a monolayer of polar, amphipathic lipids that surrounds a hydrophobic core. Each lipoprotein particle also contains one or more types of apolipoprotein (Fig. 19-1). The polar lipids that comprise the surface coat are unesterified cholesterol and phospholipid molecules arranged in a monolayer. The hydrophobic core of a lipoprotein contains cholesteryl esters (cholesterol molecules linked by an ester bond to a fatty acid) and triglycerides (three fatty acids esterified to a glycerol molecule). Apolipoproteins (also referred to as apoproteins) are amphipathic proteins that intercalate into the surface coat of lipoproteins. In addition to stabilizing the structure of lipoproteins, apolipoproteins engage in biological functions. They may act as ligands for lipoprotein receptors or may activate enzymatic activities in the plasma. The apolipoprotein composition determines the metabolic fate of the lipoprotein. For example, each LDL particle contains one apoB100 molecule, which is a ligand for the low-density lipoprotein receptor (discussed below); in turn, binding of LDL to the LDL receptor promotes cholesterol uptake into cells. From a metabolic perspective, lipoprotein particles can be divided into lipoproteins that participate in the delivery of triglyceride molecules to muscle and fat tissue (the

Assembly of ApoB-Containing Lipoproteins The cellular mechanisms by which chylomicrons and VLDL are assembled are quite similar. Regulation of the assembly process depends on the availability of apolipoprotein B and triglycerides, as well as the activity of microsomal triglyceride transfer protein (MTP). The gene that encodes apoB is transcribed principally in the intestine and the liver. Apart from this tissue-specific expression, there is little transcriptional regulation of the apoB gene. In contrast, a key regulatory event that differentiates chylomicron metabolism from VLDL metabolism is the editing of apoB mRNA (Fig. 19-2). Within enterocytes but not hepatocytes, a protein named apoB editing complex-1 (apobec-1) is expressed. This protein constitutes the catalytic subunit of the apoB editing complex, which deaminates

apoB gene Transcription

Liver and intestine

apoB mRNA

Editing

Small intestine

No editing

Liver

Stop codon mRNA Translation Protein apoB48

apoB100

FIGURE 19-2. Editing of apoB mRNA. The apoB gene, with exons represented by rectangles and introns by lines, is transcribed in both the intestine and the liver. In the intestine, but not the liver, a protein complex containing apobec-1 modifies a single nucleotide in the apoB mRNA. As a result, the codon containing this nucleotide is converted to a premature stop codon, as indicated by the “X.” The protein that is synthesized in the intestine (apoB48) is only 48% as long as the full-length protein that is synthesized in the liver (apoB100).

314 Principles of Cardiovascular Pharmacology

a cytosine at position 6666 of the apoB mRNA molecule. Deamination converts the cytosine to uridine. As a result, the codon containing this nucleotide is converted from glutamine to a premature stop codon. When translated, the intestinal form apoB48 is 48% as long as the full-length protein that is expressed in the liver and referred to as apoB100. As a consequence, chylomicrons, the apoB-containing lipoprotein produced by the intestine, contain apoB48, whereas VLDL particles produced by the liver contain apoB100. Figure 19-3 illustrates the cellular mechanisms for the assembly and secretion of apoB-containing lipoproteins. As the apoB protein is synthesized by ribosomes, it crosses into the endoplasmic reticulum. Within the endoplasmic reticulum, triglyceride molecules are added co-translationally to the elongating apoB protein (i.e., apoB is lipidated) by the action of a cofactor protein, MTP. Once apoB has been fully synthesized, the nascent lipoprotein is enlarged in the Golgi apparatus; during this process, MTP adds additional triglycerides to the core of the particle. By unclear mechanisms, cholesteryl esters are also added to the core. This entire assembly process produces lipoprotein particles, each containing a single molecule of apoB. Because the diet is the main source of triglycerides in chylomicrons (Fig. 19-4), the assembly, secretion, and metabolism of these particles are collectively referred to as the exogenous pathway of lipoprotein metabolism. By contrast, cholesteryl esters in chylomicrons are derived mainly (approximately 75%) from biliary cholesterol, with the remainder contributed by dietary sources. During digestion, cholesteryl esters and triglycerides in food are hydrolyzed to form unesterified cholesterol, free fatty acids, and monoglycerides. Bile acids, phospholipids, and cholesterol are secreted by the liver into bile and stored in the gallbladder during fasting as micelles and vesicles, which are macromolecular lipid aggregates that form due to the detergent properties of bile acid molecules. The stimulus of eating a meal promotes emptying of gallbladder bile into the small intestine, where the micelles and vesicles solubilize the digested lipids. Enterocyte or hepatocyte

Cytosol

Cholesteryl ester

Ribosome

Lipidation

MTP

MTP

Chylomicron (enterocyte) or VLDL (hepatocyte)

ApoB

Endoplasmic reticulum Triglyceride

FIGURE 19-3. Assembly and secretion of apolipoprotein B-containing lipoproteins. Chylomicrons and VLDL particles are assembled and secreted by similar mechanisms in the enterocyte and hepatocyte, respectively. The apoB protein (i.e., apoB48 or apoB100) is synthesized by ribosomes and enters the lumen of the endoplasmic reticulum. If triglycerides are available, the apoB protein is lipidated by the action of microsomal triglyceride-transfer protein (MTP) in two distinct steps, accumulating triglyceride as well as cholesteryl ester molecules. The resulting chylomicron or VLDL particle is secreted by exocytosis into the lymphatics by enterocytes or into the plasma by hepatocytes. In the absence of triglycerides, the apoB protein is degraded (not shown).

Monoglyceride Cholesterol Phospholipid Fatty acid Bile salt

Basolateral membrane

Apical membrane Ezetimibe Micelle NPC1L1

Cholesterol ACAT

Cholesteryl ester ABCG5/G8

Monoglyceride

DGAT Triglyceride id Fatty acid

FIGURE 19-4.

Absorption of cholesterol and triglycerides. Exogenous cholesterol and triglycerides are simultaneously absorbed from the intestinal lumen by different mechanisms. Cholesterol is taken up from micelles across a regulatory channel named NPC1L1. A fraction of the cholesterol is pumped back into the lumen by ABCG5/G8, a heterodimeric ATP-dependent plasma membrane protein. The remainder of the cholesterol is converted to cholesteryl esters by ACAT. Triglycerides are taken up as fatty acids and monoglycerides, which are re-esterified to triglycerides by DGAT.

Lipid absorption into enterocytes of the duodenum and jejunum is facilitated mainly by micelles. Long-chain fatty acids and monoglycerides are taken up separately into the enterocyte by carrier-mediated transport and then re-esterified to form triglycerides by the enzyme diacylglycerol acyltransferase (DGAT). By contrast, medium-chain fatty acids are absorbed directly into the portal blood and metabolized by the liver. Dietary and biliary cholesterol from micelles enter the enterocyte via a protein channel named Niemann-Pick C1-like 1 protein (NPC1L1). Some of this cholesterol is immediately pumped back into the intestinal lumen by the ATP-dependent action of a heterodimeric protein, ABCG5/ABCG8 (ABCG5/G8). The fraction of cholesterol that remains is esterified to a long-chain fatty acid by acetyl-CoA:cholesterol acyltransferase (ACAT). Once triglycerides and cholesteryl esters are packaged together with apoB48, apoA1 is added as an additional structural apolipoprotein and the chylomicron particle is exocytosed into the lymphatics for transport to the circulation via the thoracic duct. The plasma concentration of triglyceride-rich chylomicrons varies in proportion to dietary fat intake.

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 315

Very-low-density lipoproteins (VLDL) contain triglycerides that are assembled by the liver using plasma fatty acids derived from adipose tissue or synthesized de novo. For this reason, the assembly, secretion, and metabolism of VLDL are often referred to as the endogenous pathway of lipoprotein metabolism. Hepatocytes synthesize triglycerides in response to increased free fatty acid flux to the liver. This typically occurs in response to fasting, thereby ensuring a continuous supply of fatty acids for delivery to muscle in the absence of triglycerides from the diet. Interestingly, dietary saturated fats as well as carbohydrates also stimulate the synthesis of triglycerides within the liver. By cellular mechanisms that are similar to those that produce chylomicrons (Fig. 19-3), MTP in hepatocytes lipidates apoB100 to form nascent VLDL particles. Under the continued influence of MTP, the nascent VLDL particles coalesce with larger triglyceride droplets and are secreted directly into the circulation. VLDL particles may also acquire apoE, apoCI, apoCII, and apoCIII within the hepatocyte prior to secretion. However, these apolipoproteins may also be transferred to VLDL from HDL in the circulation. The synthesis of apoB48 in the intestine and apoB100 in the liver is constitutive. This permits the immediate

production of chylomicrons and VLDL particles when triglyceride molecules are available. In the absence of triglycerides, such as in enterocytes during fasting, apoB is degraded by a variety of cellular mechanisms. Intravascular Metabolism of ApoB-Containing Lipoproteins Within the circulation, chylomicrons and VLDL particles must be activated in order to target triglyceride delivery to muscle and adipose tissue (Fig. 19-5). Activation requires the addition of an optimal complement of apoCII molecules, which occurs by aqueous transfer of apoCII from HDL particles. Because there is an inherent delay in the transfer of apoCII to chylomicrons and VLDL particles, there is time for widespread circulation of triglyceride-rich particles throughout the body. Lipoprotein lipase (LPL) is a lipolytic enzyme expressed on the endothelial surface of capillaries in muscle and fat tissue. LPL is a glycoprotein that is anchored in place by electrostatic interactions with a separate glycoprotein on the endothelial cell membrane. Once chylomicrons and VLDL particles acquire apoCII, they can bind to LPL, which hydrolyzes triglycerides from the core of the lipoprotein (Fig. 19-5). LPL-mediated lipolysis liberates free fatty acids and glycerol.

HDL

apoA-I apoE

apoC-II

Liver

Intestines apoC-II

apoB100

apoC-II

apoB48

VLDL

Chylomicron

Plasma Muscle or adipose tissue

Muscle or adipose tissue apoC-II

apoB48 apoC-II

Fatty acids

VLDL Lipoprotein lipase

FIGURE 19-5.

Capillary endothelium

apoB100

Fatty acids

Chylomicron Capillary endothelium

Lipoprotein lipase

Intravascular metabolism of apoB-containing lipoproteins. Following secretion, chylomicrons and VLDL particles are activated for lipolysis when they encounter HDL particles in the plasma and acquire the exchangeable apolipoprotein apoCII. When chylomicrons and VLDL circulate into capillaries of muscle or adipose tissue, apoCII promotes binding of the particle to lipoprotein lipase, which is bound to the surface of endothelial cells. Lipoprotein lipase mediates hydrolysis of triglycerides, but not cholesteryl esters, from the core of the lipoprotein particle. The resulting fatty acids are taken up into muscle or adipose tissue.

316 Principles of Cardiovascular Pharmacology VLDL remnant (IDL)

A

Chylomicron remnant

B

apoB48

Chylomicron or VLDL remnant

Triglyceride

Hepatic sinusoidal endothelium

apoB100

apoC-II

apoE

apoC-II

Heparan sulfate proteoglycan

apoB48 Cholesteryl ester apoB48

HDL Sequestration apoE apoE

Hepatic lipase

apoA-I

Space of Disse

HDL Fatty acid

apoB48

Lipolysis

apoC-II apoA-I

apoE

VLDL remnant (IDL)

Chylomicron remnant apoB100

LDL-R

LRP HSPG

Uptake

apoE

Hepatocyte

apoE apoB48

FIGURE 19-6.

Formation and hepatic uptake of remnant particles. A. Upon completion of hydrolysis, chylomicrons and VLDL lose affinity for lipoprotein lipase. When an HDL particle is encountered, apoCII is transferred back to HDL particles in exchange for apoE. The resulting particles are chylomicron and VLDL remnants. B. The activity of lipoprotein lipase results in remnant lipoprotein particles that are small enough to enter the space of Disse. Remnant lipoproteins are sequestered in the space of Disse by binding to high-molecular-weight heparan sulfate proteoglycan (HSPG) molecules. This is followed by the action of hepatic lipase, which promotes lipolysis of some residual triglycerides in the core of the remnant lipoproteins and the release of fatty acids. Uptake of remnant lipoprotein particles into hepatocytes is mediated by the LDL receptor (LDL-R), the LDL-receptor-related protein (LRP), a complex formed between LRP and HSPG, or HSPG alone.

The free fatty acids are then taken up by the neighboring parenchymal cells. The expression level and intrinsic activity of LPL in muscle and adipose tissue are regulated according to the fed/fasting state, allowing the body to direct the delivery of fatty acids preferentially to muscle during fasting and to adipose after a meal. The rate of lipolysis of chylomicron and VLDL triglycerides is also controlled by apoCIII, which is an inhibitor of LPL activity. LPL inhibition by apoCIII may be an additional mechanism promoting widespread distribution of triglyceride-rich particles in the circulation. Receptor-Mediated Clearance of ApoB-Containing Lipoproteins As LPL continues to hydrolyze triglycerides from chylomicrons and VLDL, the particles become progressively depleted of triglycerides and relatively enriched in cholesterol. Once approximately 50% of the triglycerides have been removed, the particles lose their affinity for LPL and dissociate from the enzyme. The exchangeable apolipoproteins apoAI and

apoCII (as well as apoCI and apoCIII) are then transferred to HDL in exchange for apoE (Fig. 19-6A), which serves as a high-affinity ligand for receptor-mediated clearance of the particles. Upon acquiring apoE, the particles are termed chylomicron or VLDL remnants. Remnants of chylomicrons and VLDL are taken up by the liver in a three-step process (Fig. 19-6B). The first step is sequestration of the particles within the space of Disse between the fenestrated endothelium of the liver sinusoids and the sinusoidal (basolateral) plasma membrane of the hepatocytes. Sequestration requires that the remnant particles become small enough during lipolysis to fit between the endothelial cells. Once in the space of Disse, remnants are bound and sequestered by large heparan sulfate proteoglycans. The next step is particle remodeling within the space of Disse by the action of hepatic lipase, a lipolytic enzyme that is similar to LPL but is expressed by hepatocytes. Hepatic lipase appears to optimize the triglyceride content of remnant

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 317

particles so that they can be cleared efficiently by receptormediated mechanisms. The final phase of remnant clearance is receptor-mediated particle uptake. This is accomplished by one of four pathways. At the sinusoidal hepatocyte plasma membrane, remnant particles may be bound and taken up by the LDL receptor, the LDL-receptor-related protein (LRP), or heparan sulfate proteoglycans. A fourth pathway is mediated by the combined activities of LRP and heparan sulfate proteoglycans. These redundant mechanisms allow for efficient particle clearance, so that the half-life of remnants in the plasma is approximately 30 minutes. Formation and Clearance of LDL Particles ApoB48-containing chylomicron remnants are completely cleared from the plasma. In contrast, the presence of apoB100 alters the metabolism of VLDL remnants so that only

Hepatic lipase

A

approximately 50% are cleared by the pathways for remnant particles. The difference is manifested during the metabolism of the remnant particles by LPL. VLDL remnants are avidly metabolized by LPL, becoming an increment smaller, relatively more deficient in triglycerides, and relatively enriched in cholesteryl esters. When converted to remnants following exchange of apolipoproteins with HDL, these more dense particles are called intermediate-density lipoproteins (IDL). Because IDL contains apoE, a fraction of these particles (approximately 50%) may be cleared into the liver by remnant receptor pathways (Fig. 19-6). However, the remainder are converted to LDL by hepatic lipase, which further hydrolyzes triglycerides in the core of IDL. The further reduction in size of the particle results in the transfer of apoE to HDL. As a result, LDL is a distinct, cholesteryl ester-enriched lipoprotein with apoB100 as its only apolipoprotein (Fig. 19-7A).

apoC-II apoB100

Liver

apoB100

Fatty acid apoE

LDL

IDL

apoE apoC-II apoB100

B LDL

apoA-I

Cholesteryl linoleate

HDL

LDL binding to LDL receptor

LDL-R

Internalization LDL receptor recycling

Lysosome

Hydrolysis

Cholesterol

Hydrolysis

Amino acids

1

ER

2 HMG CoA reductase

3 ACAT

LDL receptors Cholesteryl oleate

FIGURE 19-7. Formation and clearance of LDL particles. A. Formation of LDL occurs when IDL particles interact with hepatic lipase to become denser and cholesteryl ester-enriched. As a result, both apoE and apoCII lose affinity for the particle and are transferred to HDL, leaving only apoB100. B. Binding of apolipoprotein B100 to LDL receptors on hepatocytes or other cell types promotes LDL internalization into endocytic vesicles and fusion of the vesicles with lysosomes. LDL receptors are recycled to the cell surface, whereas lipoprotein particles are hydrolyzed to amino acids (from apoB100) and free cholesterol (from cholesteryl esters). Intracellular cholesterol has three regulatory effects on the cell. First, cholesterol decreases the activity of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Second, cholesterol activates acetyl-CoA:cholesterol acyltransferase (ACAT), an enzyme that esterifies free cholesterol into cholesteryl esters for intracellular storage or export. Third, cholesterol inhibits the transcription of the gene encoding the LDL receptor, and thereby decreases further uptake of cholesterol by the cell.

Vessel lumen

Circulating monocytes

Endothelial dysfunction

Native LDL

Endothelial injury

D

B SR-A

A Resident monocytemacrophage

C E

Cell-mediated oxidation

Foam cell Oxidized LDL

Subendothelial space

Foam cell necrosis

A

LCAT

Liver

PLTP Cholesterol

Phospholipid Cholesteryl ester

Pre-β-HDL ABCA1

SR-BI

Fatty acid

apoA-I

Plasma

α-HDL Tissues Cholesterol

Hepatic lipase

CETP LCAT PLTP

B Plasma

Chylomicron or VLDL remnant

Albumin

Lyso-PC Cell LCAT PLTP

CETP

PC

Lyso-PC

LCAT

Cholesterol

α-HDL

Cholesteryl ester

FIGURE 19-9.

Reverse cholesterol transport. A. The process of reverse cholesterol transport begins when apoAI is secreted from the liver. ApoAI in plasma interacts with ATP binding cassette protein AI (ABCA1), which incorporates a small amount of phospholipid and unesterified cholesterol from hepatocyte plasma membranes to form a discoidal-shaped pre-␤-HDL particle. Due to the activity of lecithin:cholesterol acyltransferase (LCAT) in plasma, pre-␤-HDL particles mature to form spherical ␣-HDL. Spherical ␣-HDL particles function to accept excess unesterified cholesterol from the plasma membranes of cells in a wide variety of tissues. The unesterified cholesterol is transferred from the cell to nearby HDL particles by diffusion through the plasma. As explained in Panel B, LCAT and phospholipid transfer protein (PLTP) increase the capacity of HDL to accept unesterified cholesterol molecules from cells by allowing for expansion of the core and the surface coat of the particle. Cholesteryl ester transfer protein (CETP) removes cholesteryl ester molecules from HDL and replaces them with triglycerides from remnant particles. HDL particles interact with scavenger receptor, class B type I (SR-BI), which mediates selective hepatic uptake of cholesteryl esters, but not apoAI. This process is facilitated when hepatic lipase hydrolyzes triglycerides from the core of the particle. The remaining apoAI molecules may begin the cycle of reverse cholesterol transport again. B. LCAT, PLTP, and CETP promote the removal of excess cholesterol from the plasma membranes of cells. LCAT removes a fatty acid from a phosphatidylcholine molecule in the surface coat of ␣- (or pre-␤-) HDL and esterifies an unesterified cholesterol molecule on the surface of the particle. The resulting lysophosphatidylcholine (lyso-PC) becomes bound to albumin in the plasma, whereas the cholesteryl ester migrates spontaneously into the core of the lipoprotein particle. The unesterified cholesterol molecules that are consumed by LCAT are replaced by unesterified cholesterol from cells. HDL phospholipids that are consumed by LCAT action are replaced with excess phospholipids from remnant particles by the activity of PLTP. As described in Panel A, CETP increases the efficiency of cholesterol movement to the liver by transporting cholesteryl ester molecules from ␣-HDL to VLDL remnants in exchange for triglycerides. Unlike phospholipids, triglycerides, and cholesteryl esters, unesterified cholesterol and lyso-PC move by diffusion through the plasma. 319

320 Principles of Cardiovascular Pharmacology

incorporates a small amount of membrane phospholipid and unesterified cholesterol into the apoAI molecule. The resulting small, disk-shaped particle, which consists mainly of phospholipid and apolipoprotein AI, is referred to as nascent or pre-␤HDL, due to its characteristic migration on agarose gels. Intravascular Maturation of HDL Because disk-shaped pre-␤-HDL particles are relatively inefficient at removing excess cholesterol from cell membranes, these particles must mature into spherical particles in the plasma. HDL maturation occurs as a result of the activity of two distinct circulating proteins (Fig. 19-9A, B). Lecithin:cholesterol acyltransferase (LCAT) binds preferentially to disk-shaped HDL and converts cholesterol molecules within the particle to cholesteryl esters. This is accomplished by transesterification of a fatty acid from a phosphatidylcholine molecule on the surface of the HDL to the hydroxyl group of a cholesterol molecule. The reaction also creates a lysophosphatidylcholine molecule, which dissociates from the particle and binds to serum albumin. Because they are highly insoluble, cholesteryl esters migrate into the core of the HDL particle. The development of a hydrophobic core converts the pre-␤-HDL to a spherical ␣-HDL particle. The second important protein that contributes to HDL maturation in the plasma is phospholipid transfer protein (PLTP). PLTP transfers phospholipids from the surface coat of apoB-containing remnant particles to the surface coat of HDL. During LPL-mediated lipolysis of apoB-containing lipoproteins, the particles become smaller as triglycerides are removed from the core. This leaves a relative excess of phospholipids on the surface of the particle. Because phospholipids are highly insoluble and cannot otherwise dissociate from a particle, PLTP removes excess phospholipids and thereby maintains the appropriate surface concentration for the shrinking core. By transferring phospholipids to the surface of HDL, PLTP also replaces the molecules that are consumed by the LCAT reaction. This allows the core of HDL to continue to enlarge. HDL-Mediated Cholesterol Efflux from Cells Cellular cholesterol efflux is the mechanism by which excess insoluble cholesterol molecules are removed from cells. This occurs when unesterified cholesterol is transferred from the plasma membrane of cells to an HDL particle. The mechanism of cholesterol efflux varies depending on the cell type and the type of HDL particle. Lipid-poor pre-␤-HDL particles can promote cholesterol efflux by interacting with ABCA1. This process is not only important in HDL formation by the liver, but is also a mechanism for removing excess cholesterol from cells within the subendothelial space and for protecting macrophages from cholesterol-induced cytotoxicity. Spherical HDL very efficiently stimulates cholesterol efflux by several different mechanisms. First, the interaction of apoAI on HDL with SR-BI on the plasma membrane promotes cholesterol efflux. Second, macrophages express not only ABCA1 and SR-BI but also ABCG1, which also mediates cholesterol efflux to spherical HDL. Finally, spherical HDL particles may promote cholesterol efflux in the absence of binding to a specific cell-surface protein. Although cholesterol has very low monomeric solubility, it can dissociate in appreciable amounts and travel short distances through the plasma to acceptor particles that are enriched

with phospholipids on their surfaces. Quantitatively, efflux to spherical HDL particles accounts for most of the removal of excess cholesterol from cells. This capacity of HDL to remove cellular cholesterol is enhanced by the activities of LCAT and PLTP, which prevent the surface coat of the particle from becoming saturated with cholesterol. Delivery of HDL Cholesterol to the Liver When mature HDL particles circulate to the liver, they interact with SR-BI, the principal HDL receptor (Fig. 19-9A). SR-BI is highly expressed on the sinusoidal plasma membranes of hepatocytes. In contrast to its action on most nonhepatic cells, where SR-BI mediates efflux of excess cholesterol from the membrane, SR-BI in the liver promotes selective uptake of lipids. In this process, the cholesterol and cholesteryl esters of HDL particles are taken up into the hepatocyte in the absence of uptake of apolipoproteins. During SR-BI–mediated selective lipid uptake, apoAI is liberated to participate in pre-␤HDL formation. The “lifespan” of an HDL particle is 2–5 days, suggesting that each apoAI molecule can participate in many cycles of reverse cholesterol transport. Among the nonhepatic tissues that express high levels of SR-BI are the adrenal glands and gonads, presumably reflecting the requirement of these organs for cholesterol to support steroidogenesis. Delivery of cholesterol from extrahepatic tissues to the liver is optimized by two additional proteins, cholesterol ester transfer protein (CETP) and hepatic lipase. CETP is a plasma protein that transfers cholesteryl esters from mature spherical HDL to the cores of remnant lipoproteins in exchange for a triglyceride molecule, which is inserted into the core of the HDL particle (Fig. 19-9B). This process allows the body to utilize remnant particles that have completed their function of triglyceride transport for purposes of transporting cholesterol to the liver. Removal of cholesteryl ester molecules from HDL appears to serve two functions. First, it further increases the capacity of HDL to take on additional cholesterol molecules from cells. Second, it makes the process of selective uptake by SR-BI more efficient. This is because hydrolysis of triglycerides by hepatic lipase on the hepatocyte surface facilitates the activity of SR-BI (Fig. 19-9A). As noted above, reverse cholesterol transport is the overall process by which HDL removes cholesterol from macrophages and other extrahepatic tissues and returns it to the liver. The concept that increased plasma concentrations of HDL cholesterol may reflect increased rates of reverse cholesterol transport provides a possible explanation for the inverse relationship between plasma HDL levels and risk of cardiovascular disease. HDL particles also exert direct beneficial effects on vascular tissue, including enhancement of antioxidant enzyme activities that inhibit oxidation of LDL. HDL also inhibits the expression of inflammatory mediators (e.g., intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]) by vascular cells. Increased understanding of HDL metabolism may lead to the development of novel biochemical targets for increasing reverse cholesterol transport in order to slow or even reverse the progression of atherosclerosis.

Biliary Lipid Secretion Once cholesterol is delivered to the liver by the process of reverse cholesterol transport, it is eliminated by biliary secretion. An essential step occurs when a fraction of the cholesterol is converted to bile acids (Fig. 19-10A). Cholesterol 7␣-hydroxylase (CYP7A1), an enzyme expressed only in

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 321

A HO

Cholesterol 7α-hydroxylase

COO-

(liver)

Cholesterol Balance HO

HO

Cholesterol

OH

Bile acid (Cholate)

B

Blood

into the small intestine. As described above, bile facilitates the digestion and absorption of fats, in addition to promoting the elimination of endogenous cholesterol.

Bile

Sinusoidal membrane ABCG5/G8

Cholesterol Micelle

Hepatocyte ABCB11

Bile acid Bile salt ABCB4

Phospholipid

Canalicular membrane

FIGURE 19-10.

Biliary lipid secretion. A. Within hepatocytes, a portion of cholesterol is converted to bile acids. This process is rate-limited by cholesterol 7␣-hydroxylase, which is expressed only in hepatocytes. Cholate is the most abundant bile acid synthesized by the human liver. B. Within the canalicular (apical) membranes, an ATP-dependent pump ABCB11 drives the secretion of bile acids out of the cell against a concentration gradient. Bile acids then stimulate the activities of two other proteins, ABCB4 and a heterodimer of ABCG5 and ABCG8 (ABCG5/G8), to secrete phospholipids and cholesterol, respectively, into bile. Within bile, the interactions among bile acids, phospholipids, and cholesterol result in the formation of micelles.

hepatocytes, catalyzes the rate-limiting step in the catabolism of cholesterol to bile acids. Bile acids, unlike cholesterol, are highly soluble in water. Moreover, bile acids are biological detergents that promote the formation of micelles (Fig. 19-10B). These macromolecular aggregates, which are rich in phospholipids derived from hepatocyte membranes, solubilize cholesterol in bile for transport from the liver to the small intestine. In this way, micelles serve as a functional counterpart to HDL particles in plasma. Bile formation begins when bile acids are pumped into bile by the action of a canalicular membrane transport pump known as ABCB11 (Fig. 19-10B). In turn, these bile acids stimulate the biliary secretion of phospholipids and cholesterol. Phospholipid and cholesterol secretion are mediated by two additional transporters, ABCB4 for phospholipids and a heterodimer of ABCG5 and ABCG8 for cholesterol. Large amounts of bile acids, phospholipids, and cholesterol are secreted into bile at approximate rates of 24, 11, and 1.2 grams each day, respectively. Biliary lipids are stored in the gallbladder during fasting. The stimulus of a fatty meal leads to gallbladder contraction, which propels its contents

Because cholesterol is converted by the liver to bile acids and secreted unmodified into bile, overall cholesterol balance depends on the disposition of both cholesterol and bile acids. Most bile acid molecules are not lost in the feces after participating in cholesterol transport and fat digestion; instead, they are taken up and recycled by high-affinity transport proteins in the distal ileum. Bile acids enter the portal circulation and are transported back to the liver, where they are cleared from the blood by hepatocytes with high firstpass efficiency. Bile acids are then re-secreted into bile. This process of recycling bile acids between the liver and intestine is referred to as enterohepatic circulation. The enterohepatic circulation is highly efficient, allowing ⬍5% of secreted bile acids to be lost in the feces. However, because bile acids are secreted in such large amounts, the small fractional loss of bile acids amounts to about 0.4 grams per day. Considering that cholesterol is the substrate for bile acid synthesis, fecal bile acids represent a source of cholesterol loss from the body. Sensitive nuclear hormone receptors within the liver are capable of detecting the rate of loss of bile acids into the feces. These receptors tightly regulate transcription of bile acid synthetic genes. As a result, the liver synthesizes precisely the amount of bile acids that is sufficient to replace what is lost in the feces. In addition to the 1.2 grams of cholesterol that are secreted into bile each day, the average American diet contributes approximately 0.4 grams each day to intestinal cholesterol. Therefore, dietary cholesterol represents only a minor fraction (25%) of the total (i.e., biliary and dietary) cholesterol that passes through the intestine. The extent to which intestinal cholesterol is absorbed appears to be genetically regulated. Each individual absorbs a fixed percentage of intestinal cholesterol. In the population, percentages range from as low as 20% to more than 80%. For example, when an average individual absorbs 50% of intestinal cholesterol, this will amount to half of the 1.6 grams (i.e., 1.2 grams of biliary cholesterol plus 0.4 grams of dietary cholesterol), and the other half (0.8 grams) will be lost in the feces. Combined with a loss of 0.4 grams per day of cholesterol in the form of fecal bile acids, this yields a total cholesterol loss from the body of 1.2 grams each day. Taking into account intestinal absorption of dietary cholesterol and reabsorption of biliary cholesterol, total body cholesterol synthesis is approximately 0.8 grams per day (i.e., cholesterol synthesis ⫽ fecal loss of cholesterol plus bile acids – dietary cholesterol intake). Thus, the amount of endogenous cholesterol synthesis is about twofold greater than the amount consumed in the average diet.

 PATHOPHYSIOLOGY Numerous studies have demonstrated a definitive link between elevated plasma lipid concentrations and the risk of cardiovascular disease. Increased risk of cardiovascular mortality is most closely linked to elevated levels of LDL cholesterol and decreased levels of HDL cholesterol. In addition, hypertriglyceridemia represents an independent risk factor. The risk is further increased when hypertriglyceridemia is associated with low HDL-cholesterol concentrations, even if

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 325 Increased LDL-R expression and uptake of plasma LDL

Acetyl CoA + Acetoacetyl CoA

Statins HMG CoA HMG CoA reductase Mevalonate

Increased LDL receptor expression

5-pyrophosphomevalonate Isopentylpyrophosphate 3,3-dimethylallylpyrophosphate Isoprenoids Geranylpyrophosphate Farnesylpyrophosphate Squalene Lanosterol Cholesterol Cholesterol

Protease activation SREBP (inactive)

SREBP (active)

Nucleus LDL-R gene

SRE

FIGURE 19-11. Mechanism of LDL lowering by statins. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. Decreased cellular cholesterol concentrations lead to protease activation and cleavage of the sterol regulatory element binding protein (SREBP), which is a transcription factor that normally resides in the cytoplasm. The cleaved SREBP diffuses into the nucleus, where it binds to sterol response elements (SRE), leading to up-regulation of LDL receptor gene transcription. This leads to increased cellular LDL receptor expression. This promotes uptake of LDL particles and results in reduced LDL-cholesterol concentrations in the plasma.

decrease mortality even in the absence of overt cardiovascular disease, which is called primary prevention. Despite these convincing percentage risk reductions in both secondary and primary prevention trials, it should be noted that statin use is associated with a greater absolute risk reduction in secondary prevention; the reason may be that patients in this

treatment group have a greater absolute risk of death and therefore display the largest benefit from statins. It is also important to note that statins have proven to be effective in reducing cardiovascular disease risk for high-risk patients (e.g., diabetic patients) with average, or even below average, LDL-cholesterol levels. The magnitude of LDL-cholesterol lowering depends on the efficacy and dose of the statin that is administered. In general, statins reduce LDL-cholesterol concentrations by up to about 60%. Statins increase HDL-cholesterol concentrations by an average of 10%, and reduce triglyceride concentrations by up to about 40%, depending on statin dose and degree of hypertriglyceridemia. The effect of statins on triglyceride levels is mediated by decreased VLDL production and increased clearance of remnant lipoproteins by the liver. The dose–response relationship of statins is nonlinear: the largest effect occurs with the starting dose. Each subsequent doubling of the dose produces, on average, an additional 6% LDL reduction. This is sometimes referred to as the “rule of 6’s.” In addition to reducing LDL-cholesterol concentrations, statins have a number of other pharmacologic consequences. These are collectively referred to as pleiotropic effects, which include: decreased inflammation, reversal of endothelial dysfunction, decreased thrombosis, and improved stability of atherosclerotic plaques. Evidence for diminished inflammation during statin therapy includes decreases in acute-phase reactants, which are plasma proteins that are increased during inflammatory states and may play a role in the destabilization of atherosclerotic plaques. The best characterized of the acutephase reactants is C-reactive protein (CRP). Importantly, a recent large randomized clinical trial has shown that, among patients with a moderate risk of developing cardiovascular disease and with elevated baseline CRP levels, use of a statin reduces cardiovascular morbidity and mortality, even when the patients do not have elevated LDL-cholesterol concentrations. Evidence for reversal of endothelial dysfunction during statin therapy includes an improved vasodilatory response of endothelium to NO. Improved vasodilation could help prevent ischemia. Evidence for decreased thrombosis during statin therapy includes a decrease in prothrombin activation and a decrease in tissue factor production. Because thrombosis is at the root of most acute coronary syndromes, its reduction could contribute to the survival benefit of statins. Finally, plaque stability is enhanced during statin therapy because the fibrous cap that overlies the lipid-rich plaque becomes thicker. This effect may be attributable to decreased macrophage infiltration and inhibition of vascular smooth muscle proliferation. It is important to emphasize that most of these pleiotropic effects of statins have been demonstrated only in vitro or in animal models, and their relevance in humans is unclear. Clinical data indicate that the reductions in cardiovascular morbidity and mortality due to statins are primarily attributable to the lowering of LDL-cholesterol concentrations in the plasma. Seven statins—lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin— are currently approved for use in hypercholesterolemia and mixed dyslipidemia. They are considered first-line therapy for increased LDL levels, and their use is supported by numerous trials showing that statins decrease both cardiovascularrelated and total mortality. Stroke is also reduced. All of the statins are believed to act by the same mechanism. The main differences are attributable to potency and pharmacokinetic

Fibrates

PPARα activation

apoA-I, apoA-II synthesis in hepatocytes

apoC-III synthesis in hepatocytes and Lipoprotein lipase expression in muscle vascular beds

Fatty acid oxidation in hepatocytes

Fatty acid uptake in muscle cells and Fatty acid oxidation in muscle cells

Triglyceride synthesis

Plasma HDL

Plasma triglycerides

Adipose tissue

Liver Decreased plasma free fatty acids

Hormone-sensitive lipase

Peripheral cells Decreased plasma VLDL

Decreased synthesis of triglycerides Cholesterol delivery

Niacin receptor Niacin

Decreased plasma LDL

Increased excretion of cholesterol in bile

Increased plasma HDL Decreased apoA-I clearance

Niacin

Cholesterol delivery

Cholesterol removal

CHAPTER 19 / Pharmacology of Cholesterol and Lipoprotein Metabolism 329

patients. Rarely, niacin may cause myopathy. Concurrent administration of niacin with a statin slightly increases the risk of myopathy. Niacin is indicated for patients with elevations of both triglycerides and cholesterol, usually in combination with a statin. Because niacin is currently the most effective agent available for raising HDL, it may also be the drug of choice for patients with modestly elevated LDL and decreased HDL. It is not clear whether both the LDL-lowering and HDL-raising effects of niacin contribute to improved clinical outcomes.

Omega-3 Fatty Acids The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), also referred to as fish oils, are effective at reducing plasma triglycerides by up to 50% in patients with hypertriglyceridemia. The likely mechanism of triglyceride lowering involves regulation of nuclear transcription factors, including SREBP-1c and PPAR␣, to cause reduced triglyceride biosynthesis and increased fatty acid oxidation in the liver. Omega-3 fatty acids are available over the counter as nutritional supplements in the form of fatty acid ethyl esters. Lovaza®, a prescription-strength form of omega-3 fatty acids, has also become available. Lovaza® is enriched (84%) in EPA and DHA, whereas most dietary supplements contain 13–63% fish oils. The recommended dose of Lovaza® is 4 grams, once a day. Omega-3 fatty acids are generally added to therapy when plasma triglyceride concentrations exceed 500 mg/dL. The influence of omega-3 fatty acid use on clinical outcomes is uncertain.

 CONCLUSION AND FUTURE DIRECTIONS LDL reduction by the available lipid-lowering drugs— particularly the statins—represents an important advance in reducing cardiovascular disease mortality. Future drug trials will examine the possible benefits of raising HDL and lowering triglyceride levels on cardiovascular disease. Also under development are pharmacologic therapies for new biochemical targets such as CETP and MTP. Inhibition of CETP raises HDL and lowers LDL by inhibiting the transfer of cholesterol from HDL to remnant particles, whereas inhibition of MTP reduces VLDL secretion.

Suggested Reading Adult Treatment Panel III. Executive summary of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. JAMA 2001;285:2486–2497. (Clinical guidelines for cholesterol-lowering therapy.) Ballantyne CM, ed. Clinical lipidology: a companion to Braunwald’s heart disease. Philadelphia: Saunders/Elsevier; 2009; 584 pp. (Concise chapters cover all aspects of lipoprotein metabolism and pharmacology.) Duffy D, Rader DJ. Emerging therapies targeting high-density lipoprotein metabolism and reverse cholesterol transport. Circulation 2006;113: 1140–1150. (Future directions in pharmacology of HDL metabolism.) Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J Am Coll Cardiol 2004;44:720–732. (Supplemental clinical guidelines for cholesterol-lowering therapy with lower LDL-cholesterol goals for high-risk patients.) Tunaru S, Kero J, Schaub A, et al. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect. Nat Med 2003;9: 352–355. (Identification of the G protein-coupled receptor ligand for pharmacologic effects of niacin.)

20 Pharmacology of Volume Regulation Mallar Bhattacharya and Seth L. Alper

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 332-333 PHYSIOLOGY OF VOLUME REGULATION . . . . . . . . . . . . . . . 332 Determinants of Intravascular Volume . . . . . . . . . . . . . . . 332 Volume Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Volume Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 Renin-Angiotensin–Aldosterone System . . . . . . . . . . . 334 Natriuretic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Antidiuretic Hormone . . . . . . . . . . . . . . . . . . . . . . . . . 337 Renal Sympathetic Nerves . . . . . . . . . . . . . . . . . . . . . 337 Renal Control of Na⫹ Excretion . . . . . . . . . . . . . . . . . . . . 337 Proximal Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Thick Ascending Limb of the Loop of Henle . . . . . . . . . 338 Distal Convoluted Tubule . . . . . . . . . . . . . . . . . . . . . . . 339 Collecting Duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 PATHOPHYSIOLOGY OF EDEMA FORMATION . . . . . . . . . . . 340 Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

Cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 342 Agents That Modify Volume Regulators . . . . . . . . . . . . . . 342 Inhibitors of the Renin–Angiotensin System . . . . . . . . 342 B-Type Natriuretic Peptide . . . . . . . . . . . . . . . . . . . . . 344 Vasopressin Receptor Antagonists and Agonists . . . . . 344 Agents That Decrease Renal Naⴙ Reabsorption . . . . . . . . 344 Carbonic Anhydrase Inhibitors . . . . . . . . . . . . . . . . . . . 345 Osmotic Diuresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Loop Diuretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Thiazides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 Collecting Duct (Potassium-Sparing) Diuretics. . . . . . . 347 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 347 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

 INTRODUCTION

Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure.

Coordinated regulation of volume homeostasis and vascular tone maintains adequate tissue perfusion in response to varying environmental stimuli. This chapter discusses the pharmacologically relevant physiology of volume regulation, with emphasis on the hormonal pathways and renal mechanisms that modulate systemic volume. (Control of vascular tone is discussed in Chapter 21, Pharmacology of Vascular Tone.) Dysregulation of volume homeostasis can result in edema, the pathologic accumulation of fluid in the extravascular space. Pharmacologic modulation of volume is targeted at reducing volume excess; this is an effective treatment for hypertension and heart failure (HF), as well as for cirrhosis and the nephrotic syndrome. The two broad classes of pharmacologic agents used to modify volume status are modulators of neurohormonal regulators (e.g., angiotensin converting enzyme [ACE] inhibitors) and diuretics, agents that increase renal Na⫹ excretion. Drugs that modify volume regulation also have many other clinically important effects on the body, because these volume regulators act as diverse hormonal modulators in multiple physiologic pathways. Many of the clinical applications of these agents are discussed further in Chapter 25, Integrative Cardiovascular 332

 PHYSIOLOGY OF VOLUME REGULATION An intricate set of mechanisms sense, signal, and modulate changes in plasma volume. Volume sensors are located throughout the vascular tree, including in the atria and in the kidneys. Many of the volume regulators activated by these sensors include systemic and autocrine hormones, while others involve neural circuits. The integrated result of these signaling mechanisms is to alter vascular tone and to regulate renal Na⫹ reabsorption and excretion. Vascular tone maintains end-organ tissue perfusion; changes in renal Na⫹ excretion alter total volume status.

Determinants of Intravascular Volume Intravascular volume is a small proportion of total body water, but the amount of fluid in the vascular compartment critically determines the extent of tissue perfusion. Approximately 2/3 of total body water is intracellular, while 1/3 is extracellular. Of the extracellular fluid (ECF), approximately 3/4 resides in the interstitial space, while 1/4 of ECF is plasma.

334 Principles of Cardiovascular Pharmacology

Volume Regulators

A

πc Arterial end

Pc

πc

Pif

πif

Pc

Pif

πif

Venous end

Flow

B

Net fluid movement

Out

0

In Arterial end

Position along capillary

Venous end

FIGURE 20-1. Capillary fluid filtration. The balance of hydrostatic pressure and oncotic pressure determines fluid filtration along the capillary. The example shown here is for a hypothetical capillary where fluid filtration exceeds fluid reabsorption. A. At the arterial end of the capillary, the capillary hydrostatic pressure (Pc ) is large (long arrow ), and the sum of Pc and interstitial oncotic pressure (␲if) exceeds the sum of interstitial hydrostatic pressure (Pif) and capillary oncotic pressure (␲c ). Therefore, fluid moves out of the capillary into the interstitial space. As fluid continues to filter along the length of the capillary, the increased fluid filtration results in decreased Pc and increased ␲c, thus decreasing the driving force for fluid filtration from the capillary to the interstitium. Throughout the length of the capillary, Pif and ⌸if remain relatively constant. B. A graphic representation of net fluid movement along the capillary length shows the decreasing driving force for fluid filtration into the interstitium. In the hypothetical capillary shown here, fluid is filtered into the interstitium along the entire capillary length; lymphatic vessels eventually return the excess interstitial fluid to the systemic circulation (not shown).

Together, the low-pressure and high-pressure feedback systems integrate neurohumoral volume signals to maintain volume homeostasis in the face of volume perturbations. The neurohormonal response to a change in volume status is controlled by four main systems: the renin-angiotensin–aldosterone system (RAAS), natriuretic peptides, ADH, and renal sympathetic nerves. The RAAS, ADH, and renal sympathetic nerves are active in situations of intravascular volume depletion, while natriuretic peptides are released in response to intravascular volume overload. Renin-Angiotensin–Aldosterone System Renin is an aspartyl protease produced and secreted by the juxtaglomerular apparatus, a specialized set of smooth muscle cells that line the afferent and efferent arterioles of the renal glomerulus. The ultimate result of renin secretion is vasoconstriction and Na⫹ retention, actions that maintain tissue perfusion and increase extracellular fluid volume (Fig. 20-2). At least three mechanisms are thought to control juxtaglomerular cell renin release (Fig. 20-3). First, a direct pressure-sensing mechanism of the afferent arteriole increases juxtaglomerular cell release of renin in response to decreased arteriolar wall tension. The detailed molecular mechanism of

Angiotensinogen (secreted by liver)

Renin (secreted by kidney)

Angiotensin I Angiotensin converting enzyme (expressed in lung endothelium)

Angiotensin II

Renal proximal tubule

Renal efferent arterioles

(increased NaCl absorption)

(vasoconstriction; maintains GFR)

Adrenal cortex (zona glomerulosa)

the atria and pulmonary vasculature transmit a signal to noradrenergic neurons in the medulla of the central nervous system (CNS). This signal is relayed to the hypothalamus, resulting in increased secretion by the posterior pituitary gland of antidiuretic hormone (ADH, also known as vasopressin). Together with increased peripheral sympathetic tone, ADH maintains distal tissue perfusion. In response to increased wall stress (e.g., caused by increased intravascular volume), cells of the atria produce and secrete natriuretic peptide, promoting vasodilation and natriuresis (increased renal Na⫹ excretion). The high-pressure system consists of specialized baroreceptors in the aortic arch, carotid sinus, and juxtaglomerular apparatus. These sensors modulate hypothalamic control of ADH secretion and sympathetic outflow from the brainstem. In addition, sympathetic input stimulates the juxtaglomerular apparatus to secrete renin, a proteolytic enzyme that activates the renin-angiotensin–aldosterone system (see below).

Hypothalamus (thirst; increased ADH secretion)

Aldosterone (increased NaCl absorption) acting at 1. Medullary thick ascending limb of Henle 2. Distal tubule 3. Collecting duct

FIGURE 20-2.

The renin-angiotensin–aldosterone axis. Angiotensinogen is a prohormone secreted into the circulation by hepatocytes. Renin, an aspartyl protease secreted by juxtaglomerular cells of the kidney, cleaves angiotensinogen to angiotensin I. Angiotensin converting enzyme (ACE), a protease expressed on pulmonary capillary endothelium (and elsewhere), cleaves angiotensin I to angiotensin II. Angiotensin II has four actions that increase intravascular volume and maintain tissue perfusion. First, angiotensin II stimulates zona glomerulosa cells of the adrenal cortex to secrete aldosterone, a hormone that increases renal NaCl reabsorption at multiple segments along the nephron. Second, angiotensin II directly stimulates renal proximal tubule reabsorption of NaCl. Third, angiotensin II causes efferent arteriolar vasoconstriction, an action that increases intraglomerular pressure and thereby increases GFR. Fourth, angiotensin II stimulates hypothalamic thirst centers and promotes ADH secretion.

CHAPTER 20 / Pharmacology of Volume Regulation 335

Macula densa cell

Juxtaglomerular cell Adenosine (A)

Tubular lumen

β1-agonist

Na+ ClK+

β1-AR A1 receptor ? Gs

Na+ 2Cl[Na+]

NKCC2

K+

[Cl-]

Gi

COX-2 cAMP Prostaglandins (PG) Renin

Gs

PG receptor

FIGURE 20-3.

Modulation of renin release. Renin is released by juxtaglomerular cells in response to diverse stimuli that signal volume depletion. First, decreased pressure in the afferent arteriole (not shown) stimulates increased renin release, possibly by releasing prostaglandins. Second, juxtaglomerular cells express ␤1-adrenergic receptors (␤1-AR) coupled to Gs, which stimulates adenylyl cyclase to increase the intracellular level of cAMP, which is a stimulus for renin release. Third, cells lining the diluting segments of the nephron modulate renin release based on the extent of luminal NaCl flux. In cases of decreased NaCl flux, decreased Cl⫺ entry through the Na⫹/2Cl⫺/K⫹ transporter (NKCC2) on the apical membrane of macula densa cells in the distal convoluted tubule stimulates cyclooxygenase-2 (COX-2) activity, which increases prostaglandin production. The prostaglandins diffuse to and activate juxtaglomerular-cell prostaglandin (PG) receptors, which stimulate release of renin by increasing cAMP production. In contrast, increased cortical thick ascending limb (TAL) NaCl delivery leads, through still-debated mechanisms, to increased generation of adenosine in the juxtaglomerular mesangial interstitium. Activation of Gi-coupled A1 adenosine receptors of the juxtaglomerular cell decreases intracellular cAMP, which leads to decreased renin release.

this sensory transduction is unknown, but may involve autocrine prostaglandin and purinergic signaling. Second, sympathetic innervation of juxtaglomerular cells promotes renin release via ␤1-adrenoceptor signaling. Third, an autoregulatory mechanism known as tubuloglomerular feedback senses distal nephron chloride (and/or sodium) delivery and modulates renin release. Nephron anatomy is organized such that the distal end of the cortical thick ascending limb (TAL) of each nephron is closely apposed to the juxtaglomerular mesangium of that same nephron. This spatial proximity allows rapid integrative regulation of afferent arteriolar diameter and glomerular mesangial contractility by distal nephron electrolyte concentration and/or salt load. Macula densa cells of the cortical thick ascending limb respond to

increased luminal NaCl delivery by increasing extracellular adenosine in the juxtaglomerular interstitium and thereby activating A1 receptors on the juxtaglomerular mesangial cells to decrease renin release. Conversely, decreased luminal NaCl delivery activates a mesangial prostaglandin signaling cascade that culminates in increased renin release. Macula densa cells sense luminal NaCl delivery by monitoring both luminal NaCl concentration and luminal fluid flow rate as sensed by shear stress. The former may be directly sensed by receptors in the apical sensory monocilia of the macula densa cells; the latter by direct bending of the monocilia. Molecular components in the extraciliary apical membrane likely also contribute to these signal transduction processes. After renin is secreted, it acts as a protease to cleave the circulating 14-amino-acid hepatic prohormone angiotensinogen to generate the decapeptide angiotensin I. Angiotensin I is then cleaved to the active octapeptide angiotensin II (AT II) by the carboxypeptidase angiotensin converting enzyme (ACE I) located on the endothelial cell surface. Although ACE is expressed primarily in the pulmonary vascular endothelium and coronary circulation, ACE activity regulates local production of AT II in all vascular beds. Indeed, an incompletely understood “local” renin– angiotensin system is also expressed in the vasculature, producing these substances as autocrine factors independently of the kidney and liver. ACE has a broad proteolytic substrate specificity that includes bradykinin and other kinins that are venodilatory autacoids released in response to inflammation. For this reason, ACE is also known as kininase II. Kininase activity has important pharmacologic consequences, as discussed below. AT II binding to the AT II receptor subtype I (the G protein-coupled AT1 receptor, AT1R) produces at least four physiologic responses: (1) stimulation of aldosterone secretion by zona glomerulosa cells of the adrenal glands; (2) increased reabsorption of NaCl from the proximal tubule and other nephron segments; (3) central stimulation of thirst and ADH secretion; and (4) arteriolar vasoconstriction. All four of these actions increase intravascular volume and therefore help to maintain perfusion pressure: aldosterone secretion increases distal-tubule Na⫹ reabsorption; proximal-tubule NaCl reabsorption increases the fraction of filtered Na⫹ that is reabsorbed; stimulation of thirst increases the free water absorbed into the vasculature; secretion of ADH increases collecting-duct free water absorption; and arteriolar vasoconstriction maintains blood pressure. The actions of AT II are best understood in vascular smooth muscle cells, where AT1R activates phospholipase C, leading to the release of Ca2⫹ from intracellular stores and activation of protein kinase C. Inhibition of AT1R can decrease vascular smooth muscle cell contractility, and thereby decrease systemic vascular resistance and blood pressure (see below). A second G protein-coupled AT II receptor, AT2R, is expressed more prominently in fetal than in adult tissue. AT2R appears to have a vasodilatory role. Natriuretic Peptides Natriuretic peptides are hormones released by atria, ventricles, and vascular endothelium in response to volume overload. The classical natriuretic peptides are A-type, B-type, and C-type natriuretic peptides. A-type natriuretic peptide (ANP) is released primarily by the atria, while B-type natriuretic peptide (BNP) is released mainly by the ventricles.

336 Principles of Cardiovascular Pharmacology

C-type natriuretic peptide (CNP) is released by vascular endothelial cells. The natriuretic peptide uroguanylin (UGN) is released by enterocytes in response to dietary ingestion of salt. Vascular natriuretic peptides are released in response to increased intravascular volume, an effect that may be signaled by increased stretch of natriuretic peptide-secreting cells. Circulating natriuretic peptides bind to one of three receptors, termed NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are transmembrane proteins with cytoplasmic guanylyl cyclase domains (see Chapter 1, Drug–Receptor Interactions); activation of these receptors increases intracellular cGMP levels. NPR-C lacks an intracellular guanylyl cyclase domain and may serve as a “decoy” or “buffer” receptor to reduce the level of circulating natriuretic peptides available to bind to the two signaling receptors. Both ANP and BNP bind with high affinity to NPR-A, while only CNP binds to NPR-B. All three natriuretic peptides bind to NPR-C (Fig. 20-4A). UGN binds to and activates transmembrane guanylyl cyclase C in both renal proximal tubule cells and enterocytes, and binds to an undefined receptor in the renal collecting duct. Natriuretic peptides affect the cardiovascular system, the kidney, and the central nervous system. Integration of natriuretic peptide-derived signals serves to decrease volume overload and its sequelae. ANP relaxes vascular smooth muscle by increasing intracellular cGMP, which causes

A

dephosphorylation of myosin light chain and subsequent vasorelaxation (see Chapter 21). ANP also increases capillary endothelial permeability; this effect reduces blood pressure by favoring fluid filtration from the plasma into the interstitium (see Equation 20-1). In the kidney, natriuretic peptides promote both increased glomerular filtration rate (GFR) and natriuresis. GFR is increased because of constriction of the efferent arteriole and dilation of the afferent arteriole, resulting in higher intraglomerular pressure and therefore increased plasma filtration. The natriuretic effects on the kidney result from antagonism of ADH action in the collecting ducts and antagonism of Na⫹ reabsorption in multiple nephron segments. The central effects of natriuretic peptides are less well understood, but they include decreased perception of thirst (and therefore decreased fluid intake), decreased release of antidiuretic hormone, and decreased sympathetic tone. The signaling mechanisms mediating these actions are uncertain, but may be via CNP, as this natriuretic peptide is expressed at high levels in the brain. Although many of the effects of natriuretic peptides are still not understood completely, these hormones appear to play an important role in regulating the pathophysiology of volume excess. Much interest has recently focused on the relationship between natriuretic peptides and heart failure. The physiology and pharmacology of natriuretic peptides and their receptors remain subjects for active investigation.

B

GTP

Biological effects, including increased natriuresis

Apical membrane

Basolateral membrane

NPR-A

Vasopressin/ ADH Lumen of collecting duct

cGMP

Vesicle containing AQP2

ANP/BNP

GDP

V2-receptor

AQP2 NPR-C Degradation

Internalization Water

GTP

cAMP Translocation/insertion

Water

ATP

Adenylyl cyclase

FIGURE 20-4. Natriuretic peptide and antidiuretic hormone signaling pathways. A. A-type and B-type natriuretic peptides (ANP and BNP) are hormones secreted in response to volume overload. These peptides bind to natriuretic peptide receptor-A (NPR-A) and natriuretic peptide receptor-C (NPR-C). NPR-A is a transmembrane receptor with intrinsic guanylyl cyclase activity associated with its cytoplasmic domain. Increased intracellular cGMP levels mediate the effects of natriuretic peptides, including increased natriuresis. NPR-C is believed to be a “decoy receptor,” because the protein lacks the intracellular catalytic domain. Binding of natriuretic peptide to NPR-C may result in receptor internalization and in degradation of the internalized receptor together with the bound natriuretic peptide. A third natriuretic peptide, CNP, is expressed by vascular endothelial cells and binds to NPR-B (not shown). B. Antidiuretic hormone (ADH), also known as vasopressin, is secreted by the hypothalamus in response to increased osmolality and volume depletion. ADH mediates renal collecting-duct water reabsorption by activating the Gs-coupled V2 vasopressin receptor. Activation of Gs leads to increased adenylyl cyclase activity and increased cAMP levels. cAMP increases collecting-duct water reabsorption by promoting the translocation and insertion of aquaporin 2 water channel (AQP2)-containing vesicles into the collecting duct apical membrane. The increased apical membrane AQP2 results in increased water flux across the collecting duct, and therefore greater reabsorption of filtered water. Hydrolysis of cAMP by phosphodiesterase leads to removal of AQP2 from the luminal membrane by endocytosis of AQP2-containing vesicles (not shown).

CHAPTER 20 / Pharmacology of Volume Regulation 337

Antidiuretic Hormone Antidiuretic hormone (ADH, or vasopressin) is a nonapeptide hormone secreted by the posterior pituitary gland in response to increased plasma osmolality or severe hypovolemia. ADH constricts the peripheral vasculature and promotes water reabsorption in the renal collecting duct. Its actions are mediated by two distinct G protein-coupled receptors. The V1 receptor, present in vascular smooth muscle cells, stimulates vasoconstriction through a Gq-mediated mechanism. The V2 receptor, expressed in collecting duct principal cells, stimulates water reabsorption by a Gs-mediated mechanism (Fig. 20-4B). This Gs signal increases cytosolic cAMP, which leads to activation of protein kinase A (PKA). PKA phosphorylates the water channel aquaporin 2 and activates transport and fusion of aquaporin 2-containing vesicles into the apical membrane of the principal cell. Increased aquaporin 2 expression at the apical membrane promotes increased water reabsorption. Regulation of renal water reabsorption in the collecting duct modulates urine and plasma osmolality, and serves as a reserve mechanism for increasing intravascular volume in situations of severe dehydration. Renal Sympathetic Nerves Renal sympathetic nerves innervate both afferent and efferent arterioles. In response to a decrease in intravascular volume, the renal sympathetic nerves decrease GFR by stimulating constriction of the afferent arteriole to a greater degree than the efferent arteriole. The decreased GFR resulting from preferential constriction of the afferent arteriole ultimately leads to decreased natriuresis. Renal sympathetic nerves also increase renin production by stimulation of ␤1-adrenergic receptors on juxtaglomerular mesangial cells, and increase proximal-tubule NaCl reabsorption. Since transplanted kidneys function normally, and these kidneys lack sympathetic nerve input, it appears that renal innervation is not required for clinically normal kidney function.

Renal Control of Naⴙ Excretion Over the course of 24 hours, the kidneys filter approximately 180 L of fluid. To increase or decrease body fluid volume, the kidneys must increase or decrease renal Na⫹ reabsorption from the large daily volume of glomerular filtrate. For this reason, the neurohormonal mechanisms controlling extracellular volume status have important actions on the kidney. An understanding of the renal control of Na⫹ excretion is crucial to understanding the role of the kidney in regulation of body fluid volume. The renal glomerulus produces an ultrafiltrate of plasma that flows through and is processed by the nephron, the functional unit of the kidney (Fig. 20-5). The postglomerular nephron is responsible for solute and water reabsorption from the filtrate, as well as for excretion of metabolic waste products and xenobiotics, including drugs. The renal tubular epithelial cells of the postglomerular nephron enclose a lengthy tubular lumen, the “urinary space,” which leads to the ureters, urinary bladder, and urethra. The initial glomerular ultrafiltrate contains solutes of low molecular weight at concentrations similar to those in the plasma. As the ultrafiltrate passes through the nephron, substrate-specific transporters and channels in the luminal (apical) membrane of polarized renal tubular epithelial cells sequentially alter the solute concentrations of the tubular fluid. The function of these transporters and channels is, in turn, influenced by changes in solute

Carbonic anhydrase inhibitors Glomerulus

1 PCT

JG Afferent arteriole Efferent arteriole

CCD DCT

3 4 Thiazide diuretics CTAL Loop diuretics

2

Potassiumsparing diuretics

OMCD

MTAL

IMCD ATL

TDL

FIGURE 20-5.

Nephron anatomy and sites of action of diuretics. Nephron fluid filtration begins at the glomerulus, where an ultrafiltrate of the plasma enters the renal epithelial (urinary) space. This ultrafiltrate then flows sequentially through four axially distinct nephron segments (1–4). From the glomerulus, ultrafiltrate travels to the proximal convoluted tubule (PCT) (1), then to the loop of Henle (2), which includes the thin descending limb (TDL), ascending thin limb (ATL), medullary thick ascending limb (MTAL), and cortical thick ascending limb (CTAL) of Henle. The distal convoluted tubule (DCT ) (3) includes the macula densa and juxtaglomerular (JG) apparatus. The collecting duct (4) consists of the cortical collecting duct (CCD ), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). Pharmacologic agents inhibit specific solute transporters within each segment of the nephron. Carbonic anhydrase inhibitors act primarily at the proximal convoluted tubule; loop diuretics act at the medullary and cortical thick ascending limbs; thiazide diuretics inhibit solute transport in the distal convoluted tubule; and potassium-sparing diuretics inhibit collecting-duct Na⫹ reabsorption.

concentrations in the cells themselves, as regulated in part by channels and transporters on the contraluminal (basolateral) side of the cells. Systemic volume regulation by the kidney is accomplished by tubular solute reabsorption through integrated action of ion channels and ion transporters in the apical and basolateral membranes of tubular epithelial cells and by the accompanying reabsorption of water. The nephron beyond the glomerulus exhibits remarkable heterogeneity along its length. Four segments of the nephron are especially relevant to the pharmacology of body volume regulation (Fig. 20-5). These are the proximal tubule, the thick ascending limb (TAL) of the loop of Henle, the distal convoluted tubule (DCT), and the cortical collecting duct (CCD). In each tubular segment, a complex but tightly choreographed group of segment-specific ion transporters and channels collaborate in the reabsorption of NaCl from the

338 Principles of Cardiovascular Pharmacology

lumen across the cellular monolayer of tubular epithelium into the interstitial space. NaCl reabsorption is key for systemic water retention. Solute and water transport across each segment requires coordination of transporter function in the luminal and basolateral membranes. In addition, paracellular transport of ions across the tight junctions between cells requires regulated communication between adjacent cells of the tubular epithelium. Integration of the transcellular and paracellular components of transepithelial transport requires integration of signals transmitted by sensors of extracellular and intracellular ion concentrations and of intracellular, local extracellular, and systemic volume. Alteration of ion transport by drugs in any nephron segment can induce compensatory regulation locally and in more distal nephron segments. Proximal Tubule The proximal tubule (PT) is the first reabsorptive site in the nephron. It is responsible for approximately two-thirds of sodium reabsorption, 85–90% of bicarbonate reabsorption, and approximately 60% of chloride reabsorption (Fig. 20-6). Specific sodium-coupled symporters in the proximal tubule apical membrane drive renal reabsorption of all glucose, amino acids, phosphate, and sulfate from the glomerular filtrate. The proximal tubule also mediates secretion and reabsorption of organic weak acids and weak bases coupled to processes of sodium or proton symport or antiport, or to anion exchange Apical membrane

Basolateral membrane

Lumen of proximal convoluted tubule 3HCO3-

Na+

NBCe1

NHE3

Na+

H+

vH+ ATPase

H+ + HCO3H2CO3

H2CO3 3Na+

H+

Na+/K+

CAII CAIV

Acetazolamide

ATPase

2K+ Acetazolamide

CO2 + H2O

CO2 + H2O

FIGURE 20-6.

Proximal convoluted tubule cell. A significant percentage of proximal convoluted tubule Na⫹ is reabsorbed via the NHE3 Na⫹/H⫹ antiporter. The action of this antiporter, together with that of an apical membrane vacuolar ATPase (vH⫹ ATPase), results in significant H⫹ extrusion into the proximal convoluted tubule urinary space. H⫹ extrusion is coupled to HCO3⫺ reabsorption by the action of an apical membrane carbonic anhydrase IV (CAIV) that catalyzes the cleavage of HCO3⫺ into OH⫺ and CO2. OH⫺ combines with H⫹ to form water, while CO2 diffuses into the cytoplasm of the epithelial cell. The cytoplasmic enzyme carbonic anhydrase II (CAII) catalyzes the formation of HCO3⫺ from CO2 and OH⫺; the HCO3⫺ is then transported into the interstitium together with Na⫹. The net result of this process is reabsorption of HCO3⫺ and Na⫹ by the basolateral cotransporter NBCe1. Acetazolamide inhibits both isoforms of carbonic anhydrase; the decreased carbonic anhydrase activity results in decreased Na⫹ and HCO3⫺ absorption.

mechanisms. Among these weak acids and bases are many of the drugs used to regulate systemic volume (see below). Bicarbonate reabsorption requires the coordinated action of apical and basolateral ion transporters together with apical and intracellular enzymatic activities (Fig. 20-6). At the luminal surface of the proximal tubule, filtered bicarbonate encounters active proton secretion across the proximal tubule brush-border microvilli. Two-thirds of the proton efflux is in exchange for influx of Na⫹, largely via the NHE3 Naⴙ/ Hⴙ exchanger. The remaining third of proton efflux is mediated by the vacuolar Hⴙ-ATPase (vHⴙ ATPase). The HCO3⫺ permeability of the luminal membrane of the proximal tubular cell is low. However, the outer leaflet of the luminal membrane harbors the glycosylphosphatidylinositollinked exoenzyme carbonic anhydrase IV (CAIV). CAIV converts luminal HCO3⫺ to CO2 and OH⫺. The OH⫺ is rapidly hydrated to water by the abundance of local protons, and the CO2 freely diffuses into the cytoplasm of the proximal tubular epithelial cell. The intracellular CO2 is rapidly rehydrated to HCO3⫺ by cytoplasmic carbonic anhydrase II (CAII); this reaction consumes the intracellular OH⫺ accumulated as a result of the H⫹-extruding activities of apical NHE3 and vH⫹ ATPase. The HCO3⫺ produced by the CAII reaction is then cotransported with Na⫹ across the basolateral membrane of the epithelial cell, accounting for the net reabsorption of sodium and bicarbonate. The Na⫹/HCO3⫺ co-transporter NBCe1 mediates electrogenic basolateral efflux of three HCO3⫺ ions with each co-transported Na⫹ ion. Basolateral K⫹ channels maintain an inside-negative membrane potential to enhance the driving force for net efflux of two negative charges per NBCe1 transport cycle. Emerging evidence also suggests the presence of several types of transmembrane ecto-carbonic anhydrases in the basolateral membrane that help dissipate the local accumulation of bicarbonate within the small interstitial space between the epithelial cells and peritubular capillaries. Solute absorption in the proximal tubule is iso-osmotic— water accompanies reabsorbed ions to maintain osmotic balance. In the past, water flow was assumed to be largely paracellular. However, data from mice genetically modified to lack the aquaporin water channel AQP1 (and from rare cases of humans lacking AQP1) demonstrate that most water reabsorption across the proximal tubule—and, beyond that, across the thin descending limb of Henle—is transcellular. Aquaporins are central to transepithelial water permeability in all water-permeable nephron segments. Thus, the transition from the water-permeable thin descending limb of Henle to the water-impermeable ascending thin limb is paralleled by decreased AQP1 expression. Thick Ascending Limb of the Loop of Henle The tubular fluid emerging from the ascending thin limb is hypertonic and has an elevated NaCl concentration. The three nephron segments into which this fluid flows, the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule or segment (CNT), together constitute “the diluting segment.” The apical membrane of the thick ascending limb of Henle is devoid of aquaporins, as is the apical membrane of the rest of the diluting segment; therefore, these nephron segments reabsorb NaCl and urea without accompanying water (Fig. 20-7), thus diluting the solutes of the tubular fluid. Reabsorption of NaCl and urea across the TAL provides the interstitial solute that generates and maintains the corticomedullary osmotic gradient of the

CHAPTER 20 / Pharmacology of Volume Regulation 339 Apical membrane Lumen of medullary thick ascending limb

Basolateral membrane

Loop diuretics

3Na+ Na+/K+ ATPase

Na+ 2Cl-

reabsorption of luminal calcium and magnesium ions across cation-selective channels, called paracellins or claudins, that reside among components of the tight junctions between the apical membranes of adjacent TAL epithelial cells.

2K+

NKCC2

K+

ROMK

K+

Cl-

CLC-K2

Ca2+ Mg2+ Na+ 10mV

FIGURE 20-7.

Medullary thick ascending limb cell. The medullary thick ascending limb of the loop of Henle absorbs Na⫹ through an apical membrane Na⫹/K⫹/2Cl⫺ (NKCC2) transporter. The Na⫹/K⫹-ATPase pumps sodium from the cytoplasm into the interstitium, and a basolateral Cl⫺ channel (CLC-K2) transports Cl⫺ into the interstitium. K⫹ is primarily recycled into the urinary space via a luminal K⫹ channel (ROMK). The combined activities of apical ROMK and basolateral CLC-K2 result in a lumen-positive transepithelial potential difference (approximately 10 mV) that drives paracellular absorption of cations, including Ca2⫹ and Mg2⫹. Loop diuretics inhibit NKCC2, resulting in significantly increased renal sodium excretion. Disruption of the positive transepithelial potential by loop diuretics also increases the excretion of Ca2⫹ and Mg2⫹.

kidney, allowing operation of the “countercurrent multiplier” that can concentrate the urine of humans to 1,200 mOsM and that of desert rodents to 4,000 mOsM. The TAL reabsorbs between 25% and 35% of the filtered Na⫹ load by means of the luminal membrane Na⫹-K⫹-2Cl⫺ co-transporter, NKCC2. The Cl⫺ imported by NKCC2 exits the basolateral side of the cell via CLC-K2 chloride channels. The Na⫹ imported from the lumen via NKCC2 leaves the basolateral side of the cell via the Na⫹/K⫹-ATPase. Because Cl⫺ carries a negative charge, exit of unaccompanied Cl⫺ through basolateral CLC-K2 depolarizes the cell. The stoichiometry of the Na⫹/K⫹ ATPase, 3Na⫹ outward per 2 K⫹ inward, partly counters this depolarization; additional repolarization of the cell is accomplished by the apical K⫹ channel ROMK, which recycles back into the lumen the K⫹ imported into the cell via NKCC2. The coordinated operation of these apical and basolateral transporters and channels generates a lumen-positive electrical potential across the TAL. This transepithelial potential difference drives the paracellular reabsorption of additional Na⫹ from lumen to interstitium. The paracellular component of Na⫹ reabsorption, approximately 50% of the Na⫹ reabsorbed by the TAL, effectively reduces by 50% the energetic cost to TAL epithelial cells (measured as ATP consumption), because Na⫹/K⫹ transport consumes most of the ATP in the TAL cell. Even with the energy conserved by the paracellular Na⫹ absorptive pathway, the TAL working at maximal capacity can consume up to 25% of the body’s total ATP production, or approximately 65 moles per day at rest. The lumen-positive transepithelial potential of the TAL also drives paracellular

Distal Convoluted Tubule This continuation of the diluting segment actively reabsorbs between 2% and 10% of the filtered NaCl load, while remaining impermeable to luminal water (Fig. 20-8). Luminal Na⫹ enters the epithelial cells of the distal convoluted tubule via the electroneutral, K⫹-independent NCC Na⫹-Cl⫺ co-transporter. Basolateral exit of Na⫹ is mediated by Na⫹/K⫹-ATPase, while the imported Cl⫺ exits by basolateral anion pathways that include both electrogenic Cl⫺ channels and (at least in the mouse) electroneutral K⫹-Cl⫺ co-transport. The distal convoluted tubule (DCT) also mediates transepithelial reabsorption of luminal calcium and magnesium ions via ion-specific, regulated TRPV5 calcium channels and TRPM6 magnesium channels in the apical membrane. The reabsorbed calcium crosses the DCT cell basolateral membrane via specific NCX Na⫹/Ca2⫹ exchangers and Ca2⫹-ATPases. The basolateral exit pathway(s) for magnesium remain undefined, but the recent molecular cloning of several Mg2⫹ transporters suggests that these pathways will soon be identified. Collecting Duct This terminal portion of the nephron is divided into cortical, outer medullary, and inner medullary collecting duct (CD) segments (Fig. 20-9). The cortical and outer medullary CD segments consist of two cell types, principal cells and

Apical membrane

Lumen of distal convoluted tubule

Basolateral membrane

Ca2+ NCX1

Ca2+

3Na+

TRPV5

3Na+ Na+/K+ ATPase

Na+

2K+

NCC

Cl-

Cl-

gCl-

Thiazides

FIGURE 20-8. Distal convoluted tubule cell. Distal convoluted tubule cells absorb Na⫹ via an apical membrane NaCl co-transporter (NCC). Na⫹ is then transported across the basolateral membrane into the interstitium via the Na⫹/K⫹ATPase, and Cl⫺ is transported from the cytosol into the interstitium via Cl⫺ channels (gCl⫺) and perhaps by K⫹-Cl⫺ co-transporters (not shown). Renal epithelial cells of the distal convoluted tubule also absorb Ca2⫹ via apical membrane Ca2⫹ channels (TRPV5), and Ca2⫹ is transported across the basolateral membrane into the interstitium by the Na⫹/Ca2⫹ exchanger NCX1 and by the Ca2⫹-ATPase PMCA (not shown). Thiazides inhibit NCC, resulting in increased Na⫹ excretion. Thiazides also increase epithelial cell absorption of Ca2⫹ by an unknown mechanism (not shown).

340 Principles of Cardiovascular Pharmacology

Principal cell

Amiloride, Triamterene

ENaC expression

3Na+

Na+/K+ ATPase expression Na+

IC secrete protons via the apical H⫹-ATPase and reabsorb bicarbonate through the basolateral Cl⫺/HCO3⫺ exchanger (also known as “kidney AE1”). Type B IC secrete HCO3⫺ through the apical Cl⫺/HCO3⫺ exchanger pendrin and reabsorb protons via the basolateral H⫹-ATPase. Pendrin also likely mediates Cl⫺ reabsorption via a third IC type, the “non-A, non-B” IC. In addition, intercalated cells mediate K⫹ absorption by electroneutral luminal H⫹/K⫹-ATPases, flow-sensitive K⫹ secretion by Ca2⫹-activated maxi-K⫹ channels, and NH4⫹ secretion by proteins related to the erythroid Rhesus (Rh) antigens.

Basolateral membrane

Apical membrane

Na+/K+ ATPase

ENaC

2K+ Nucleus

+ K channel

K+ Mineralocorticoid receptor

Lumen of cortical collecting duct Spironolactone

Aldosterone

O

O

O

H

OH

HO

H

O

O

H

H

H

S

H

O

O

FIGURE 20-9.

Cortical collecting duct principal cell. Cortical collecting duct principal cells absorb Na⫹ via an apical membrane Na⫹ channel (ENaC). Cytoplasmic Na⫹ is then transported across the basolateral membrane via the Na⫹/K⫹-ATPase. In addition, collecting duct cells express apical membrane K⫹ channels that allow K⫹ to exit into the urinary space. ENaC expression and apical surface localization is modulated by aldosterone. Aldosterone binds to the mineralocorticoid receptor, which then increases transcription of the gene encoding ENaC as well as genes encoding other proteins involved in Na⫹ reabsorption (such as Na⫹/K⫹-ATPase). The collecting duct principal cell is the site of action of the two classes of potassium-sparing diuretics. Mineralocorticoid receptor antagonists such as spironolactone competitively inhibit the interaction of aldosterone with the mineralocorticoid receptor, and thereby decrease expression of ENaC. Direct inhibitors of ENaC, such as amiloride and triamterene, inhibit Na⫹ influx through ENaC channels at the apical plasma membrane.

intercalated cells. Principal cells reabsorb between 1% and 5% of the filtered sodium load, depending on plasma aldosterone levels (aldosterone increases sodium reabsorption and water retention, see below). Luminal Na⫹ enters the principal cells of the cortical collecting duct via heterotrimeric epithelial Na⫹ channels, ENaC, in the apical membrane. Intracellular Na⫹ exits the basolateral side of the cell via the Na⫹/K⫹-ATPase. Principal cells also secrete K⫹ into the lumen to maintain tight control of plasma [K⫹], as well as to minimize the transepithelial potential difference resulting from Na⫹ reabsorption. In addition, cortical and outer medullary principal cells, as well as cells of the inner medullary collecting duct, express vasopressin (ADH)-responsive water channels. ADH activates water reabsorption by stimulating a Gs protein-coupled V2 receptor in the basolateral membrane; in turn, Gs protein signaling promotes the reversible insertion into the apical membrane of intracellular vesicles containing aquaporin 2 (AQP2) water channels (Fig. 20-4B). At least two subtypes of intercalated cells (IC) contribute to systemic acid–base balance through cell type-specific polarized expression of the vacuolar H⫹-ATPase. Type A

 PATHOPHYSIOLOGY OF EDEMA FORMATION Edema is defined as the accumulation of fluid in the interstitial space. Edema can be either exudative (having a high protein content) or transudative (having a low protein content, essentially a plasma ultrafiltrate). Exudative edema occurs as part of the acute inflammatory response (see Chapter 41, Principles of Inflammation and the Immune System). The type of edema considered here is transudative edema, which can result from pathologic renal retention of Na⫹. Under physiologic conditions, any increased fluid filtration across the capillary membrane is quickly counterbalanced by homeostatic mechanisms. This return to a physiologic setpoint is mediated by three factors: osmotic forces, lymphatic drainage, and long-term modulation of volume by physiologic sensors and signals. Osmotic forces play an immediate role in fluid shifts between compartments. For example, increased fluid shift to the interstitial space will result in increased interstitial hydrostatic pressure and increased plasma oncotic pressure. Both of these variables favor fluid shift back into the intravascular space (Fig. 20-1). The lymphatic system can also increase return of filtered fluid dramatically, thereby decreasing the amount of filtered fluid that remains in the interstitial space. Over a period of days to weeks, volume sensors and signals respond to changes in volume by altering the extent of natriuresis or sodium reabsorption necessary to maintain a constant intravascular volume. These combined systems closely monitor and regulate intravascular volume. Therefore, the pathophysiology of transudative edema formation almost always requires an element of pathologic renal Na⫹ retention. The three most common clinical situations resulting in edema formation are heart failure, cirrhosis, and nephrotic syndrome. All of these diseases manifest deranged Na⫹ reabsorption caused by pathologic alterations in volume regulation. Understanding the pathophysiology of edema formation in these diseases provides a rationale for the therapeutic use of natriuretic agents.

Heart Failure Heart failure (HF) is defined by the inability of the heart to perfuse tissues and organs adequately. Insufficient cardiac output and subsequent decreased blood flow through the arterial vascular bed leads to congestion in the venous “capacitance” vessels. The resulting increase in capillary hydrostatic pressure favors fluid transudation into tissue interstitial spaces. Right heart failure leads initially to peripheral edema, whereas left heart failure can lead first to pulmonary edema. In the introductory case, Mr. R’s compromised cardiac function

Compromised cardiac function

Decreased arterial blood pressure

Fluid transudation

Systemic and pulmonary congestion, decreased lymphatic drainage

Extracellular fluid volume expansion

Elevated cardiac atrial pressure

Chronic dilation of cardiac chambers

Attenuation of natriuretic response

Renal sodium retention

Renal sensors perceive decreased volume

342 Principles of Cardiovascular Pharmacology A "Underfill model"

B "Overflow model"

Hepatic venous outflow obstruction

Liver injury leading to post-sinusoidal obstruction

Ascites formation

Hepato-renal reflex

Decreased intravascular volume

Primary renal Na+ retention

Low venous filling pressure

Increased plasma volume

Low cardiac output

Ascites formation

Activation of baroreceptors Renal Na+ retention

FIGURE 20-11. Proposed mechanisms of Na⫹ retention in cirrhosis. The postsinusoidal obstruction in cirrhosis is associated with renal Na⫹ retention as well as the accumulation of ascites fluid. Two models have been proposed to explain the mechanisms of these effects. A. Hepatic venous outflow obstruction causes increased hydrostatic pressure, which initiates ascites formation. The accumulation of ascites fluid decreases intravascular volume, leading to low venous filling pressure, decreased cardiac output, and subsequent activation of arterial baroreceptors that initiate renal Na⫹ retention. B. Postsinusoidal obstruction activates the hepato-renal reflex, an autonomic response involving the liver and kidney that initiates renal Na⫹ reabsorption by a poorly understood mechanism. The renal Na⫹ retention leads to an expansion of plasma volume, increased hydrostatic pressure in the portal circuit, and the formation of ascites. pathologic Na⫹ retention leads to intravascular volume expansion, increased portal hydrostatic pressure, and formation of ascites. Although not well understood, this mechanism is consistent with a number of experimental model systems demonstrating that renal Na⫹ retention in cirrhosis occurs before the development of ascites. Ascites formation may well involve elements of both the underfill and overflow models. Both models begin with the observation that cirrhosis leads to significant hepatic outflow obstruction, and both must consider compromised portal hemodynamics, decreased hepatic synthetic and secretory functions leading to decreased plasma oncotic pressure, and poorly characterized neural or hormonal interactions between the liver and the kidney. Elucidation of the mechanism of the hepato-renal reflex may lead in the future to more effective pharmacologic interventions to manage the development of ascites in cirrhosis.

Nephrotic Syndrome Nephrotic syndrome is characterized by massive proteinuria (⬎3.5 g/day), edema, hypoalbuminemia, and often hypercholesterolemia. The primary cause of nephrotic syndrome is glomerular dysfunction, which may be due to immune complex disease, diabetes, lupus, amyloidosis, or other conditions affecting glomerular function. A classical explanation of edema formation in nephrotic syndrome follows this sequence. First, massive proteinuria leads to decreased plasma oncotic pressure, reducing the

forces favoring fluid retention in the capillary and leading to fluid transudation into the interstitium. The increased net fluid transudation decreases intravascular volume, activating volume sensors to enhance renal Na⫹ retention. The resulting expansion in fluid volume, in the absence of adequate compensatory albumin synthesis, maintains low plasma oncotic pressure and continued edema formation. In this view, renal Na⫹ retention is secondary to decreased renal arterial perfusion. However, the edema of nephrotic syndrome may also be caused by intrinsic changes in capillary junctional permeability and/or by primary renal Na⫹ retention. The postulated primary Na⫹ retention of nephrotic syndrome may be localized to the distal nephron, arising from resistance to natriuretic peptides, increased sympathetic nervous system activity, or increased ENaC activation by luminal proteases. Although treatment of nephrotic syndrome can include diuretics to counter renal Na⫹ retention, correction of edema typically requires correction of the underlying glomerular disorder, eventually leading to decreased proteinuria and correction of the edema. The glucocorticoids and immunosuppressants used to treat some forms of nephrotic syndrome can themselves promote further sodium retention. Diuretics are used in the short term to minimize edema formation.

 PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic modulators of extracellular fluid volume can be divided into agents that modify neurohormonal volume regulators and agents that act directly on the nephron segments to alter renal Na⫹ handling. The former category includes agents that interrupt the renin–angiotensin axis, alter circulating levels of natriuretic peptides, or interrupt ADH signaling. The latter category includes the various classes of diuretics, which directly target renal ion transporter or channel function or expression to increase renal Na⫹ excretion. Neurohormonal volume regulators may also act directly on Na⫹ reabsorption through mechanisms less well understood than those of the diuretics.

Agents That Modify Volume Regulators Inhibitors of the Renin–Angiotensin System There are four clinically available pharmacologic strategies for interruption of the renin-angiotensin–aldosterone system (RAAS). First, inhibition of the enzymatic activity of renin prevents generation of angiotensin I. Second, ACE inhibitors interrupt the conversion of angiotensin I to angiotensin II. Third, angiotensin receptor antagonists are competitive antagonists of the AT1 receptor, and thus inhibit the targetorgan effects of angiotensin II. Fourth, antagonists of the mineralocorticoid receptor block aldosterone action at the nephron collecting duct. The first three classes of agents are discussed here; antagonists of aldosterone action are considered diuretics and are addressed below (see “PotassiumSparing Diuretics”). Renin Inhibitors

Aliskiren is the first approved inhibitor of the enzymatic activity of renin. By blocking the activity of renin, aliskiren prevents the conversion of angiotensinogen to angiotensin I. Aliskiren is an effective antihypertensive and can be used in hypertensive patients with renal insufficiency. Aliskiren may

Angiotensinogen

Kininogen

Renin inhibitor

Renin Angiotensin I ACE

Kallikrein Bradykinin

ACE inhibitor

Angiotensin II

Kininase II

Inactive

AT1 receptor antagonists

Aldosterone secretion (mediated by AT1 receptors)

Increased Na+ and H2O reabsorption

Vasoconstriction

Vasodilation

(mediated by AT1 receptors)

Increased peripheral vascular resistance

Increased blood pressure

Decreased peripheral vascular resistance

Decreased blood pressure

344 Principles of Cardiovascular Pharmacology

hemodynamics. ACE inhibitors may be used in combination with aliskiren if required. ACE inhibitors are contraindicated in pregnancy (including for treatment of pregnancy-associated hypertension), since they have been associated with an increased risk of major fetal malformations. Angiotensin Receptor Antagonists

AT1 receptor antagonists, such as losartan and valsartan, inhibit the action of angiotensin II at its receptor (Fig. 20-12). Compared to ACE inhibitors, AT1 receptor antagonists may allow more complete inhibition of angiotensin II’s actions, because ACE is not the only enzyme that can generate angiotensin II. In addition, because AT1 receptor antagonists have no effect on bradykinin metabolism, their use may minimize the incidence of drug-induced cough and angioedema. However, the inability of AT1 receptor antagonists to potentiate the vasodilatory effects of bradykinin may result in less effective vasodilation. Unlike ACE inhibitors, AT1 receptor antagonists may indirectly increase vasorelaxant AT2 receptor activity. Both ACE inhibitors and AT1 antagonists increase renin release as a compensatory mechanism; in the case of AT1 blockade, the increased angiotensin II that results could lead to increased interaction of angiotensin II with AT2 receptors. AT1 receptor antagonists are approved for the treatment of hypertension. Although these agents were initially prescribed only for patients with intolerable adverse reactions to ACE inhibitors, they are now considered first-line treatments for hypertension. AT1 receptor antagonists are also under study for the treatment of heart failure. Recent trials have suggested that the combination of an AT1 receptor antagonist and an ACE inhibitor may have some clinical benefit in severe heart failure, and studies testing such combinations in the treatment of chronic kidney disease and cardiac disease progression are currently under way. Combined therapy using AT1 receptor antagonists and aliskiren is also under investigation for treatment of hypertension, heart failure, and renal failure. AT1 receptor antagonists may protect against stroke, not only by controlling hypertension but also through beneficial secondary effects. These include reduced platelet aggregation, decreased serum uric acid levels, decreased incidence of atrial fibrillation, and antidiabetic effects. The mechanisms of these secondary effects remain to be elucidated. B-Type Natriuretic Peptide Nesiritide, a recombinant human-sequence B-type natriuretic peptide (BNP), can be used for short-term management of decompensated heart failure. Because nesiritide is a peptide, it is ineffective when given orally. In clinical trials of nesiritide in acute heart failure, the drug decreased pulmonary capillary wedge pressure (a measure of hydrostatic pressure in the pulmonary system), decreased systemic vascular resistance, and improved cardiac hemodynamic parameters such as stroke volume. Although nesiritide was not more efficacious in these trials than the more commonly used dobutamine (see Chapter 25), nesiritide may be associated with a lower incidence of arrhythmias than dobutamine. At low doses, nesiritide appears to promote water excretion to a greater degree than sodium excretion. Hypotension is a major adverse effect of nesiritide, reflecting the vasorelaxant properties of the natriuretic peptides. The risk of hypotension is increased by co-administration of nesiritide with an ACE inhibitor. Nesiritide treatment is also associated with an increased risk of renal dysfunction. These

adverse effects have not been reported in preliminary clinical trials of an investigational peptide related to ANP, which exhibits powerful natriuretic as well as diuretic properties. Vasopressin Receptor Antagonists and Agonists The tetracycline analogue demeclocycline has long been used in the treatment of syndromes of inappropriate ADH secretion (SIADH), when dietary water restriction is not feasible or sufficient. Its mechanism of action is uncertain. Conivaptan is the first specific nonpeptide vasopressin receptor antagonist approved for treatment of euvolemic hyponatremias (SIADH). Its disadvantages include a requirement for intravenous administration and some V1 receptor antagonist activity. However, the V2-selective receptor antagonist tolvaptan is orally bioavailable. In clinical trials, V2 receptor antagonists have also shown benefit in the treatment of other conditions associated with inappropriate ADH-induced water retention, including heart failure and cirrhotic ascites. V2 receptor antagonists are also showing promise as agents to retard vasopressin-driven renal cyst growth in autosomal dominant polycystic kidney disease. Congenital nephrogenic diabetes insipidus may result from mutations in either the V2 receptor or the collectingduct principal-cell aquaporin AQP2. Some V2 receptor mutations are associated with trapping of newly synthesized receptor polypeptides inside the principal cell. Vasopressin receptor antagonists may act as molecular chaperones for a subset of these mutant receptors; in these cases, antagonist binding presumably promotes a receptor conformation that allows insertion of the mutant protein into the apical membrane of the cell. Cell-permeant, vasopressin-mimetic small molecules have also been shown to activate mutant V2 receptors inside cells, generating sufficient cAMP to mobilize aquaporin 2 water channels to the apical surface. This strategy is thus far the most promising approach to the treatment of V2 receptor-linked nephrogenic diabetes insipidus. Similar strategies are being adopted for many hereditary diseases of G protein-coupled receptors. Terlipressin is an investigational vasopressin analog with moderate V1 receptor agonist activity and specificity. It may have potential clinical application in reducing portal hypertension and improving renal hemodynamics in liver failure and ascites.

Agents That Decrease Renal Naⴙ Reabsorption As discussed above, the kidney modifies the ionic composition of the glomerular filtrate by the concerted action of ion transporters and channels in both apical and basolateral membranes of renal tubular epithelial cells. This transepithelial ion transport can be modulated pharmacologically by the actions of diuretic drugs to regulate urinary volume and composition. Pharmacologic inhibition of ion reabsorption leads to reduction of the osmotic driving force that favors water reabsorption in the water-permeable segments of the nephron. Diuretics target sodium reabsorption along four segments of the nephron: the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and collecting duct. The kidney concentrates and secretes these drugs into the tubule lumen, allowing diuretics to reach higher concentrations in the tubule than in the blood. Because of this concentrating effect, therapeutic diuretic doses are often accompanied by low blood levels of diuretics and by mild extrarenal adverse effects.

CHAPTER 20 / Pharmacology of Volume Regulation 345

Carbonic Anhydrase Inhibitors Carbonic anhydrase inhibitors, exemplified by acetazolamide, inhibit sodium reabsorption by noncompetitively and reversibly inhibiting proximal-tubule cytoplasmic carbonic anhydrase II and luminal carbonic anhydrase IV (Fig. 20-6). Inhibition of carbonic anhydrase leads to increased delivery of sodium bicarbonate to more distal segments of the nephron. Much of this sodium bicarbonate is initially excreted, resulting in an acute decrease in plasma volume (diuresis). However, over the course of several days of therapy, the diuretic effect of the drug is diminished by compensatory up-regulation of NaHCO3 reabsorption and by increased NaCl reabsorption across more distal nephron segments (by incompletely understood mechanisms). Use of carbonic anhydrase inhibitors is often associated with mild-to-moderate metabolic acidosis, arising not only from inhibition of proximal tubular H⫹ secretion, but also from inhibition of carbonic anhydrase in acid-secreting intercalated cells of the collecting duct. The alkalinized urine resulting from carbonic anhydrase inhibition increases the urinary excretion of organic acid anions, including aspirin. The clinical use of carbonic anhydrase inhibitors is primarily restricted to several carbonic anhydrase-dependent conditions (see below). In addition, carbonic anhydrase inhibitors are occasionally used to restore acid–base balance in heart failure patients with metabolic alkalosis due to treatment with loop diuretics. Carbonic anhydrase inhibitors also have ophthalmologic applications. The ciliary process epithelium of the anterior chamber of the eye secretes sodium chloride into the aqueous humor. This NaCl secretion requires carbonic anhydrase activity, because a portion of the basolateral Cl⫺ uptake by the ciliary epithelium requires coupled Cl⫺-HCO3⫺ and Na⫹-H⫹ exchange as well as Na⫹-HCO3⫺ symport. The basolateral membrane Na⫹-K⫹-2Cl⫺ co-transporter NKCC1 mediates most remaining Cl⫺ uptake by ciliary epithelial cells. Glaucoma is characterized by increased pressure in the anterior chamber of the eye. This is usually attributed to partially obstructed outflow of aqueous humor, but in some cases, overproduction of aqueous humor may also contribute. Inhibition of carbonic anhydrase in the ciliary process epithelium reduces secretion of aqueous humor and may thereby reduce elevated intraocular pressure. Topical lipophilic carbonic anhydrase inhibitors are often used in concert with topical ␤-adrenergic antagonists in the treatment of glaucoma (see Chapter 10, Adrenergic Pharmacology). Ascent to altitudes greater than 3,000 m above sea level predisposes several body organs, including the brain, to edema and ionic disequilibria. Symptoms of acute mountain sickness can include nausea, headache, dizziness, insomnia, pulmonary edema, and confusion. Carbonic anhydrase is involved in the secretion of chloride and bicarbonate into the cerebrospinal fluid by the choroid plexus of the cerebral ventricles, and inhibition of carbonic anhydrase can be used prophylactically against acute mountain sickness. The stillcontroversial mechanism(s) of action include effects on the choroid plexus and ependyma, on the respiratory control centers of the brain, and on the blood–brain barrier. Carbonic anhydrase inhibitors are also used in the treatment of epilepsy, although the antiepileptic mechanism of some of these drugs may not require inhibition of carbonic anhydrase. One such antiepileptic drug, topiramate, can produce mild-to-moderate acidosis due to impaired renal acidification of the urine.

The treatment of hyperuricemia or gout (see Chapter 48, Integrative Inflammation Pharmacology: Gout) may involve alkalinization of the urine to increase the urinary solubility of uric acid. Increased uric acid solubility prevents uric acid precipitation in the urine, with consequent uric acid nephropathy and nephrolithiasis (kidney stones). Urinary alkalinization can be achieved by oral bicarbonate, supplemented as needed by a carbonic anhydrase inhibitor to reduce renal reabsorption of the filtered bicarbonate. Osmotic Diuresis Osmotic diuretics, such as mannitol, are small molecules that are filtered at the glomerulus but not subsequently reabsorbed in the nephron. Thus, they constitute an intraluminal osmotic force limiting reabsorption of water across water-permeable nephron segments. The effect of osmotic agents is greatest in the proximal tubule, where most isoosmotic reabsorption of water takes place. By causing water loss in excess of sodium excretion, osmotic diuresis can sometimes lead to unintended hypernatremia. Alternatively, the increased urine volume associated with osmotic diuresis can also promote vigorous natriuresis. Therefore, careful monitoring of clinical volume status and serum electrolytes is warranted. Mannitol is used primarily for rapid (emergent) treatment of increased intracranial pressure. In the setting of head trauma, brain hemorrhage, or a symptomatic cerebral mass, the increased intracranial pressure can be relieved, at least transiently, by the acute reduction in cerebral intravascular volume that follows the mannitol-induced reduction in systemic vascular volume. Osmotic diuresis can also occur as a result of pathologic states. Two common examples of this phenomenon are hyperglycemia and the use of radiocontrast dyes. In diabetic hyperglycemia, the filtered glucose load exceeds the reabsorptive capacity of the proximal tubule for glucose. As a result, significant quantities of glucose remain in the lumen of the nephron and act as an osmotic agent to increase fluid retention in the tubular lumen, thereby decreasing fluid reabsorption. Radiocontrast agents used for radiologic imaging studies are filtered at the glomerulus but not reabsorbed by the tubular epithelium. Thus, these dyes constitute an osmotic load and can produce osmotic diuresis. In patients with borderline cardiovascular status, the consequent reduction in intravascular volume can lead to hypotension or to renal and/or cardiac insufficiency secondary to reduced organ perfusion. Loop Diuretics The so-called loop diuretics act at the TAL of the loop of Henle. These agents reversibly and competitively inhibit the Na⫹-K⫹-2Cl⫺ co-transporter NKCC2 in the apical (luminal) membrane of TAL epithelial cells (Fig. 20-7). In addition to the primary effect of inhibiting Na⫹ reabsorption across the TAL, inhibition of transcellular NaCl transport secondarily reduces or abolishes the lumen-positive transepithelial potential difference across the TAL. Consequently, paracellular reabsorption of divalent cations, particularly calcium and magnesium, is also inhibited. The increased delivery of luminal calcium and magnesium to downstream reabsorptive sites in the distal convoluted tubule can lead to increased urinary excretion of calcium and magnesium. The resultant hypocalcemia and/or hypomagnesemia can be clinically significant in some patients who require prolonged

348 Principles of Cardiovascular Pharmacology

and ensures that the kidney is able to filter waste products from the plasma. Regulation of extracellular volume is accomplished by integrated neurohormonal mechanisms that respond to changes in arterial and atrial wall stress. These hormones modulate numerous steps in renal Na⫹ handling, and thereby maintain a homeostatic balance between dietary Na⫹ intake and Na⫹ excretion. Edema can develop when the capillary hydrostatic pressure gradient favoring fluid filtration exceeds the opposing oncotic forces favoring fluid entry into the intravascular space. Pharmacologic treatment of dysregulated extracellular volume involves modification of neurohormonal signaling and direct inhibition of renal Na⫹ reabsorption. ACE inhibitors prevent the conversion of angiotensin I to angiotensin II; drugs in this class have important vasodilatory actions. Angiotensin receptor antagonists and renin inhibitors are also useful in interrupting the angiotensin–aldosterone axis. Both ACE inhibitors and angiotensin receptor antagonists have beneficial effects in slowing the progression of hypertrophy and fibrosis in the heart, the kidney, and the vasculature. B-type natriuretic peptide (nesiritide) is used in the treatment of decompensated heart failure, and terlipressin is under investigation for the treatment of portal hypertension. Diuretics are agents that alter nephron Na⫹ reabsorption and secondarily alter the reabsorption and secretion of other ions. Essential to understanding diuretic mechanisms is an appreciation of the functional organization of the nephron. With the exception of osmotic diuretics, which increase urinary flow by osmotic retention of water throughout the nephron, specific classes of diuretic drugs target each of the four segments of the nephron. Carbonic anhydrase inhibitors such as acetazolamide decrease sodium and bicarbonate reabsorption in the proximal tubule; loop agents such as furosemide decrease sodium and chloride reabsorption by the apical Na⫹-K⫹-2Cl⫺ pump in the thick ascending limb of the loop of Henle; thiazides such as hydrochlorothiazide inhibit the apical Na⫹-Cl⫺ co-transporter in the distal convoluted tubule; and potassium-sparing diuretics such as spironolactone and amiloride inhibit, respectively, the aldosterone receptor and the ENaC apical Na⫹ channel in the collecting duct. The most important use of diuretics is in the treatment of hypertension; the second most important use is to treat edema of any cause. Future developments in the pharmacology of extracellular volume regulation will likely focus on interrupting or enhancing the hormonal pathways implicated in the disruption of volume homeostasis, as well as on the solute and water transporters themselves. Specific V2 vasopressin receptor antagonists will be used increasingly in hypervolemic conditions accompanied by elevated ADH levels or action. V2 receptor antagonists have also shown promise in retarding progression of cyst growth in autosomal dominant polycystic kidney disease. Aquaporin blockers (aquaretics) are under development for regulation of fluid homeostasis, and aquaglyceroporin blockers are under investigation as treatments for skin conditions and as modulators of lipid metabolism. Chloride channel blockers and potassium channel

blockers are under development to treat the volume depletion of severe toxigenic and infectious diarrhea as well as rare congenital diarrheas. Chloride channel activators and potassium channel activators are being developed to treat the pulmonary, gastrointestinal, and genitourinary hyposecretion disorders of cystic fibrosis, sicca syndromes, and inflammatory biliary cirrhosis. Carbonic anhydrase II has recently been shown to act as a nitrate reductase and thereby to generate nitric oxide at the acidic pH of ischemic or hypoxic tissue. Surprisingly, this nitrite reductase activity is activated by sulfonamide carbonic anhydrase inhibitors even as they inhibit carbonic anhydrase activity. This property may explain the vasodilation associated with use of carbonic anhydrase inhibitors and encourages consideration of new uses for this old drug class. New drugs to interrupt the renin-angiotensin–aldosterone axis may include neutral endopeptidase inhibitors, (pro) renin receptor antagonists, AT2 receptor agonists, selective endothelin receptor antagonists, and natriuretic peptides of increased potency and selectivity. The latter will likely play an increasingly important role in the management of decompensated heart failure and possibly the ascites of hepatic failure. Drugs acting on the renin-angiotensin–aldosterone axis will also likely be useful in slowing the rate of renal and cardiac fibrosis, reinforcing or improving on the actions of ACE inhibitors, AT1 receptor antagonists, and mineralocorticoid receptor blockers. These drugs also have general and cell type-specific trophic actions. One example is provided by the role of the AT1 receptor in promoting proliferation of epidermal growth factor receptor ERBB2-negative mammary tumor cells in culture and in xenografts. AT1 receptor blockers have slowed mammary cell tumor xenograft growth. Thus, AT1 blockade is a reasonable candidate adjunct therapy for mammary tumors that may not respond to more conventional therapy.

Suggested Reading Christova M, Alper SL. Core curriculum in nephrology. Tubular transport: Core curriculum 2010. Am J Kidney Dis 2010; 56:1202–1217. (Annotated review of transport by renal tubular epithelial cells.) Ernst ME, Moser M. Drug therapy: use of diuretics in patients with hypertension. N Engl J Med 2009;361:2153–2164. (Clinical pharmacology of diuretics.) Greenberg A, Verbalis JG. Vasopressin receptor antagonists. Kidney Int 2006;69:2124–2130. (Introduction to the physiology and clinical indications of this drug class.) Okusa MD, Ellison DH. Physiology and pathophysiology of diuretic action. In: Alpern RJ, Hebert SC, eds. The kidney: physiology and pathophysiology. 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2008:1051–1094; Chapter 37. (Full discussion of the physiology and pathophysiology of diuretic action.) Palmer BF, Sterns RH. Fluid, electrolyte, and acid base disturbances. NephSAP (American Society of Nephrology) 2009;8:61–165. (Updated nephrology board review summary and questions about fluid and electrolyte disorders.) Potter LR, Yoder AR, Flora DR, Antos LK, Dickey DM. Natriuretic peptides: their structures, receptors, physiological functions, and therapeutic applications. Handb Exp Pharmacol 2009;191:341–366. (Overview of natriuretic peptide physiology in volume regulation.)

21 Pharmacology of Vascular Tone Deborah Yeh Chong and Thomas Michel

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 353-354 PHYSIOLOGY OF VASCULAR SMOOTH MUSCLE CONTRACTION AND RELAXATION. . . . . . . . . . . . . 353 Vascular Resistance and Capacitance . . . . . . . . . . . . . . . 353 Vascular Smooth Muscle Contraction and Relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 Regulation of Vascular Tone . . . . . . . . . . . . . . . . . . . . . . . 356 Vascular Endothelium . . . . . . . . . . . . . . . . . . . . . . . . . 356 Autonomic Nervous System . . . . . . . . . . . . . . . . . . . . 357 Neurohormonal Mechanisms . . . . . . . . . . . . . . . . . . . 358 Local Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 358 Organic Nitrates, Inhaled Nitric Oxide, and Sodium Nitroprusside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . 359 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Pharmacologic Tolerance . . . . . . . . . . . . . . . . . . . . . . 361

Effects of Nitrates in Addition to Vasodilation . . . . . . . . 362 Contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 Phosphodiesterase Inhibitors . . . . . . . . . . . . . . . . . . . . . . 362 Ca2⫹ Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . 363 Chemical Classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 Toxicities and Contraindications . . . . . . . . . . . . . . . . . 364 K⫹ Channel Openers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Endothelin Receptor Antagonists . . . . . . . . . . . . . . . . . . . 365 Other Drugs That Modulate Vascular Tone . . . . . . . . . . . . 365 Hydralazine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 ␣1-Adrenergic Antagonists . . . . . . . . . . . . . . . . . . . . . 365 ␤-Adrenergic Antagonists . . . . . . . . . . . . . . . . . . . . . . 366 Renin–Angiotensin System Blockers . . . . . . . . . . . . . . 366 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 366 SUGGESTED READING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

 INTRODUCTION

and demand is critical for the function of all tissues, especially for the myocardium. Vascular tone is an important determinant of both myocardial O2 supply and demand. Myocardial O2 supply depends on the tone of the coronary arteries, while myocardial O2 demand depends on the tone of both the systemic arterioles (resistance vessels) and veins (capacitance vessels).

In combination with cardiac output, vascular tone (i.e., the degree of contraction of vascular smooth muscle) determines the adequacy of perfusion of the tissues of the body. The importance of vascular tone is underscored by the wide spectrum of disease states—ranging from angina pectoris to hypertension to Raynaud’s phenomenon to migraine headaches—that are associated with dysregulated vascular tone. As the major factors that govern the regulation of blood vessel diameter have become understood at the molecular level, it has become apparent that a complex array of mechanisms is required to maintain proper vascular tone in the face of diverse stimuli. Pharmacologic strategies for intervention in these regulatory pathways have already yielded many successful therapies for disorders of vascular tone, and provide hope that, in the future, even better therapies will be available to manage the multiple types of vascular disorders.

 PHYSIOLOGY OF VASCULAR SMOOTH MUSCLE CONTRACTION AND RELAXATION Vascular tone is a key regulator of tissue perfusion, which determines whether tissues receive sufficient O2 and nutrients to meet their demands. The delicate balance between O2 supply

Vascular Resistance and Capacitance The tone of the arterial portion of the circulation and the tone of the venous portion of the circulation play important yet distinctive roles in modulating the balance of myocardial O2 demand. The major determinants of myocardial O2 demand are heart rate, contractility, and ventricular wall stress. Wall stress can be expressed as:   (P  r)/2h

Equation 21-1

where ␴ is wall stress, P is ventricular pressure, r is ventricular chamber radius, and h is ventricular wall thickness. Systolic and diastolic ventricular wall stresses are influenced by systemic arteriolar and venous tone, respectively. Arteriolar tone directly controls systemic vascular resistance and, thus, arterial blood pressure: MAP  SVR  CO

Equation 21-2 353

Myocardial O2 SUPPLY

Myocardial O2 DEMAND

Perfusion of the heart

Ventricular wall stress

Vascular tone of the coronary arteries

Preload

Afterload Heart (pump)

Venous tone

Arteriolar tone

Veins

Arteries

Left circumflex coronary artery Capillaries

Right coronary artery

Left anterior descending coronary artery

Veins (capacitance vessels)

Arterioles (resistance vessels)

Vascular smooth muscle cell

Sarcoplasmic reticulum Extracellular space

Ca2+

Cytosol

Ca2+

Ca2+

Actin-myosin crossbridges Ca2+

Contraction

356 Principles of Cardiovascular Pharmacology

Contraction

Relaxation

Ca2+

NO

L-type voltage-gated Ca2+ channel

Ca2+ + CaM

Guanylyl cyclase

Guanylyl cyclase cGMP

Ca2+-CaM MLCK Myosin-LC

GTP Myosin-LC phosphatase

MLCK

Myosin-LC phosphatase Myosin-LC P

Myosin-LC

Actin-myosin crossbridges Contraction

Relaxation

Vascular smooth muscle cell

FIGURE 21-3.

Mechanism of vascular smooth muscle cell contraction and relaxation. Vascular smooth muscle cell contraction and relaxation are controlled by the coordinated action of several intracellular signaling mediators. Ca2⫹ entry through L-type voltage-gated Ca2⫹ channels (left panel) is the initial stimulus for contraction. Ca2⫹ entry into the cell activates calmodulin (CaM). The Ca2⫹-CaM complex activates myosin light chain kinase (MLCK) to phosphorylate myosin light chain (myosin-LC). The phosphorylated myosin-LC interacts with actin to form actin–myosin cross-bridges, a process that initiates vascular smooth muscle cell contraction. Relaxation (right panel) is a coordinated series of steps that act to dephosphorylate (and hence inactivate) myosin-LC. Nitric oxide (NO) diffuses into the cell and activates guanylyl cyclase. The activated guanylyl cyclase catalyzes the conversion of guanosine triphosphate (GTP) to guanosine 3⬘,5⬘-cyclic monophosphate (cGMP). cGMP stimulates cGMP-dependent protein kinase (not shown ), which activates myosin-LC phosphatase, which dephosphorylates myosin light chain, preventing actin–myosin cross-bridge formation. As a result, the vascular smooth muscle cell relaxes. The active form of each enzyme is italicized and blue.

then activates myosin light chain phosphatase. Dephosphorylation of the myosin light chain inhibits the interaction of the myosin head with actin, leading to smooth muscle relaxation (Fig. 21-3, right panel).

Regulation of Vascular Tone Vascular tone is governed by a wide variety of mechanisms. Recent research has highlighted the importance of interactions between vascular endothelial cells and vascular smooth muscle cells in the control of vascular tone. The autonomic nervous system and a number of neurohormonal mediators also control vascular smooth muscle contraction and relaxation. Many of these physiologic mechanisms provide the basis for current drug discovery research. Vascular Endothelium Research in the past two decades has elucidated several signaling modes in the vascular endothelium to control vascular tone. Endothelial cells elaborate many signaling mediators and alter the expression of many genes in response to diverse stimuli. Two of the most pharmacologically relevant targets, nitric oxide and endothelin, are discussed here. Nitric Oxide

The obligatory role of endothelial cells in regulating vascular tone was first recognized with the observation that acetylcholine causes vasoconstriction when applied directly to de-endothelialized blood vessels, but causes vasodilation when applied to normally endothelialized vessels (Fig. 21-4). It was hypothesized that muscarinic cholinergic stimulation

of the endothelium induces the production of a relaxant molecule in the endothelial cell and that this molecule then diffuses to subjacent vascular smooth muscle cells to activate guanylyl cyclase. The putative vasodilatory compound was termed endothelial-derived relaxing factor, or EDRF. Before the molecular identity of EDRF was determined to be nitric oxide (NO), nitroglycerin—an organic nitrate commonly prescribed for angina pectoris—was known to be metabolized in the body to form NO, and NO was known to cause relaxation of vascular smooth muscle. Based on these findings, it was hypothesized and later confirmed that the EDRF released from endothelial cells is NO, a gas that reacts with a wide range of biomolecules to elicit cellular responses. Although acetylcholine was the first ligand to be identified that promotes endothelial-cell synthesis of NO, a number of other mediators have since been described. Shear stress, acetylcholine, histamine, bradykinin, sphingosine 1-phosphate, serotonin, substance P, and ATP can all elicit increased NO synthesis by vascular endothelial cells. NO is synthesized by a family of Ca2⫹-CaM–activated NO synthases. The endothelial isoform of nitric oxide synthase (eNOS) is responsible for endothelial-cell NO synthesis; this enzyme plays a critical role in controlling vascular tone and platelet aggregation. The importance of NO in regulating vascular tone is underscored by the observation that eNOS-deficient mice are hypertensive. Recent evidence suggests that NO may effect vasodilation not only by activating guanylyl cyclase, but also by activating Ca2⫹-dependent K⫹ channels in vascular smooth muscle cells (Fig. 21-4). NO appears to activate these K⫹ channels directly via a guanylyl cyclase-independent mechanism, leading to

CHAPTER 21 / Pharmacology of Vascular Tone 357

Agonist (e.g., acetylcholine, bradykinin)

Ca2+

Endothelial cell

Ca2+

Ca2+ Ca2+-CaM

L-Arg

Ca2+-dependent K+ channel

Sarcoplasmic reticulum

eNOS

eNOS

NO

NO

Vascular smooth muscle cell

NO

K+

Guanylyl cyclase

Guanylyl cyclase

vascular wall is more than 100 times greater than that in the circulation, because ET-1 is secreted chiefly on the basal side of endothelial cells (Fig. 21-5). Endothelin precursors are proteolytically processed in two steps to generate the mature active peptides. First, preproendothelin is cleaved into big endothelin. Second, big endothelin is cleaved by endothelin-converting enzyme into endothelin. There are two endothelin receptor subtypes, ETA and ETB. Both ETA and ETB are G protein-coupled receptors whose effectors likely involve phospholipase C-modulated pathways. ET-1 binds to ETA receptors on vascular smooth muscle cells as well as ETB receptors on both endothelial cells and vascular smooth muscle cells. ETA receptors on vascular smooth muscle cells mediate vasoconstriction. ETB receptors are located predominantly on vascular endothelial cells, where they mediate vasodilation via the release of prostacyclin and NO. ETB receptors are also found on vascular smooth muscle cells, where they mediate vasoconstriction. Autonomic Nervous System The sympathetic nervous system is an important determinant of vascular tone. The firing of certain sympathetic postganglionic neurons releases norepinephrine from nerve terminals that end

Hyperpolarization Relaxation

Lumen

FIGURE 21-4.

Endothelial regulation of nitric oxide-mediated vascular smooth muscle relaxation. Endothelial-cell production of nitric oxide (NO) controls the extent of vascular smooth muscle cell relaxation. Production of NO is stimulated by agonists such as acetylcholine or bradykinin. Stimulation of receptors by these agonists activates Ca2⫹ second messenger systems and promotes direct entry of Ca2⫹ into the cytosol. The increased cytosolic Ca2⫹ activates a Ca2⫹-calmodulin complex that stimulates endothelial nitric oxide synthase (eNOS), an enzyme that catalyzes the formation of NO from L-arginine (L-Arg, an amino acid). NO diffuses from the endothelial cell into subjacent vascular smooth muscle cells, where it activates guanylyl cyclase, promoting smooth muscle cell relaxation (see Fig. 21-3). NO can also directly activate Ca2⫹-dependent K⫹ channels. This parallel signaling pathway contributes to relaxation by hyperpolarizing the smooth muscle cell. The active form of each enzyme is italicized and blue.

Endothelial cells

Arachidonic acid COX

L-Arg

Prostacyclin

NO

Endothelin-1

ETB

Endothelin-1 Prostacyclin

hyperpolarization of the cells and, subsequently, to vasodilation. (See below for a further explanation of how opening K⫹ channels leads to hyperpolarization and vasodilation.)

Endothelin precursors

eNOS

Endothelin-1 NO

IP

NO

ETA

ETB

Endothelin

Endothelin is a 21-amino acid vasoconstrictor peptide. It is the most potent endogenous vasoconstrictor yet discovered. Endothelin can be considered a functional “mirror-image” of NO: it is a potent endothelium-derived vasoconstrictor, while NO is a potent endothelium-derived vasodilator. In addition to its effects on the vasculature, endothelin has positive inotropic and chronotropic actions on the heart, and it contributes to remodeling within the cardiovascular system. Proposed mechanisms of endothelin-induced remodeling include neointimal proliferation and increased collagen deposition leading to fibrosis. Endothelin also plays an important role in the lungs, kidneys, and brain. Three isoforms of endothelin—ET-1, ET-2, and ET-3—have been identified. ET-1—the isoform mainly involved in cardiovascular actions—is produced by endothelial cells (and vascular smooth muscle cells under inflammatory conditions), and it appears to act locally in a paracrine or autocrine fashion. The local ET-1 concentration within the

Relaxation

Contraction

Vascular smooth muscle cells

FIGURE 21-5.

Effects of endothelin on the blood vessel wall. Endothelin mediates both contraction and relaxation of vascular smooth muscle cells. Endothelin precursors in endothelial cells are processed to endothelin-1. Endothelin-1 is secreted on the basal side of the endothelial cell, where it interacts with ETA and ETB receptors on vascular smooth muscle cells. Activation of these receptors stimulates contraction by incompletely understood mechanisms. ETB receptors are also expressed on endothelial cells. Endothelial cell ETB activation stimulates cyclooxygenase (COX), which catalyzes the formation of prostacyclin from arachidonic acid. Prostacyclin diffuses from the endothelial cell to the vascular smooth muscle cell membrane, where it binds to and activates the isoprostanoid (IP) receptor. ETB activation also stimulates endothelial nitric oxide synthase (eNOS), which catalyzes the formation of NO from arginine (L-Arg). Both prostacyclin and NO stimulate vascular smooth muscle cell relaxation.

358 Principles of Cardiovascular Pharmacology

on vascular smooth muscle cells. Activation of ␣1-adrenergic receptors on vascular smooth muscle cells causes vasoconstriction, whereas activation of ␤2-adrenergic receptors on vascular smooth muscle cells induces vasodilation. The effect of norepinephrine at ␣1-adrenergic receptors is typically greater than its effect at ␤2-adrenergic receptors, especially in organs that receive decreased blood flow during “fight or flight” responses (i.e., skin and viscera). Thus, the net effect of norepinephrine on these vascular beds is typically vasoconstrictive. Because blood vessels are not innervated by parasympathetic fibers, the parasympathetic nervous system has little influence on vascular tone. Neurohormonal Mechanisms Many neurohormonal mediators act on vascular smooth muscle cells, endothelial cells, and neurons to regulate vascular tone. For example, circulating catecholamines from the adrenal gland (i.e., epinephrine) can influence vascular tone via ␣1-adrenergic and ␤2-adrenergic receptors on vascular smooth muscle cells: as noted above, stimulation of ␣1-adrenergic receptors leads to vasoconstriction, while stimulation of ␤2adrenergic receptors leads to vasodilation. Other examples of neurohormonal mediators include angiotensin II, which stimulates the angiotensin II receptor subtype 1 (AT1) to vasoconstrict arterioles and increase intravascular volume; aldosterone, which acts via the mineralocorticoid receptor to increase intravascular volume; natriuretic peptides, which both promote renal natriuresis (sodium excretion) in situations of volume overload and cause vasodilation by stimulating guanylyl cyclase receptors on endothelial cells and vascular

smooth muscle cells; and antidiuretic hormone/arginine vasopressin, which stimulates arteriolar V1 receptors to constrict arterioles and activates renal V2 receptors to increase intravascular volume. These mediators, which also have important roles in volume regulation, are all discussed in greater detail in Chapter 20, Pharmacology of Volume Regulation. Local Mechanisms A panoply of local control mechanisms also modulate vascular tone. Autoregulation is a homeostatic mechanism in which vascular smooth muscle cells respond to increases or decreases in perfusion pressure by vasoconstriction or vasodilation, respectively, to preserve blood flow at a relatively constant level (Flow ⫽ Perfusion Pressure/Resistance). Vascular tone, and thus blood flow, is also governed by metabolites—such as H⫹, CO2, O2, adenosine, lactate, and K⫹—produced in surrounding tissue. Local mechanisms of vascular tone regulation predominate in the vascular beds of essential organs (e.g., heart, brain, lung, kidney), so that blood flow, and thus O2 supply, can be adjusted quickly to meet the demands of local metabolism in these organs.

 PHARMACOLOGIC CLASSES AND AGENTS The pharmacologic agents considered in this chapter are all vasodilators, that is, drugs that act on vascular smooth muscle and/or on the adjacent vascular endothelium to decrease vascular tone. Most vasodilators act by reducing the contractility of actin–myosin complexes in vascular smooth muscle cells. There are a number of categories of vasodilators (Fig. 21-6). Pharmacologic donors of NO—such as the

ETA, ETB antagonists

α1 antagonists

K+ channel openers Ca2+ channel blockers ACE inhibitors AT-I

ACE

NE KATP channel

L-type Ca2+ Ca2+ channel

AT-II

Hyperpolarization

Ca2+ AT1 antagonists

ACE inhibitors

ETA, ETB

Inactive

KII

Bradykinin

PDE5 inhibitors K+

AT1

α1

ET-1

GMP

Arginine PDE cGMP

eNOS NO

Bradykinin receptor

Nitrates

CaM Myosin-LC phosphatase

Ca2+-CaM MLCK Myosin-LC

MLCK

Myosin-LC phosphatase Myosin-LC P

Contraction

Myosin-LC

Relaxation

Vascular smooth muscle cell

Sites of action of vasodilators. Vasodilators act at several sites in the vascular smooth muscle cell. Left panel: Ca2⫹ channel blockers and K⫹ channel openers inhibit the entry of Ca2⫹ into vascular smooth muscle cells by decreasing activation of L-type Ca2⫹ channels. ACE inhibitors, AT1 antagonists, ␣1-antagonists, and endothelin receptor (ETA, ETB) antagonists all decrease intracellular Ca2⫹ signaling. The decreased cytosolic Ca2⫹ results in decreased vascular smooth muscle cell contraction, and, hence, in relaxation. Right panel: ACE inhibitors inhibit kininase II (KII), leading to increased levels of bradykinin. Nitrates release NO. Sildenafil and other PDE5 inhibitors inhibit phosphodiesterase (PDE). These agents all cause an increase in cGMP, an effect that promotes vascular smooth muscle relaxation. The active form of each enzyme is italicized and blue. ␣1, ␣1-adrenergic receptor; ACE, angiotensin converting enzyme; AT-I, angiotensin I; AT-II, angiotensin II; AT1, angiotensin II receptor; CaM, calmodulin; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; MLCK, myosin light chain kinase; myosin-LC, myosin light chain.

FIGURE 21-6.

Sodium nitroprusside (SNP)

Organic nitrates (RNO2) Enzymes and extracellular reductants

Spontaneous

Nitric oxide (NO)

S-Nitrosothiol (RSNO)

RNO2 Enzymes and intracellular reductants RSNO

Endothelial cell SNP

RSNO2

NO

NO

RSNO

Guanylyl cyclase

Relaxation

Vascular smooth muscle cell

360 Principles of Cardiovascular Pharmacology Heart (pump)

Veins

Capacitance vessels

Organic nitrates Arteries

Myocardial O2 supply by dilating large epicardial arteries

Resistance vessels

Organic nitrates

Organic nitrates

Preload Myocardial O2 demand

Afterload Myocardial O2 demand

FIGURE 21-8.

Sites of action of organic nitrates. Organic nitrates exert the majority of their vasodilator action on venous capacitance vessels. This selectivity results in greatly decreased preload, with resulting decreased myocardial O2 demand. Organic nitrates also mildly dilate arteriolar resistance vessels, with resulting decreased afterload and decreased myocardial O2 demand. Myocardial O2 supply is mildly increased by dilation of large epicardial arteries.

little effect on systemic blood pressure when administered by inhalation. Therapy with inhaled NO has established efficacy in the treatment of primary pulmonary hypertension of the newborn, but the therapeutic value of inhaled NO in other conditions characterized by elevated pulmonary artery pressures (including heart failure and various forms of lung disease) remains to be established. Sodium nitroprusside is a nitrate compound that consists of a nitroso group, five cyanide groups, and an iron atom (Fig. 21-10A). As with the organic nitrates, sodium nitroprusside effects vasodilation by release of NO. Unlike the organic nitrates, however, sodium nitroprusside appears to liberate NO primarily through a nonenzymatic process (Fig. 21-7). As a result of this nonenzymatic conversion to NO, sodium nitroprusside’s action does not appear to be targeted to specific types of vessels and, consequently, the drug dilates both arteries and veins. Sodium nitroprusside is used intravenously for powerful hemodynamic control in hypertensive emergencies and severe heart failure. Because of its rapid onset of action, short duration

ONO2 O2NO

Clinically, the administration of doses of organic nitrates sufficient to vasodilate the large epicardial arteries can be dangerous because such doses may also induce excessive peripheral arteriolar vasodilation and refractory hypotension. The excessive decrease in mean arterial pressure can be manifested as dizziness, lightheadedness, and occasionally, overt syncope, and can even lead to myocardial ischemia. Because coronary perfusion depends on the pressure gradient between the aorta and the endocardium during diastole, a marked decrease in diastolic aortic pressure can lead to an insufficient supply of O2 to the heart. Moreover, systemic hypotension can lead to reflex tachycardia, which also decreases myocardial O2 supply by shortening diastole and, thus, myocardial perfusion time. As noted above, reflex tachycardia can also harm the delicate myocardial O2 supply:demand balance by increasing myocardial O2 consumption. Reflex tachycardia is typically observed when baroreceptors in the aortic arch and carotid sinuses sense a decrease in blood pressure. In patients with overt heart failure, however, reflex tachycardia is rare. Thus, nitrates can often be used to decrease pulmonary congestion in patients with heart failure (by effecting venodilation and decreasing end-diastolic pressure), without eliciting significant reflex tachycardia. Several important adverse effects of nitrates are the result of excessive vasodilation; these include flushing, caused by vasodilation of cutaneous vascular beds, and headache, caused by vasodilation of cerebral arteries. Several different preparations of organic nitrates are currently available. The most commonly used organic nitrates include NTG, isosorbide dinitrate, and isosorbide 5-mononitrate (Fig. 21-9). Although these organic nitrates share a common mechanism of action, they differ in their routes of administration and pharmacokinetics, leading to important differences in their therapeutic utility in a variety of clinical settings. Inhaled nitric oxide gas can be used to selectively dilate the pulmonary vasculature. Because NO is rapidly inactivated by binding to hemoglobin in the blood, NO gas has

ONO2

Nitroglycerin (Glyceryl trinitrate)

OH

ONO2 O2NO

OH

O2NO

Glyceryl 1,2-dinitrate

ONO2

Glyceryl 1,3-dinitrate

O2NO

H O

O H

ONO2

Isosorbide dinitrate

HO

O2NO

H

H

O O

O

H

ONO2

Isosorbide 2-mononitrate

FIGURE 21-9.

O

H

OH

Isosorbide 5-mononitrate

Chemical structures and metabolism of nitroglycerin and isosorbide dinitrate. Nitroglycerin and isosorbide dinitrate are biologically active nitrates that are metabolized into active molecules with longer half-lives than their parent compounds. Nitroglycerin is denitrated into glyceryl 1,2-dinitrate and glyceryl 1,3-dinitrate; these active metabolites have a half-life of approximately 40 minutes. Isosorbide dinitrate is denitrated into isosorbide 2-mononitrate and isosorbide 5-mononitrate; these active metabolites have half-lives of 2 and 4 hours, respectively.

NO

A

NC Fe+2 NC

CN CN

CN Nitroprusside B Sodium nitroprusside

NO

Cyanide

Vasodilation

Liver Sulfhydryl donor

Thiocyanate

Renal excretion

SA node

AV node

Automaticity

Conduction

Cardiac myocytes Afterload Myocardial O2 demand

Coronary arteries Vasodilation Myocardial O2 supply

Veins

Peripheral veins Minimal venodilation

Peripheral arterioles Vasodilation Afterload Myocardial O2 demand

Arteries Heart (pump)

CHAPTER 21 / Pharmacology of Vascular Tone 365

have suggested that Ca2⫹ channel blockers increase the risk of mortality in patients with heart failure, and Ca2⫹ channel blockers are therefore contraindicated in the management of heart failure. Some reports also suggest that the short-acting agent nifedipine is associated with an increased risk of myocardial ischemia and infarction, by virtue of this drug’s tendency to disturb the myocardial O2 supply:demand balance (see above).

Kⴙ Channel Openers K⫹ channel openers cause direct arterial vasodilation by opening ATP-modulated Kⴙ channels (sometimes called KⴙATP channels) in the plasma membrane of vascular smooth muscle cells. Because these agents act by a mechanism that is entirely different from that of other vasodilators, K⫹ATP channel openers represent a powerful family of drugs that can be used to treat hypertension refractory to other antihypertensive therapeutics. What is the normal function of ATP-modulated K⫹ channels? Recall that the Nernst equilibrium potential for K⫹ is about –90 mV, while the resting membrane potential is less negative than this value. Therefore, opening K⫹ channels hyperpolarizes the membrane. If a sufficient number of K⫹ channels are open at the same time, then normal excitatory stimuli are not able to promote membrane depolarization. In the absence of depolarization, voltage-gated Ca2⫹ channels do not open, and Ca2⫹ influx and smooth muscle contraction are inhibited (Fig. 21-6). The K⫹ATP channel opener drugs include minoxidil, cromakalim, pinacidil, and nicorandil. These drugs act primarily on arterial smooth muscle cells, and therefore decrease arterial blood pressure. Adverse effects of the K⫹ATP channel openers include headache, caused by excessive dilation of cerebral arteries, and flushing, caused by excessive dilation of cutaneous arteries. When arterial vasodilators (e.g., Ca2⫹ channel blockers or K⫹ATP channel openers) are used as monotherapy, the decrease in arterial pressure often elicits reflex sympathetic discharge, leading to tachycardia and increased cardiac work. As noted above in the discussion of nifedipine, reflex sympathetic discharge can upset the balance between myocardial O2 supply and demand, precipitating myocardial ischemia; this effect is of particular concern in patients with preexisting coronary artery disease. However, the use of ␤blockers in combination with arterial vasodilators can help to block the effects of reflex sympathetic activity, and thereby preserve the therapeutic utility of the arterial vasodilators.

Endothelin Receptor Antagonists Bosentan is a competitive antagonist at ETA and ETB receptors. It is approved for use in the treatment of pulmonary hypertension. In clinical trials involving patients with severe dyspnea related to pulmonary hypertension, bosentan significantly improved 6-minute walk-test distance (i.e., the distance a patient can walk in 6 minutes) and decreased pulmonary vascular resistance relative to placebo. The major adverse effect of bosentan is an elevation in serum transaminase levels, with approximately 10% of patients having elevations that exceed three times the upper limit of normal. It is therefore necessary to monitor liver function tests monthly in patients taking bosentan. Ambrisentan is an endothelin receptor antagonist with relative specificity for the ETA receptor. As with bosentan,

patients with pulmonary hypertension have improved 6-minute walk-test distance and increased functional status when taking this medication. Ambrisentan may have less hepatotoxicity than bosentan.

Other Drugs That Modulate Vascular Tone Hydralazine Hydralazine is an orally administered arteriolar vasodilator that is sometimes used in the treatment of hypertension and, in combination with isosorbide dinitrate, in the treatment of heart failure. The mechanism of action of hydralazine remains unclear; studies have suggested that membrane hyperpolarization, K⫹ATP channel opening, and inhibition of IP3-induced Ca2⫹ release from the sarcoplasmic reticulum in vascular smooth muscle cells may all be involved. Hydralazine appears to prevent the development of nitrate tolerance, perhaps by inhibiting vascular superoxide production. A combination pill containing isosorbide dinitrate and hydralazine has recently been found to reduce morbidity and mortality in black Americans with advanced heart failure; it remains to be determined whether the benefits of this therapy extend to other patient populations. However, if the success of this hydralazine–isosorbide dinitrate combination therapy for heart failure is related to hydralazine’s prevention of nitrate tolerance, then these drugs may be broadly efficacious in heart failure treatment. The use of hydralazine has been limited because it was initially thought that the frequent dosing required for sustained blood pressure control and the rapid development of tachyphylaxis to its antihypertensive effects made chronic use of this drug impractical. As the benefits of combination therapy for hypertension and heart failure are becoming better appreciated, it may be possible for hydralazine to be used more effectively, especially in patients for whom other vasodilators (e.g., ACE inhibitors) are contraindicated. Hydralazine typically has low bioavailability because of extensive first-pass hepatic metabolism. The rate of its metabolism depends on whether the patient is a slow or fast acetylator, however. In slow acetylators (see Chapter 4), hydralazine has a slower rate of hepatic degradation and, thus, higher bioavailability and higher plasma concentrations. A rare adverse effect of hydralazine is the development of a reversible lupus erythematosus-like syndrome; this effect occurs chiefly in slow acetylators. 1-Adrenergic Antagonists Epinephrine and norepinephrine stimulate ␣1-adrenergic receptors on vascular smooth muscle, and thereby induce vasoconstriction. The ␣1-adrenergic receptor is a G proteincoupled receptor that associates with the heterotrimeric G protein Gq, which activates phospholipase C to generate inositol trisphosphate and diacylglycerol. ␣1-Adrenergic antagonists, such as prazosin, block ␣1-adrenergic receptors in arterioles and venules, leading to vasodilation. The effect of these agents is greater in arterioles than in venules. The ␣1-adrenergic antagonists cause a significant reduction in arterial pressure, and are thus useful in the treatment of hypertension. Initiation of therapy with ␣1-adrenergic antagonists can be associated with orthostatic hypotension. Like other arterial vasodilators, the ␣1-adrenergic antagonists can also cause retention of salt and water. ␤-Adrenergic blockers and diuretics may be used

366 Principles of Cardiovascular Pharmacology

together with ␣1-adrenergic antagonists to mitigate these compensatory responses. Some ␣1-adrenergic antagonists, such as terazosin, are used principally to inhibit the contraction of nonvascular smooth muscle (e.g., prostatic smooth muscle), but these agents also have some effect on the vasculature (see Chapter 10, Adrenergic Pharmacology). -Adrenergic Antagonists Activation of ␤2-adrenergic receptors on vascular smooth muscle cells leads to vasodilation. The increased intracellular cAMP induced by ␤2-receptor stimulation may cause smooth muscle relaxation by accelerating the inactivation of myosin light chain kinase and by increasing the extrusion of Ca2⫹ from the cell. Activation of ␤2-adrenergic receptors on endothelial cells also leads to vasodilation through activation of endothelial nitric oxide synthase. Despite the beneficial vasodilatory effects of ␤2- and ␤3-agonist action in the systemic circulation, ␤-adrenergic antagonists are of major clinical importance in the treatment of hypertension, angina, cardiac arrhythmias, and other conditions. Acting at cardiac ␤1-adrenergic receptors, ␤-adrenergic antagonists have negative inotropic and chronotropic effects on the heart; these actions reduce cardiac output, which is an important determinant of both myocardial O2 demand and blood pressure (see Equation 21-2). The cardiac effects of ␤-adrenergic antagonists are discussed in greater detail in Chapter 10. These drugs also have important effects on the vasculature: antagonism of ␤2-adrenergic receptors on vascular smooth muscle cells can lead to unopposed vasoconstriction mediated by ␣1-adrenergic receptors and, consequently, to an increase in systemic vascular resistance. Importantly, although some ␤-adrenergic antagonists may initially increase systemic vascular resistance, the net effect in most cases is a decrease in blood pressure. This hypotensive effect reflects the combined negative inotropic effect (leading to a decrease in cardiac output), inhibition of renin secretion, and central nervous system effects of the ␤-blockers. Renin–Angiotensin System Blockers As discussed in Chapter 20, inhibition of the renin– angiotensin system results in significant vasorelaxation. The hypotensive effect of ACE inhibitors may be caused in part by decreased catabolism of bradykinin, a vasorelaxant released in response to inflammatory stimuli. Antagonists at the AT1 receptor, which selectively inhibit angiotensin IImediated vasoconstriction at the level of the target organ, have a more direct effect. ACE inhibitors and AT1 receptor antagonists are considered “balanced” vasodilators because they affect both arterial and venous tone. Both classes of drugs are effective in the treatment of hypertension and heart failure, as discussed in Chapter 25.

 CONCLUSION AND FUTURE DIRECTIONS Vascular tone is subject to exquisite control, as would be expected for a system that must perfuse all the tissues of the body. Vascular tone represents a balance between vascular smooth muscle contraction and relaxation. Vasoconstriction occurs when an increase in intracellular Ca2⫹ activates Ca2⫹-CaM–dependent myosin light chain kinase (MLCK).

In turn, MLCK phosphorylates myosin light chains and allows the formation of actin–myosin cross-bridges. Vascular smooth muscle relaxes when the intracellular Ca2⫹ concentration returns to basal levels and myosin light chains are dephosphorylated, terminating the formation of actin–myosin cross-bridges. Vascular tone is influenced by the state of the vascular smooth muscle cells and the overlying endothelial cells, by sympathetic innervation, and by neurohormonal and local regulators. Diverse therapeutic agents modulate various components of this critical system, with important differences in molecular mechanisms and effects. Classes of vasodilators include nitrates, Ca2⫹ channel blockers, K⫹ channel openers, ␣1-adrenergic receptor antagonists, ACE inhibitors, AT1 receptor antagonists, and endothelin receptor antagonists (Fig. 21-6). Nitrates dilate primarily veins, not arteries. These agents act by releasing NO in vascular smooth muscle cells; in turn, NO activates guanylyl cyclase, which increases intracellular cGMP, which activates cGMP-dependent protein kinase, which activates myosin light chain phosphatase, which terminates the formation of actin–myosin cross-bridges. Ca2⫹ channel blockers act mainly on arteries and resistance arterioles, and may also have direct effects on the heart. These drugs cause vasodilation by blocking L-type voltage-gated Ca2⫹ channels in the plasma membrane of vascular smooth muscle cells, thereby inhibiting the Ca2⫹ influx through these channels that is necessary for contraction. K⫹ATP channel openers, as with Ca2⫹ channel blockers, are predominantly arteriodilators, not venodilators. This class of drugs opens ATP-modulated K⫹ channels, thereby hyperpolarizing vascular smooth muscle cells and preventing the activation of voltage-gated Ca2⫹ channels that is necessary for Ca2⫹ influx and muscle contraction. ␣1-Adrenergic receptor antagonists, AT1 receptor antagonists, and endothelin receptor antagonists prevent vasoconstriction by inhibiting the activation of their respective receptors by endogenous agonists. The mechanisms that control vascular tone are regulated by multiple intersecting signaling pathways. The emerging science of systems biology combines mathematical, computational, and experimental approaches to understand complex signaling pathways in a wide array of tissues and organs. This integrated approach to signaling will provide novel quantitative information regarding the interplay of intracellular signaling pathways in the vasculature, and may lead to the identification of new drug targets. For example, insights into the relationships among cGMP-modulated regulatory pathways in vascular smooth muscle cells have recently led to new therapeutic applications for PDE5 inhibitors, such as sildenafil, in the treatment of pulmonary hypertension and heart failure. A new class of drug, exemplified by ranolazine, appears to have minimal direct effects on cardiac contractility or vascular tone, yet appears to have efficacy in the treatment of angina. This drug and other new agents may exert their principal therapeutic effects on the cardiovascular system by affecting metabolic pathways in target tissues. A recently approved ␤-adrenergic blocker, nebivolol, has the interesting property that it is both a ␤1-adrenergic antagonist and a ␤3-adrenergic agonist. This drug, which appears to be as efficacious as other ␤-blockers in the treatment of hypertension, combines cardiac-selective ␤1-adrenergic blockade

CHAPTER 21 / Pharmacology of Vascular Tone 367

with stimulation of ␤3 receptors, which activate nitric oxide synthase in the vasculature. Continued elucidation of complex signaling pathways will likely lead to the identification of new points for pharmacologic intervention in the cellular milieu of the vascular wall, and will help to integrate the pharmacology of vascular tone across the spectrum of cardiovascular disease.

Suggested Reading Abrams J. Chronic stable angina. N Engl J Med 2005;352:2524–2533. (An informative case vignette and review of the pathophysiology and pharmacotherapy of angina pectoris.)

Bloch KD, Ichinose F, Roberts JD Jr, Zapol WM. Inhaled NO as a therapeutic agent. Cardiovasc Res 2007;75:339–348. (A useful review of the therapeutic applications of inhaled nitric oxide.) Cheng JW. Nebivolol: a third-generation beta-blocker for hypertension. Clin Ther 2009;31:447–462. (A review of trials that study a recently approved ␤-adrenergic blocker that combines ␤1-antagonist with ␤3agonist properties.) Mark JD, Griffiths M, Evans TW. Drug therapy: inhaled nitric oxide therapy in adults. N Engl J Med 2005;353:2683–2695. (Reviews the history of inhaled NO and current indications for this therapy.) Tsai EJ, Kass DA. Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics. Pharm Ther 2009;122:216–238. (This review explores mechanisms and therapies involving cyclic GMP in the control of vascular tone and cardiac function.)

22 Pharmacology of Hemostasis and Thrombosis April W. Armstrong and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . 372-373 PHYSIOLOGY OF HEMOSTASIS . . . . . . . . . . . . . . . . . . . . . . 372 Vasoconstriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Primary Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Platelet Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Platelet Granule Release Reaction . . . . . . . . . . . . . . . 373 Platelet Aggregation and Consolidation . . . . . . . . . . . . 375 Secondary Hemostasis: The Coagulation Cascade . . . . . . 376 Regulation of Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . 379 PATHOGENESIS OF THROMBOSIS . . . . . . . . . . . . . . . . . . . . 380 Endothelial Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Abnormal Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Hypercoagulability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 381 Antiplatelet Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Cyclooxygenase Inhibitors . . . . . . . . . . . . . . . . . . . . . . 382 Phosphodiesterase Inhibitors . . . . . . . . . . . . . . . . . . . 383 ADP Receptor Pathway Inhibitors . . . . . . . . . . . . . . . . 386 GPIIb–IIIa Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . 386

Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 Unfractionated and Low-MolecularWeight Heparins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Selective Factor Xa Inhibitors . . . . . . . . . . . . . . . . . . . 391 Direct Thrombin Inhibitors . . . . . . . . . . . . . . . . . . . . . 391 Recombinant Activated Protein C (r-APC) . . . . . . . . . . 392 Thrombolytic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Streptokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Recombinant Tissue Plasminogen Activator (t-PA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Tenecteplase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Reteplase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Inhibitors of Anticoagulation and Fibrinolysis . . . . . . . . . . 393 Protamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Serine-Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . 393 Lysine Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 393 Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

 INTRODUCTION

must also remain localized to prevent widespread clotting within intact vessels. The formation of a localized clot at the site of vessel injury is accomplished in four temporally overlapping stages (Fig. 22-1). First, localized vasoconstriction occurs as a response to a reflex neurogenic mechanism and to the secretion of endothelium-derived vasoconstrictors such as endothelin. Immediately following vasoconstriction, primary hemostasis occurs. During this stage, platelets are activated and adhere to the exposed subendothelial matrix. Platelet activation involves both a change in shape of the platelet and the release of secretory granule contents from the platelet. The secreted granule substances recruit other platelets, causing more platelets to adhere to the subendothelial matrix and to aggregate with one another at the site of vascular injury. Primary hemostasis ultimately results in the formation of a primary hemostatic plug. The goal of the final two stages of hemostasis is to form a stable, permanent plug. During secondary hemostasis, also known as the coagulation cascade, the activated endothelium and other nearby cells (see below) express a membrane-bound procoagulant factor called tissue factor, which complexes

Blood carries oxygen and nutrients to tissues and takes metabolic waste products away from tissues. Humans have developed a well-regulated system of hemostasis to keep the blood fluid and clot-free in normal vessels and to form a localized plug rapidly in injured vessels. Thrombosis describes a pathologic state in which normal hemostatic processes are activated inappropriately. For example, a blood clot (thrombus) may form as the result of a relatively minor vessel injury and occlude a section of the vascular tree. This chapter presents the normal physiology of hemostasis, the pathophysiology of thrombosis, and the pharmacology of drugs that can be used to prevent or reverse a thrombotic state. Drugs introduced in this chapter are used to treat a variety of cardiovascular diseases, such as deep vein thrombosis, stroke, and myocardial infarction.

 PHYSIOLOGY OF HEMOSTASIS An injured blood vessel must induce the formation of a blood clot to prevent blood loss and to allow healing. Clot formation 372

374 Principles of Cardiovascular Pharmacology Endothelin release by activated endothelium Reflex vasoconstriction

A

E

Site of vascular injury (denuded endothelium) Vascular smooth muscle

Resting platelets

Basement membrane Endothelial cells

2. Platelet adhesion and activation

1. Subendothelial matrix exposure

6. Platelet aggregation (hemostatic plug)

B

5. Platelet recruitment ADP 4. Platelet shape change TxA2 3. Platelet granule release Fibrin 1. Tissue factor expression on activated endothelium

C

4. Fibrin polymerization 3. Thrombin activation

Activated spread platelet

Activated contracted platelet

FIGURE 22-1.

Sequence of events in hemostasis. The hemostatic process can be divided conceptually into four stages—vasoconstriction, primary hemostasis, secondary hemostasis, and resolution—although recent evidence suggests that these stages are temporally overlapping and may be nearly simultaneous. A. Vascular injury causes endothelial denudation. Endothelin, released by activated endothelium, and neurohumoral factor(s) induce transient vasoconstriction. B. Injury-induced exposure of the subendothelial matrix (1 ) provides a substrate for platelet adhesion and activation (2 ). In the granule release reaction, activated platelets secrete thromboxane A2 (TxA2) and ADP (3 ). TxA2 and ADP released by activated platelets cause nearby platelets to become activated; these newly activated platelets undergo shape change (4 ) and are recruited to the site of injury (5 ). The aggregation of activated platelets at the site of injury forms a primary hemostatic plug (6 ). C. Tissue factor expressed on activated endothelial cells (1) and leukocyte microparticles (not shown), together with acidic phospholipids expressed on activated platelets and activated endothelial cells (2 ), initiate the steps of the coagulation cascade, culminating in the activation of thrombin (3 ). Thrombin proteolytically activates fibrinogen to form fibrin, which polymerizes around the site of injury, resulting in the formation of a definitive (secondary) hemostatic plug (4 ). D. Natural anticoagulant and thrombolytic factors limit the hemostatic process to the site of vascular injury. These factors include tissue plasminogen activator (t-PA), which activates the fibrinolytic system (1 ); thrombomodulin, which activates inhibitors of the coagulation cascade (2 ); prostacyclin, which inhibits both platelet activation and vasoconstriction (3 ); and surface heparin-like molecules, which catalyze the inactivation of coagulation factors (4 ). E. Scanning electron micrographs of resting platelets (1 ), a platelet undergoing cell spreading shortly after cell activation (2 ), and a fully activated platelet after actin filament bundling and cross-linking and myosin contraction (3 ).

2. Phospholipid complex expression

D

t-PA

PGI2

1. Release of t-PA (fibrinolysis) 2. Thrombomodulin (blocks coagulation cascade) 3. Release of prostacyclin (inhibits platelet aggregation and vasoconstriction)

4. Surface heparin-like molecules (blocks coagulation cascade)

important in mediating platelet aggregation, causing platelets to become “sticky” and adhere to one another (see below). Although strong agonists (such as thrombin and collagen) can trigger granule secretion even when aggregation is prevented, ADP can trigger granule secretion only in the presence of platelet aggregation. Presumably, this difference is caused by the set of intracellular effectors

that are coupled to the various agonist receptors. Release of Ca2⫹ ions is also important for the coagulation cascade, as discussed below. Although platelet activation can be initiated via exposure of subendothelial collagen, a separate and parallel process of platelet activation occurs without disruption of the endothelium and without the involvement of von Willebrand factor. This second pathway of platelet activation is initiated by tissue factor, a lipoprotein expressed by activated leukocytes and by microparticles derived from activated leukocytes (see below). As in the coagulation cascade, tissue factor forms a complex with factor VIIa, and the tissue factor–factor VIIa complex activates factor IX. Factor IX activation leads to a proteolytic cascade that results in the generation of thrombin (factor IIa), a multifunctional enzyme that plays a critical role in the coagulation cascade (see below). In the tissue-factor initiated pathway of platelet activation, thrombin cleaves protease-activated receptor 4 on the platelet surface and thereby causes the platelets to release ADP, serotonin, and TxA2. By activating other nearby platelets, these agonists amplify the signal for thrombus formation.

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 375

Endothelium Endothelium

Collagen

Collagen

Resting platelet Tissue factor

Platelet

TXA2

Prothrombin

Fibrinogen ADP

Thrombin

GPVI GPIIb-IIIa GPIb von Willebrand factor von Willebrand factor Collagen (subendothelium)

Activated endothelium

Collagen

Platelet adhesion and aggregation. von Willebrand factor mediates platelet adhesion to the subendothelium by binding both to the platelet membrane glycoprotein GPIb and to exposed subendothelial collagen. Platelet adhesion to the subendothelial matrix also requires a direct binding interaction between platelet membrane glycoprotein GPVI and subendothelial collagen. During platelet aggregation, fibrinogen cross-links platelets to one another by binding to GPIIb–IIIa receptors on platelet membranes.

FIGURE 22-3. Platelet activation. Platelet activation is initiated at the site of vascular injury when circulating platelets adhere to exposed subendothelial collagen and are activated by locally generated mediators. Activated platelets undergo shape change and granule release, and platelet aggregates are formed as additional platelets are recruited and activated. Platelet recruitment is mediated by the release of soluble platelet factors, including ADP and thromboxane A2 (TxA2). Tissue factor, expressed on activated endothelium, is a critical initiating component in the coagulation cascade. The membranes of activated platelets provide a surface for a number of critical reactions in the coagulation cascade, including the conversion of prothrombin to thrombin.

Platelet Aggregation and Consolidation TxA2, ADP, and fibrous collagen are all potent mediators of platelet aggregation. TxA2 promotes platelet aggregation through stimulation of G protein-coupled TxA2 receptors in the platelet membrane (Fig. 22-4). Binding of TxA2 to platelet TxA2 receptors leads to activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5bisphosphate (PI[4,5]P2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 raises the cytosolic Ca2⫹ concentration, and DAG activates protein kinase C (PKC), which in turn promotes the activation of PLA2. Through an incompletely understood mechanism, PLA2 activation induces the expression of functional GPIIb–IIIa, the membrane integrin that mediates platelet aggregation. ADP triggers platelet activation by binding to G proteincoupled ADP receptors on the platelet surface (Fig. 22-5). The two subtypes of G protein-coupled platelet ADP receptors are termed P2Y1 receptors and P2Y(ADP) receptors. P2Y1, a Gq-coupled receptor, releases intracellular calcium stores through activation of phospholipase C. P2Y(ADP), a Gicoupled receptor, inhibits adenylyl cyclase. The P2Y(ADP) receptor is the target of the antiplatelet agents ticlopidine,

clopidogrel, and prasugrel (see below). Activation of ADP receptors mediates platelet shape change and expression of functional GPIIb–IIIa. Fibrous collagen activates platelets by binding directly to platelet glycoprotein VI (GPVI). Activation of GPVI by collagen initiates signaling cascades that promote the granule release reaction and that induce conformational changes in cell-surface integrins (especially GPIIb–IIIa and ␣2␤1) that promote the direct or indirect binding of these integrins to collagen. These additional binding interactions further strengthen the adhesion of activated platelets to the subendothelial matrix. Platelets aggregate with one another through a bridging molecule, fibrinogen, which has multiple binding sites for functional GPIIb–IIIa (Fig. 22-2). Just as the vWF:GPIb interaction is important for platelet adhesion to exposed subendothelial collagen, the fibrinogen:GPIIb–IIIa interaction is critical for platelet aggregation. Platelet aggregation ultimately leads to the formation of a reversible clot, or a primary hemostatic plug. Activation of the coagulation cascade proceeds nearly simultaneously with the formation of the primary hemostatic plug, as described below. Activation of the coagulation

FIGURE 22-2.

376 Principles of Cardiovascular Pharmacology Arachidonic acid

1 Generation of thromboxane A2 by activated platelets

Cyclooxygenase

3

TXA2

G protein-mediated activation of phospholipase C

4

PIP2

TXA2-R

β

αq γ

PLC hydrolyzes PIP2 to yield IP3 and DAG

PLC

DAG

αq

PKC (active)

GTP

6

GDP

2

Activation of protein kinase C

PKC Activation of thromboxane A2 receptor

IP3 Ca2+

7

Ca2+

5 Increase in cytosolic calcium concentration

PLA2

8 Activation of GPIIb-IIIa

Activation of phospholipase A2

GP IIb -III a

9 Binding of fibrinogen to GPIIb-IIIa

Fibrinogen

10 Platelet aggregation

FIGURE 22-4.

Platelet activation by thromboxane A2. 1. Thromboxane A2 (TxA2) is generated from arachidonic acid in activated platelets; cyclooxygenase catalyzes the committed step in this process. 2. Secreted TxA2 binds to the cell-surface TxA2 receptor (TxA2-R), a G protein-coupled receptor. 3. The G␣ isoform G␣q activates phospholipase C (PLC). 4. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). 5. IP3 raises the cytosolic Ca2⫹ concentration by promoting vesicular release of Ca2⫹ into the cytosol. 6. DAG activates protein kinase C (PKC). 7. PKC activates phospholipase A2 (PLA2). 8. Through an incompletely understood mechanism, activation of PLA2 leads to the activation of GPIIb–IIIa. 9. Activated GPIIb–IIIa binds to fibrinogen. 10. Fibrinogen crosslinks platelets by binding to GPIIb–IIIa receptors on other platelets. This cross-linking leads to platelet aggregation and formation of a primary hemostatic plug.

cascade leads to the generation of fibrin, initially at the periphery of the primary hemostatic plug. Platelet pseudopods attach to the fibrin strands at the periphery of the plug and contract. Platelet contraction yields a compact, solid, irreversible clot, or a secondary hemostatic plug.

Secondary Hemostasis: The Coagulation Cascade Secondary hemostasis is also termed the coagulation cascade. The goal of this cascade is to form a stable fibrin clot at the site of vascular injury. Details of the coagulation cascade are presented schematically in Figure 22-6. Several general principles should be noted. First, the coagulation cascade is a sequence of enzymatic events. Most plasma coagulation factors circulate as inactive proenzymes, which are synthesized by the liver. These proenzymes are proteolytically cleaved, and thereby activated, by the activated factors that precede them in the cascade. The activation reaction is catalytic and not stoichiometric. For

example, one “unit” of activated factor X can potentially generate 40 “units” of thrombin. This robust amplification process rapidly generates large amounts of fibrin at a site of vascular injury. Second, the major activation reactions in the cascade occur at sites where a phospholipid-based protein–protein complex has formed (Fig. 22-7). This complex is composed of a membrane surface (provided by activated platelets, activated endothelial cells, and possibly activated leukocyte microparticles [see below]), an enzyme (an activated coagulation factor), a substrate (the proenzyme form of the downstream coagulation factor), and a cofactor. The presence of negatively charged phospholipids, especially phosphatidylserine, is critical for assembly of the complex. Phosphatidylserine, which is normally sequestered in the inner leaflet of the plasma membrane, translocates to the outer leaflet of the membrane in response to agonist stimulation of platelets, endothelial cells, or leukocytes. Calcium is required for the enzyme, substrate, and cofactor to adopt the proper conformation for the proteolytic cleavage of a coagulation factor proenzyme to its activated form.

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 377 Thrombin

ADP

4

Adenylyl cyclase

1

αi

P2Y(ADP) receptor

β

GDP

β γ αq

αi γ

GTP

6

2

GTP

β αq γ GDP

PDE

PKA

5 P2Y1 receptor

GDP

AMP

PLC

αq

ATP cAMP

ADP

Thrombin receptor

7 Increased PLC activity leads to platelet activation

3 Decreased PKA activity leads to platelet activation

FIGURE 22-5.

Platelet activation by ADP and thrombin. Left panel: 1. Binding of ADP to the P2Y(ADP) receptor activates a Gi protein, which inhibits adenylyl cyclase. 2. Inhibition of adenylyl cyclase decreases the synthesis of cAMP, and hence decreases protein kinase A (PKA) activation (dashed arrow). cAMP is metabolized to AMP by phosphodiesterase (PDE). 3. PKA inhibits platelet activation through a series of poorly understood steps. Therefore, the decreased PKA activation that results from ADP binding to the P2Y(ADP) receptor causes platelet activation. Right panel: 4. Thrombin proteolytically cleaves the extracellular domain of its receptor. This cleavage creates a new N-terminus, which binds to an activation site on the thrombin receptor to activate a Gq protein. 5. ADP also activates Gq by binding to the P2Y1 receptor. 6. Gq activation (by either thrombin or ADP) activates phospholipase C (PLC). 7. PLC activity leads to platelet activation, as shown in Figure 22-4. Note that ADP can activate platelets by binding to either the P2Y(ADP) receptor or the P2Y1 receptor, although evidence suggests that full platelet activation requires the participation of both receptors.

Third, the coagulation cascade has been divided traditionally into the intrinsic and extrinsic pathways (Fig. 22-6). This division is a result of in vitro testing and is essentially arbitrary. The intrinsic pathway is activated in vitro by factor XII (Hageman factor), while the extrinsic pathway is initiated in vivo by tissue factor, activated endothelial cells, subendothelial smooth muscle cells, and subendothelial fibroblasts at the site of vascular injury. Although these two pathways converge at the activation of factor X, there is also much interconnection between the two pathways. Because factor VII (activated by the extrinsic pathway) can proteolytically activate factor IX (a key factor in the intrinsic pathway), the extrinsic pathway is regarded as the primary pathway for the initiation of coagulation in vivo. Fourth, both the intrinsic and extrinsic coagulation pathways lead to the activation of factor X. In an important reaction that requires factor V, activated factor X proteolytically cleaves prothrombin (factor II) to thrombin (factor IIa) (Fig. 22-8). Thrombin acts in the coagulation cascade in four important ways: (1) it converts the soluble plasma protein fibrinogen into fibrin, which then forms long, insoluble polymer fibers; (2) it activates factor XIII, which cross-links the fibrin polymers into a highly stable meshwork or clot; (3) it amplifies the clotting cascade by catalyzing the feedback activation of factors VIII and V; and (4) it strongly activates

platelets, causing granule release, platelet aggregation, and platelet-derived microparticle generation. In addition to its procoagulant properties, thrombin acts to modulate the coagulation response. Thrombin binds to thrombin receptors on the intact vascular endothelial cells adjacent to the area of vascular injury, and stimulates these cells to release the platelet inhibitors prostacyclin (PGI2) and nitric oxide (NO), the profibrinolytic protein tissue plasminogen activator (t-PA), and the endogenous t-PA modulator plasminogen activator inhibitor 1 (PAI-1) (see below). The thrombin receptor, a protease-activated G proteincoupled receptor, is expressed in the plasma membrane of platelets, vascular endothelial cells, smooth muscle cells, and fibroblasts. Activation of the thrombin receptor involves proteolytic cleavage of an extracellular domain of the receptor by thrombin. The new NH2-terminal-tethered ligand binds intramolecularly to a discrete site within the receptor and initiates intracellular signaling. Activation of the thrombin receptor results in G protein-mediated activation of PLC (Fig. 22-5) and inhibition of adenylyl cyclase. Finally, evidence from intravital (in vivo) microscopy experiments suggests that microparticles have an important role in coupling platelet plug formation (primary hemostasis) to fibrin clot formation (secondary hemostasis). Microparticles

378 Principles of Cardiovascular Pharmacology Intrinsic pathway

Proteolytic cleavage (activation) of factor X

Extrinsic pathway Tissue injury

XII Kallikrein

VII IX

HMWK

XI

IXa

Prekallikrein

XIIa

XIa

VIIIa Ca

Thrombin (IIa)

2+

Xa

X

VIIa

IXa

Ca2+

2+

Xa

Ca2+

VIIIa

IX Xa a IXa

VIIIa Ca

VIIa

Thrombin (IIa) VIII

Proteolytic cleavage (activation) of prothrombin

X

Prothrombin (II)

Va

Tissue factor

Ca2+

Thrombin (IIa)

Va

Ca2+

Ca2+ Ca2+

Common pathway Xa Thrombin (IIa) V

Va Xa Ca2+ XIII

Prothrombin (II)

Thrombin (IIa)

Ca2+

XIIIa

Ca2+

Fibrinogen

Fibrin

FIGURE 22-7. Coagulation factor activation on phospholipid surfaces. Surface catalysis is critical for a number of the activation reactions in the coagulation cascade. Each activation reaction consists of an enzyme (e.g., factor IXa), a substrate (e.g., factor X), and a cofactor or reaction accelerator (e.g., factor VIIIa), all of which are assembled on the phospholipid surface of activated platelets, endothelial cells, and leukocytes. Ca2⫹ allows the enzyme and substrate to adopt the proper conformation in each activation reaction. In the example shown, factor VIIIa and Ca2⫹ act as cofactors in the factor IXa-mediated cleavage of factor X to factor Xa. Factor Va and Ca2⫹ then act as cofactors in the factor Xa-mediated cleavage of prothrombin to thrombin.

Fibrin polymer

Crosslinked fibrin polymer Resting endothelial Activated cells endothelial cells

V Va

FIGURE 22-6.

Coagulation cascade. The coagulation cascade is arbitrarily divided into the intrinsic pathway, the extrinsic pathway, and the common pathway. The intrinsic and extrinsic pathways converge at the level of factor X activation. The intrinsic pathway is largely an in vitro pathway, while the extrinsic pathway accounts for the majority of in vivo coagulation. The extrinsic pathway is initiated at sites of vascular injury by the expression of tissue factor on several different cell types, including activated endothelial cells, activated leukocytes (and leukocyte microparticles), subendothelial vascular smooth muscle cells, and subendothelial fibroblasts. Note that Ca2⫹ is a cofactor in many of the steps, and that a number of the steps occur on phospholipid surfaces provided by activated platelets, activated endothelial cells, and activated leukocytes (and leukocyte microparticles). Activated coagulation factors are shown in blue and indicated with a lower case “a.” HMWK, high-molecular-weight kininogen.

are vesicular structures derived from leukocytes, monocytes, platelets, endothelial cells, and smooth muscle cells; they display proteins of the cells from which they were derived. For example, a subpopulation of microparticles is released from monocytes that are activated in the context of tissue injury and inflammation. These microparticles appear to express both tissue factor and P-selectin glycoprotein ligand-1 (PSGL-1). In turn, PSGL-1 on the microparticles binds to the P-selectin adhesion receptor expressed on activated platelets. By recruiting tissue factor-bearing microparticles throughout the developing platelet plug (primary hemostasis), thrombin generation and fibrin clot formation (secondary hemostasis) could be greatly accelerated within the plug itself. Indeed, it seems that both vessel-wall tissue factor (expressed by activated endothelial cells, subendothelial

VII VIIa VIII VIIIa XI

XIa

Prothrombin (II)

Va Xa Ca2+ Resting platelets

PL

Thrombin (IIa) Activated platelets

Fibrinogen g XIII

XIIIa

Fibrin n

Crosslinked fibrin polymer

FIGURE 22-8. Central role of thrombin in the coagulation cascade. In the coagulation cascade, prothrombin is cleaved to thrombin by factor Xa; factor Va and Ca2⫹ act as cofactors in this reaction, and the reaction takes place on an activated (phosphatidylserine-expressing) phospholipid surface (PL). Thrombin converts the soluble plasma protein fibrinogen to fibrin, which spontaneously polymerizes. Thrombin also activates factor XIII, a transglutaminase that cross-links the fibrin polymers into a highly stable meshwork or clot. Thrombin also activates cofactors V and VIII, as well as coagulation factors VII and XI. In addition, thrombin activates both platelets and endothelial cells. Finally, thrombin stimulates the release of several antithrombotic factors—including PGI2, NO, and t-PA—from resting (intact) endothelial cells near the site of vascular injury; these factors limit primary and secondary hemostasis to the injured site (not shown).

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 379

fibroblasts, and smooth muscle cells) and microparticle tissue factor are important for the formation of a stable clot.

After vascular injury, the endothelium surrounding the injured area participates in five separate mechanisms that limit the initiation and propagation of the hemostatic process to the immediate vicinity of the injury. These mechanisms involve prostacyclin (PGI2), antithrombin III, proteins C and S, tissue factor pathway inhibitor (TFPI), and tissue-type plasminogen activator (t-PA). Prostacyclin (PGI2) is an eicosanoid (i.e., a metabolite of arachidonic acid) that is synthesized and secreted by the endothelium. By acting through Gs protein-coupled plateletsurface PGI2 receptors, this metabolite increases cAMP levels within platelets and thereby inhibits platelet aggregation and platelet granule release. PGI2 also has potent vasodilatory effects; this mediator induces vascular smooth muscle relaxation by increasing cAMP levels within the vascular smooth muscle cells. (Note that these mechanisms are physiologically antagonistic to those of TxA2, which induces platelet activation and vasoconstriction by decreasing intracellular cAMP levels.) Therefore, PGI2 both prevents platelets from adhering to the intact endothelium that surrounds the site of vascular injury and maintains vascular patency around the site of injury. Antithrombin III inactivates thrombin and other coagulation factors (IXa, Xa, XIa, and XIIa, where “a” denotes an “activated” factor) by forming a stoichiometric complex with the coagulation factor (Fig. 22-9). These interactions are

Regulation of Hemostasis Hemostasis is exquisitely regulated for two major reasons. First, hemostasis must be restricted to the local site of vascular injury. That is, activation of platelets and coagulation factors in the plasma should occur only at the site of endothelial damage, tissue factor expression, and procoagulant phospholipid exposure. Second, the size of the primary and secondary hemostatic plugs must be restricted so that the vascular lumen remains patent. After vascular injury, intact endothelium in the immediate vicinity of the injury becomes “activated.” This activated endothelium presents a set of procoagulant factors that promote hemostasis at the site of injury, and anticoagulant factors that restrict propagation of the clot beyond the site of injury. The procoagulant factors, such as tissue factor and phosphatidylserine, tend to be membrane-bound and localized to the site of injury—these factors provide a surface on which the coagulation cascade can proceed. In contrast, the anticoagulant factors are generally secreted by the endothelium and are soluble in the blood. Thus, the activated endothelium maintains a balance of procoagulant and anticoagulant factors to limit hemostasis to the site of vascular injury. A ATIII

+

Heparin

Endogenous heparin-like molecules or exogenous unfractionated heparin

Antithrombin III

ATIII Heparin

B Active coagulation factors

Inactive coagulation factors

Thrombin

Thrombin

Thrombin

ATIII

ATIII Heparin

Xa

ATIII

+

Xa

ATIII

Xa

ATIII

+

Heparin

IXa Heparin

XIa

XIIa

FIGURE 22-9.

Heparin

IXa XIa XIIa

ATIII

IXa XIa XIIa

ATIII

Heparin

Antithrombin III action. Antithrombin III (ATIII) inactivates thrombin and factors IXa, Xa, XIa, and XIIa by forming a stoichiometric complex with these coagulation factors. These reactions are catalyzed physiologically by heparin-like molecules expressed on healthy endothelial cells; sites of vascular injury do not express heparin-like molecules because the endothelium is denuded or damaged. Pharmacologically, these reactions are catalyzed by exogenously administered heparin. In more detail, the binding of heparin to ATIII induces a conformational change in ATIII (A) that allows the ATIII to bind thrombin or coagulation factors IXa, Xa, XIa, or XIIa. The stoichiometric complex between ATIII and the coagulation factor is highly stable, allowing heparin to dissociate without breaking up the complex (B).

380 Principles of Cardiovascular Pharmacology Tissue-type or urokinase-type plasminogen activator (inactive)

Tissue-type or urokinase-type plasminogen activator

Plasminogen activator inhibitor 1 or 2

Plasminogen activator inhibitor 1 or 2 Inactivated plasmin

Plasminogen Plasmin

fibrin polymer

+

Fibrin degradation products

FIGURE 22-10. The fibrinolytic system. Plasmin is formed by the proteolytic cleavage of plasminogen by tissue-type or urokinase-type plasminogen activator. Plasmin formation can be inhibited by plasminogen activator inhibitor 1 or 2, which binds to and inactivates plasminogen activators. In the fibrinolytic reaction, plasmin cleaves cross-linked fibrin polymers into fibrin degradation products. ␣2-Antiplasmin, which circulates in the bloodstream, neutralizes free plasmin in the circulation. enhanced by a heparin-like molecule that is expressed at the surface of intact endothelial cells, ensuring that this mechanism is operative at all locations in the vascular tree except where endothelium is denuded at the site of vascular injury. (These endothelial cell surface proteoglycans are referred to as “heparin-like” because they are the physiologic equivalent of the pharmacologic agent heparin, discussed below.) Heparin-like molecules on the endothelial cells bind to and activate antithrombin III, which is then primed to complex with (and thereby inactivate) the activated coagulation factors. Protein C and protein S are vitamin K-dependent proteins that slow the coagulation cascade by inactivating coagulation factors Va and VIIIa. Protein C and protein S are part of a feedback control mechanism, in which excess thrombin generation leads to activation of protein C, which, in turn, helps to prevent the enlarging fibrin clot from occluding the vascular lumen. Specifically, the endothelial cell-surface protein thrombomodulin is a receptor for both thrombin and protein C in the blood. Thrombomodulin binds these proteins in such a way that thrombomodulin-bound thrombin cleaves protein C to activated protein C (also known as protein Ca). In a reaction that requires the cofactor protein S, activated protein C then inhibits clotting by cleaving (and thereby inactivating) factors Va and VIIIa. Tissue factor pathway inhibitor (TFPI), as its name indicates, limits the action of tissue factor (TF). The coagulation cascade is initiated when factor VIIa complexes with TF at the site of vascular injury (Fig. 22-6). The resulting VIIa:TF complex catalyzes the activation of factors IX and X. After limited quantities of factors IXa and Xa are generated, the VIIa:TF complex is feedback inhibited by TFPI in a two-step reaction.

First, TFPI binds to factor Xa and neutralizes its activity in a Ca2⫹-independent reaction. Subsequently, the TFPI:Xa complex interacts with the VIIa:TF complex via a second domain on TFPI, so that a quaternary Xa:TFPI:VIIa:TF complex is formed. The molecular “knots” of the TFPI molecule hold the quaternary complex tightly together and thereby inactivate the VIIa:TF complex. In this manner, TFPI prevents excessive TF-mediated activation of factors IX and X. Plasmin exerts its anticoagulant effect by proteolytically cleaving fibrin into fibrin degradation products. Because plasmin has powerful antithrombotic effects, the formation of plasmin has intrigued researchers for many years, and a number of pharmacologic agents have been developed to target the plasmin formation pathway (Fig. 22-10). Plasmin is generated by the proteolytic cleavage of plasminogen, a plasma protein that is synthesized in the liver. The proteolytic cleavage is catalyzed by tissue plasminogen activator (t-PA), which is synthesized and secreted by the endothelium. Plasmin activity is carefully modulated by three regulatory mechanisms in order to restrict plasmin action to the site of clot formation. First, t-PA is most effective when it is bound to a fibrin meshwork. Second, t-PA activity can be inhibited by plasminogen activator inhibitor (PAI). When local concentrations of thrombin and inflammatory cytokines (such as IL-1 and TNF-␣) are high, endothelial cells increase the release of PAI, preventing t-PA from activating plasmin. This ensures that a stable fibrin clot forms at the site of vascular injury. Third, ␣2-antiplasmin is a plasma protein that neutralizes free plasmin in the circulation and thereby prevents random degradation of plasma fibrinogen. Plasma fibrinogen is important for platelet aggregation in primary hemostasis (see above), and it is also the precursor for the fibrin polymer that is required to form a stable clot.

 PATHOGENESIS OF THROMBOSIS Thrombosis is the pathologic extension of hemostasis. In thrombosis, coagulation reactions are inappropriately regulated so that a clot uncontrollably enlarges and occludes the lumen of a blood vessel. The pathologic clot is now termed a thrombus. Three major factors predispose to thrombus formation—endothelial injury, abnormal blood flow, and hypercoagulability. These three factors influence one another and are collectively known as Virchow’s triad (Fig. 22-11).

Endothelial injury

Thrombosis

Abnormal blood flow

Hypercoagulability

FIGURE 22-11. Virchow’s triad. Endothelial injury, abnormal blood flow, and hypercoagulability are three factors that predispose to thrombus formation. These three factors are interrelated; endothelial injury predisposes to abnormal blood flow and hypercoagulability, while abnormal blood flow can cause both endothelial injury and hypercoagulability.

384 Principles of Cardiovascular Pharmacology Membrane phospholipids

Phospholipase A2

NSAIDs (aspirin, others)

COOH

Arachidonic acid

Cyclooxygenase

O

Lipoxygenase

OOH

COOH

COOH

O OOH 5-HPETE

Prostaglandin G2 Peroxidase

Dehydrase

O

O

COOH

COOH

O OH

Leukotriene A 4

Prostaglandin H2

COOH PGI2 synthase

GlutathioneS-transferase Prostaglandin synthases

OH

O

COOH

H

H Other prostaglandins

OH

S H N

OH

COOH

HN

Prostacyclin (PGI2)

PGE2 synthase

H2N

TxA2 synthase

O O COOH

O

Leukotriene C 4

COOH

COOH O O OH Thromboxane A 2

HO

OH Prostaglandin E2

FIGURE 22-12. Overview of prostaglandin synthesis. Membrane phospholipids are cleaved by phospholipase A2 to release free arachidonic acid. Arachidonic acid can be metabolized through either of two major pathways, the cyclooxygenase pathway or the lipoxygenase pathway. The cyclooxygenase pathway, which is inhibited by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), converts arachidonic acid into prostaglandins and thromboxanes. Platelets express TxA2 synthase and synthesize the pro-aggregatory mediator thromboxane A2; endothelial cells express PGI2 synthase and synthesize the anti-aggregatory mediator prostacyclin. The lipoxygenase pathway converts arachidonic acid into leukotrienes, which are potent inflammatory mediators. (See Chapter 42, Pharmacology of Eicosanoids, for a detailed discussion of the lipoxygenase and cyclooxygenase pathways.) Aspirin inhibits cyclooxygenase by covalent acetylation of the enzyme near its active site. Because platelets lack the capability to synthesize new proteins, aspirin inhibits thromboxane synthesis for the life of the platelet.

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 385

A Arachidonic acid NSAIDs (aspirin, others)

Cyclooxygenase

TXA2 (released by activated platelets) PLC

PIP2

TXA2-R

DAG

αq

β

αq γ

PKC (active)

GTP

GDP PKC IP3 Ca2+

Ca2+ PLA2

GP IIb -III a

Abciximab, eptifibatide, tirofiban

Fibrinogen

Thrombin

B ADP

Adenylyl cyclase

Clopidogrel, ticlopidine, prasugrel P2Y(ADP) receptor

αi

β

GDP

β γ αq

αi γ

GTP

GDP

ATP

ADP

Thrombin receptor

PLC

P2Y1 receptor

αq GTP

cAMP

Dipyridamole

β αq γ GDP

PDE

PKA AMP

Platelet activation

Platelet activation

FIGURE 22-13.

Mechanism of action of antiplatelet agents. A. NSAIDs and GPIIb–IIIa antagonists inhibit steps in thromboxane A2 (TxA2)-mediated platelet activation. Aspirin inhibits cyclooxygenase by covalent acetylation of the enzyme near its active site, leading to decreased TxA2 production. The effect is profound because platelets lack the ability to synthesize new enzyme molecules. GPIIb–IIIa antagonists, such as the monoclonal antibody abciximab and the small-molecule antagonists eptifibatide and tirofiban, inhibit platelet aggregation by preventing activation of GpIIb–IIIa (dashed line), leading to decreased platelet cross-linking by fibrinogen. B. Clopidogrel, ticlopidine, prasugrel, and dipyridamole inhibit steps in ADP-mediated platelet activation. Clopidogrel, ticlopidine, and prasugrel are antagonists of the P2Y(ADP) receptor. Dipyridamole inhibits phosphodiesterase (PDE), thereby preventing the breakdown of cAMP and increasing cytoplasmic cAMP concentration.

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 387

Anticoagulants As with antiplatelet agents, anticoagulants are used both to prevent and treat thrombotic disease. There are four classes of anticoagulants: warfarin, unfractionated and low-molecularweight heparins, selective factor Xa inhibitors, and direct thrombin inhibitors. Anticoagulants target various factors in the coagulation cascade, thereby interrupting the cascade and preventing the formation of a stable fibrin meshwork (secondary hemostatic plug). In this section, the four classes of anticoagulants are discussed in order of selectivity, from the least selective agents (warfarin and unfractionated heparin) to the most selective agents (selective factor Xa inhibitors and direct thrombin inhibitors). Recombinant activated protein C also has anticoagulant activity, although its clinical indication is severe sepsis. Because of the mechanisms of action of these drugs, bleeding is an adverse effect common to all anticoagulants. Warfarin In the early 1900s, farmers in Canada and the North Dakota plains adopted the practice of planting sweet clover instead of corn for fodder. In the winter months of 1921 to 1922, a fatal hemorrhagic disease was reported in cattle that had foraged on the sweet clover. In almost every case, it was found that the affected cattle had foraged on sweet clover that had been spoiled by the curing process. After an intensive investigation, scientist K. P. Link reported that the spoiled clover contained the natural anticoagulant 3,3⬘-methylenebis-(4-hydroxycoumarin) or “dicumarol.” Dicumarol and warfarin (a potent synthetic congener) were introduced during the 1940s as rodenticides and as oral anticoagulants. Because the oral anticoagulants act by affecting vitamin Kdependent reactions, it is important to understand how vitamin K functions. Mechanism of Action of Vitamin K

Vitamin K (“K” is derived from the German word “Koagulation”) is required for the normal hepatic synthesis of four coagulation factors (II, VII, IX, and X), protein C, and protein S. The coagulation factors, protein C, and protein S are biologically inactive as unmodified polypeptides following protein synthesis on ribosomes. These proteins gain biological activity by post-translational carboxylation of their 9 to 12 amino-terminal glutamic acid residues. The ␥-carboxylated glutamate residues (but not the unmodified glutamate residues) are capable of binding Ca2⫹ ions. Ca2⫹ binding induces a conformational change in these proteins that is required for efficient binding of the proteins to phospholipid surfaces. The binding of Ca2⫹ to the ␥-carboxylated molecules increases the enzymatic activity of coagulation factors IIa, VIIa, IXa, Xa, and protein Ca by approximately 1,000-fold. Thus, vitamin K-dependent carboxylation is crucial for the enzymatic activity of the four coagulation factors and protein C, and for the cofactor function of protein S. The carboxylation reaction requires (1) a precursor form of the target protein with its 9 to 12 amino-terminal glutamic acid residues, (2) carbon dioxide, (3) molecular oxygen, and (4) reduced vitamin K. The carboxylation reaction is schematically presented in Figure 22-14. During this reaction, vitamin K is oxidized to the inactive 2,3-epoxide. An enzyme, vitamin K epoxide reductase (also called VKORC1), is then required to convert the inactive 2,3-epoxide into the

active, reduced form of vitamin K. Thus, the regeneration of reduced vitamin K is essential for the sustained synthesis of biologically functional clotting factors II, VII, IX, and X, all of which are critical components of the coagulation cascade. Mechanism of Action of Warfarin

Warfarin acts on the carboxylation pathway, not by inhibiting the carboxylase directly, but by blocking the epoxide reductase that mediates the regeneration of reduced vitamin K (Fig. 22-14). Because depletion of reduced vitamin K in the liver prevents the ␥-carboxylation reaction that is required for the synthesis of biologically active coagulation factors, the onset of action of the oral anticoagulants parallels the half-life of these coagulation factors in the circulation. Of the four affected clotting factors (II, VII, IX, and X), factor VII has the shortest half-life (6 hours). Thus, the pharmacologic effect of a single dose of warfarin is not manifested for approximately 18–24 hours (i.e., for 3–4 factor VII half-lives). This delayed action is one pharmacologic property that distinguishes the warfarin class of anticoagulants from all the other classes of anticoagulants. Evidence from studies of long-term rodenticide use and of anticoagulant use supports the hypothesis that the epoxide reductase is the molecular target of oral anticoagulant action. The use of oral anticoagulants as rodenticides has been a widespread practice in farming communities. In some areas of the United States, heavy rodenticide use has selected for a population of wild rodents that is resistant to 4-hydroxycoumarins. In vitro studies of tissues from these rodents have demonstrated a mutation in the rodent epoxide reductase that renders the enzyme resistant to inhibition by the anticoagulant. Similarly, a small population of patients is genetically resistant to warfarin because of mutations in their epoxide reductase gene. These patients require 10–20 times the usual dose of warfarin to achieve the desired anticoagulant effect. More generally, genetic variation in the VKORC1 gene has recently been associated with approximately 25–30% of the variance in warfarin maintenance dose in patients taking this drug (see Chapter 6, Pharmacogenomics). Clinical Uses of Warfarin

Warfarin is often administered to complete a course of anticoagulation that has been initiated with heparin (see below) and to prevent thrombosis in predisposed patients. Orally administered warfarin is nearly 100% bioavailable, and its levels in the blood peak at 0.5–4 hours after administration. In the plasma, 99% of racemic warfarin is bound to plasma protein (albumin). Warfarin has a relatively long elimination half-life (approximately 36 hours). The drug is hydroxylated by the cytochrome P450 system in the liver to inactive metabolites that are subsequently eliminated in the urine (see Fig. 6-4). Drug–drug interactions must be carefully considered in patients taking warfarin. Because warfarin is highly albumin-bound in the plasma, co-administration of warfarin with other albumin-bound drugs can increase the free (unbound) plasma concentrations of both drugs. In addition, because warfarin is metabolized by P450 enzymes in the liver, coadministration of warfarin with drugs that induce and/or compete for P450 metabolism can affect the plasma concentrations of both drugs. Tables 22-2 and 22-3 list some of the major interactions between warfarin and other drugs.

388 Principles of Cardiovascular Pharmacology

FIGURE 22-14.

O H N

O

N H COOH

H N N H COOH

COOH

γ-Carboxyglutamate residue in

Glutamate residue in coagulation factor

coagulation factor

CO2 Vitamin K-dependent carboxylase

Mechanism of action of warfarin. Vitamin K is a necessary cofactor in the post-translational carboxylation of glutamate residues on factors II, VII, IX, and X. During the carboxylation reaction, vitamin K is oxidized to the inactive 2,3epoxide. The enzyme vitamin K epoxide reductase (also called VKORC1) converts the inactive vitamin K 2,3-epoxide into the active, reduced form of vitamin K. The regeneration of reduced vitamin K is essential for the sustained synthesis of biologically functional coagulation factors II, VII, IX, and X. Warfarin acts on the carboxylation pathway by inhibiting the epoxide reductase that is required for the regeneration of reduced (active) vitamin K. Dicumarol is the natural anticoagulant formed in spoiled clover. Both warfarin and dicumarol are orally bioavailable and are often termed “oral anticoagulants.”

O2

O

OH R

R O

OH

O

Vitamin K-reduced (active form)

Vitamin K 2,3-epoxide (inactive form)

Epoxide reductase

NAD+

NADH Oral anticoagulants

O OH

O

OO Dicumarol

OH

OH

O

O

O

Warfarin

Among the adverse effects of warfarin, bleeding is the most serious and predictable toxicity. Withdrawal of the drug may be recommended for patients who suffer from repeated bleeding episodes at otherwise therapeutic drug concentrations. For severe hemorrhage, patients should promptly receive fresh frozen plasma, which contains biologically functional clotting factors II, VII, IX, and X. Warfarin should never be administered to pregnant women because it can cross the placenta and cause a hemorrhagic disorder in the fetus. In addition, newborns exposed to warfarin in utero may have serious congenital defects characterized by abnormal bone formation (note that certain bone matrix proteins are ␥-carboxylated). Rarely, warfarin causes skin necrosis as a result of widespread thrombosis in the microvasculature. The fact that warfarin can cause thrombosis may seem

paradoxical. Recall that, in addition to inhibiting the synthesis of biologically active coagulation factors II, VII, IX, and X, warfarin also prevents the synthesis of biologically active proteins C and S, which are natural anticoagulants. In patients who are genetically deficient in protein C or protein S (most commonly, patients who are heterozygous for protein C deficiency), an imbalance between warfarin’s effects on coagulation factors and its effects on proteins C and S may lead to microvascular thrombosis and skin necrosis. Because warfarin has a narrow therapeutic index and participates in numerous drug–drug interactions, the pharmacodynamic (functional) effect of chronic warfarin therapy must be monitored regularly (on the order of every 2–4 weeks). Monitoring is most easily performed using the prothrombin time (PT), which is a simple test of the

390 Principles of Cardiovascular Pharmacology Anticoagulant class

Effect on Thrombin

Effect on Factor Xa

Unfractionated heparin (about 45 saccharide units, MW ~ 13,500)

Xa

Thrombin

ATIII

ATIII Heparin

Heparin

Binds to antithrombin III (ATIII) and thrombin (inactivates thrombin)

Binds to antithrombin III (ATIII) via pentasaccharide (sufficient to inactivate Xa)

Low molecular weight (LMW) heparins Xa

Thrombin

(about 15 saccharide units, MW ~ 4,500)

ATIII

ATIII

LMWH

Binds to antithrombin III (ATIII) but not to thrombin (poorly inactivates thrombin)

LMWH

Binds to antithrombin III (ATIII) via pentasaccharide (sufficient to inactivate Xa)

Xa

Selective factor Xa inhibitors

ATIII

No effect on thrombin

Fondaparinux Binds to antithrombin III (ATIII) via pentasaccharide (sufficient to inactivate Xa) Substrate recognition site (Exosite) Thrombin

Direct thrombin inhibitors

Heparinbinding site

Catalytic site (Active site) No effect on Xa Argatroban

Thrombin

Lepirudin Thrombin

Selectively inactivate thrombin

FIGURE 22-15.

Differential effects of unfractionated heparin, low-molecular-weight heparins, selective factor Xa inhibitors, and direct thrombin inhibitors on coagulation factor inactivation. Effect on thrombin: To catalyze the inactivation of thrombin, heparin must bind both to antithrombin III via a high-affinity pentasaccharide unit and to thrombin via an additional 13-saccharide unit. Low-molecular-weight heparin (LMWH) does not contain a sufficient number of saccharide units to bind thrombin, and therefore is a poor catalyst for thrombin inactivation. Selective factor Xa inhibitors do not inactivate thrombin, while direct thrombin inhibitors selectively inactivate thrombin. Argatroban and dabigatran (not shown ) bind only to the active (catalytic) site of thrombin, while lepirudin and bivalirudin (not shown) bind to both the active site and the substrate-recognition site of thrombin. Effect on factor Xa: Inactivation of factor Xa requires only the binding of antithrombin III to the high-affinity pentasaccharide unit. Since unfractionated heparin, low-molecular-weight heparin, and fondaparinux all contain this pentasaccharide, these agents are all able to catalyze the inactivation of factor Xa. Direct thrombin inhibitors have no effect on factor Xa.

392 Principles of Cardiovascular Pharmacology

Thus, direct thrombin inhibitors would be expected to have profound effects on coagulation. The currently approved direct thrombin inhibitors include lepirudin, desirudin, bivalirudin, argatroban, and dabigatran. These agents are specific inhibitors of thrombin, with negligible anti-factor Xa activity (Fig. 22-15). Lepirudin, a recombinant 65-amino-acid polypeptide derived from the medicinal leech protein hirudin, is the prototypical direct thrombin inhibitor. For years, surgeons have used medicinal leeches to prevent thrombosis in the fine vessels of reattached digits. Lepirudin binds with high affinity to two sites on the thrombin molecule—the enzymatic active site and the “exosite,” a region of the thrombin protein that orients substrate proteins. Lepirudin binding to thrombin prevents the thrombin-mediated activation of fibrinogen and factor XIII. Lepirudin is a highly effective anticoagulant because it can inhibit both free and fibrin-bound thrombin in developing clots and because lepirudin binding to thrombin is essentially irreversible. It is approved for use in the treatment of heparin-induced thrombocytopenia. Lepirudin has a short half-life, is available parenterally, and is renally excreted. It can be administered with relative safety to patients with hepatic insufficiency. As with all direct thrombin inhibitors, bleeding is the major adverse effect of lepirudin, and clotting times must be monitored closely. A small percentage of patients may develop anti-hirudin antibodies, limiting the long-term effectiveness of this agent as an anticoagulant. Another recombinant formulation of hirudin, desirudin, has been approved for prophylaxis against deep vein thrombosis in patients undergoing hip replacement. Bivalirudin is a synthetic 20-amino-acid peptide that, like lepirudin and desirudin, binds to both the active site and exosite of thrombin and thereby inhibits thrombin activity. Thrombin slowly cleaves an arginine–proline bond in bivalirudin, leading to reactivation of the thrombin. Bivalirudin is approved for anticoagulation in patients undergoing coronary angiography and angioplasty and may reduce rates of bleeding relative to heparin for this indication. The drug is excreted renally and has a short half-life (25 minutes). Argatroban is a small-molecule inhibitor of thrombin that is approved for the treatment of patients with heparin-induced thrombocytopenia. Unlike other direct thrombin inhibitors, argatroban binds only to the active site of thrombin (i.e., it does not interact with the exosite). Also unlike other direct thrombin inhibitors, argatroban is excreted by biliary secretion and can therefore be administered with relative safety to patients with renal insufficiency. Argatroban has a short halflife and is administered by continuous intravenous infusion. Dabigatran is an orally available direct thrombin inhibitor that has recently been approved for prevention of thromboembolism in patients with non-valvular atrial fibrillation (i.e., atrial fibrillation not related to mitral stenosis or a prosthetic heart valve). Dabigatran is a prodrug that is metabolized to an active species that, like argatroban, binds competitively to the active site of thrombin. Like other anticoagulants, dabigatran may cause significant bleeding. One advantage relative to warfarin is that plasma levels of dabigatran do not need to be monitored. Recombinant Activated Protein C (r-APC) As described above, endogenously activated protein C (APC) exerts an anticoagulant effect by proteolytically cleaving factors Va and VIIIa. APC also reduces the amount of circulating

plasminogen activator inhibitor 1, thereby enhancing fibrinolysis. Finally, APC reduces inflammation by inhibiting the release of tumor necrosis factor ␣ (TNF-␣) by monocytes. Because enhanced coagulability and inflammation are both hallmarks of septic shock, APC has been tested both in animal models of this disorder and in humans. Recombinant activated protein C (r-APC) has been found to significantly reduce mortality in patients at high risk of death from septic shock, and the U.S. Food and Drug Administration (FDA) has approved r-APC for the treatment of patients with severe sepsis who demonstrate evidence of acute organ dysfunction, shock, oliguria, acidosis, and hypoxemia. r-APC is not indicated for the treatment of patients with severe sepsis and a lower risk of death, however. As is the case with other anticoagulants, r-APC increases the risk of bleeding. This agent is therefore contraindicated in patients who have recently undergone a surgical procedure and in those with chronic liver failure, kidney failure, or thrombocytopenia.

Thrombolytic Agents Although warfarin, unfractionated and low-molecular-weight heparins, selective factor Xa inhibitors, and direct thrombin inhibitors are effective in preventing the formation and propagation of thrombi, these agents are generally ineffective against preexisting clots. Thrombolytic agents are used to lyse already-formed clots, and thereby to restore the patency of an obstructed vessel before distal tissue necrosis occurs. Thrombolytic agents act by converting the inactive zymogen plasminogen to the active protease plasmin (Fig. 22-10). As noted above, plasmin is a relatively nonspecific protease that digests fibrin to fibrin degradation products. Unfortunately, thrombolytic therapy has the potential to dissolve not only pathologic thrombi, but also physiologically appropriate fibrin clots that have formed in response to vascular injury (systemic fibrinolysis). Thus, the use of thrombolytic agents can lead to hemorrhage of varying severity. Streptokinase Streptokinase is a protein produced by ␤-hemolytic streptococci as a component of that organism’s tissue-destroying machinery. The pharmacologic action of streptokinase involves two steps—complexation and cleavage. In the complexation reaction, streptokinase forms a stable, noncovalent 1:1 complex with plasminogen. The complexation reaction produces a conformational change in plasminogen that exposes this protein’s proteolytically active site. Streptokinasecomplexed plasminogen, with its active site exposed and available, can then proteolytically cleave other plasminogen molecules to plasmin. In fact, the thermodynamically stable streptokinase:plasminogen complex is the most catalytically efficient plasminogen activator in vitro. Although streptokinase exerts its most dramatic and potentially beneficial effects in fresh thrombi, its use has been limited by two factors. First, streptokinase is a foreign protein that is capable of eliciting antigenic responses in humans upon repeated administration. Previous administration of streptokinase is a contraindication to its use, because of the risk of anaphylaxis. Second, the thrombolytic actions of streptokinase are relatively nonspecific and can result in systemic fibrinolysis. Currently, streptokinase is approved for treatment of ST elevation myocardial infarction and for treatment of life-threatening pulmonary embolism.

CHAPTER 22 / Pharmacology of Hemostasis and Thrombosis 393

Recombinant Tissue Plasminogen Activator (t-PA) An ideal thrombolytic agent would be nonantigenic and would cause local fibrinolysis only at the site of a pathologic thrombus. Tissue plasminogen activator (t-PA) approximates these goals. t-PA is a serine protease produced by human endothelial cells; therefore, t-PA is not antigenic. t-PA binds to newly formed (fresh) thrombi with high affinity, causing fibrinolysis at the site of a thrombus. Once bound to the fresh thrombus, t-PA undergoes a conformational change that renders it a potent activator of plasminogen. In contrast, t-PA is a poor activator of plasminogen in the absence of fibrin-binding. Recombinant DNA technology has allowed the production of recombinant t-PA, generically referred to as alteplase. Recombinant t-PA is effective at recanalizing occluded coronary arteries, limiting cardiac dysfunction, and reducing mortality following an ST elevation myocardial infarction. At pharmacologic doses, however, recombinant t-PA can generate a systemic lytic state and (as with other thrombolytic agents) cause unwanted bleeding, including cerebral hemorrhage. Thus, its use is contraindicated in patients who have had a recent hemorrhagic stroke. Like streptokinase, t-PA is approved for use in the treatment of patients with ST elevation myocardial infarction or life-threatening pulmonary embolism. It is also approved for the treatment of acute ischemic stroke. Tenecteplase Tenecteplase is a genetically engineered variant of t-PA. The molecular modifications in tenecteplase increase its fibrin specificity relative to t-PA and make tenecteplase more resistant to plasminogen activator inhibitor 1. Large trials have shown that tenecteplase is identical in efficacy to t-PA, with similar (and possibly decreased) risk of bleeding. Additionally, tenecteplase has a longer half-life than t-PA. This pharmacokinetic property allows tenecteplase to be administered as a single weight-based bolus, thus simplifying administration. Reteplase Similar to tenecteplase, reteplase is a genetically engineered variant of t-PA with longer half-life and increased specificity for fibrin. Its efficacy and adverse-effect profile are similar to those of streptokinase and t-PA. Because of its longer half-life, reteplase can be administered as a “double bolus” (two boluses, 30 minutes apart).

Inhibitors of Anticoagulation and Fibrinolysis Protamine Protamine, a low-molecular-weight polycationic protein, is a chemical antagonist of heparin. This agent rapidly forms a stable complex with the negatively charged heparin molecule through multiple electrostatic interactions. Protamine is administered intravenously to reverse the effects of heparin in situations of life-threatening hemorrhage or great heparin excess (for example, at the conclusion of coronary artery bypass graft surgery). Protamine is most active against the large heparin molecules in unfractionated heparin and it can partially reverse the anticoagulant effects of low-molecular-weight heparins, but it is inactive against fondaparinux. Serine-Protease Inhibitors Aprotinin, a naturally occurring polypeptide, is an inhibitor of the serine proteases plasmin, t-PA, and thrombin.

By inhibiting fibrinolysis, aprotinin promotes clot stabilization. Inhibition of thrombin may also promote platelet activity by preventing platelet hyperstimulation. At higher doses, aprotinin may also inhibit kallikrein and thereby (paradoxically) inhibit the coagulation cascade. Clinical trials have demonstrated decreased perioperative bleeding and erythrocyte transfusion requirement in patients treated with aprotinin during cardiac surgery. However, these positive findings have been tempered by recent evidence suggesting that, compared to other antifibrinolytic agents, aprotinin may increase the risk of postoperative acute renal failure. Lysine Analogues Aminocaproic acid and tranexamic acid are analogues of lysine that bind to and inhibit plasminogen and plasmin. Like aprotinin, these agents are used to reduce perioperative bleeding during coronary artery bypass grafting. Unlike aprotinin, these agents may not increase the risk of postoperative acute renal failure.

 CONCLUSION AND FUTURE DIRECTIONS Hemostasis is a highly regulated process that maintains the fluidity of blood in normal vessels and initiates rapid formation of a stable fibrin-based clot in response to vascular injury. Pathologic thrombosis results from endothelial injury, abnormal blood flow, and hypercoagulability. Antiplatelet agents, anticoagulants, and thrombolytic agents target different stages of thrombosis and thrombolysis. Antiplatelet agents interfere with platelet adhesion, the platelet release reaction, and platelet aggregation; these agents can provide powerful prophylaxis against thrombosis in susceptible individuals. Anticoagulants primarily target plasma coagulation factors and disrupt the coagulation cascade by inhibiting crucial intermediates. After a fibrin clot has been established, thrombolytic agents mediate dissolution of the clot by promoting the conversion of plasminogen to plasmin. These classes of pharmacologic agents can be administered either individually or in combination, to prevent or disrupt thrombosis and to restore the patency of blood vessels occluded by thrombus. Future development of new antiplatelet, anticoagulant, and thrombolytic agents will be forced to contend with two major constraints. First, for many clinical indications in this field, highly effective, orally bioavailable, and inexpensive therapeutic agents are already available: these include the antiplatelet drug aspirin and the anticoagulant warfarin. Second, virtually every antithrombotic and thrombolytic agent is associated with the mechanism-based toxicity of bleeding, and this adverse effect is likely to plague new agents under development. Nonetheless, opportunities remain for the development of safer and more effective therapies. It is likely that pharmacogenomic techniques (see Chapter 6) will be capable of identifying individuals in the population who carry an elevated genetic risk of thrombosis, and such individuals may benefit from long-term antithrombotic treatment. Combinations of antiplatelet agents, low-molecular-weight heparins, orally bioavailable direct thrombin inhibitors, and new agents that target currently unexploited components of hemostasis (such as inhibitors of the factor VIIa/tissue factor pathway) could all be useful in these settings. At the other end of the spectrum, there

394 Principles of Cardiovascular Pharmacology

remains a great need for new agents that can achieve rapid, noninvasive, convenient, and selective lysis of acute thromboses associated with life-threatening emergencies such as ST elevation myocardial infarction and stroke. Carefully designed clinical trials will be critical to optimize the indications, dose, and duration of treatment for such drugs and drug combinations.

Suggested Reading Angiolillo DJ, Bhatt DL, Gurbel PA, Jennings LK. Advances in antiplatelet therapy: agents in clinical development. Am J Cardiol 2009; 103(3 Suppl):40A–51A. (Reviews new antiplatelet agents, including prasugrel, cangrelor, ticagrelor, and SCH530348.) Bates SM, Ginsberg JS. Treatment of deep-vein thrombosis. N Engl J Med 2004;351:268–277. (Reviews treatment options for deep vein thrombosis.)

Brass LF. The molecular basis for platelet activation. In: Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology: basic principles and practice. 5th ed. Philadelphia: Churchill Livingstone; 2008. (Detailed and mechanistic description of platelet activation.) Di Nisio M, Middeldorp S, Buller HR. Direct thrombin inhibitors. N Engl J Med 2005;353:1028–1040. (Reviews mechanism of action and clinical indications for direct thrombin inhibitors.) Franchini M, Veneri D, Salvagno GL, et al. Inherited thrombophilia. Crit Rev Clin Lab Sci 2006;43:249–290. (Reviews epidemiology, pathophysiology, and treatment of hypercoagulable states.) Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med 2008;359:938–949. (Reviews mechanisms of hemostasis and thrombosis.) Grosser T, Fries S, FitzGerald GA. Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities. J Clin Invest 2006;116:4–15. (Reviews effects of COX-2 inhibition in cellular, animal, and human studies.) Levy JH. Hemostatic agents. Transfusion 2004;44:58S–62S. (Reviews aprotinin, aminocaproic acid, and tranexamic acid.)

23 Pharmacology of Cardiac Rhythm Ehrin J. Armstrong, April W. Armstrong, and David E. Clapham

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 401-402 ELECTRICAL PHYSIOLOGY OF THE HEART . . . . . . . . . . . . . 401 Pacemaker and Nonpacemaker Cells . . . . . . . . . . . . . . . . 401 Cardiac Action Potentials . . . . . . . . . . . . . . . . . . . . . . . . . 402 Determination of Firing Rate . . . . . . . . . . . . . . . . . . . . . . 406 PATHOPHYSIOLOGY OF ELECTRICAL DYSFUNCTION . . . . . . 406 Defects in Impulse Formation (SA Node) . . . . . . . . . . . . . 406 Altered Automaticity . . . . . . . . . . . . . . . . . . . . . . . . . . 406 Triggered Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 Defects in Impulse Conduction . . . . . . . . . . . . . . . . . . . . . 407 Re-entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Conduction Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 Accessory Tract Pathways. . . . . . . . . . . . . . . . . . . . . . 408 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 408 General Mechanisms of Action of Antiarrhythmic Agents . . . . . . . . . . . . . . . . . . . . . . . . . 408

Classes of Antiarrhythmic Agents . . . . . . . . . . . . . . . . . . . 409 Class I Antiarrhythmic Agents: Fast Naⴙ Channel Blockers . . . . . . . . . . . . . . . . . . . . . 409 Class II Antiarrhythmic Agents: ␤-Adrenergic Antagonists . . . . . . . . . . . . . . . . . . . . . . 413 Class III Antiarrhythmic Agents: Inhibitors of Repolarization . . . . . . . . . . . . . . . . . . . . . 413 Class IV Antiarrhythmic Agents: Ca 2ⴙ Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . 415 Other Antiarrhythmic Agents . . . . . . . . . . . . . . . . . . . . 416 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 416 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

 INTRODUCTION

of cell depolarization and impulse conduction. Once initiated, a cardiac action potential is a spontaneous event that proceeds based on the characteristic responses of ion channels to changes in membrane voltage. At the completion of a cycle, the spontaneous depolarization of pacemaker cells ensures that the process repeats without interruption.

The human heart is both a mechanical and an electrical organ. To perfuse the body adequately with blood, the mechanical and electrical components of the heart must work in precise concert with each other. The mechanical component pumps the blood; the electrical component controls the rhythm of the pump. When the mechanical component fails despite a normal rhythm, heart failure can result (see Chapter 25, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure). When the electrical component goes awry (called an arrhythmia), cardiac myocytes fail to contract in synchrony, and effective pumping is compromised. Changes in the membrane potential of cardiac cells directly affect cardiac rhythm, and most antiarrhythmic drugs act by modulating the activity of ion channels in the plasma membrane. This chapter discusses the ionic basis of electric rhythm formation and conduction in the heart, the pathophysiology of electrical dysfunction, and the pharmacologic agents used to restore a normal cardiac rhythm.

 ELECTRICAL PHYSIOLOGY OF THE HEART Electrical activity in the heart, leading to rhythmic cardiac contraction, is a manifestation of the heart’s exquisite control

Pacemaker and Nonpacemaker Cells The heart contains two types of cardiac myocytes—those that can spontaneously initiate action potentials and those that cannot. Cells possessing the ability to initiate spontaneous action potentials are termed pacemaker cells. All pacemaker cells possess automaticity, the ability to depolarize above a threshold voltage in a rhythmic fashion. Automaticity results in the generation of spontaneous action potentials. Pacemaker cells are found in the sinoatrial node (SA node), the atrioventricular node (AV node), and the ventricular conducting system (bundle of His, bundle branches, and Purkinje fibers). Together, the pacemaker cells constitute the specialized conducting system that governs the electrical activity of the heart. The second type of cardiac cells, the nonpacemaker cells, includes the atrial and ventricular myocytes. The nonpacemaker cells contract in response to depolarization and are responsible for the majority of cardiac 401

CHAPTER 23 / Pharmacology of Cardiac Rhythm 403

160

ECa +150 mV

120 80

Em (mV)

Depolarize

A SA node cell

myocyte remains near EK until the cell is stimulated by a wave of depolarization that is initiated by nearby pacemaker cells. The five phases of the ventricular myocyte action potential result from an intricately woven cascade of channel openings and closings; the phases are numbered from 0 to 4 (Fig. 23-3 and Table 23-1).

ENa +70 mV

40 +10 0

Repolarize

Phases of SA Node Action Potential

-40

-55

-80 -120 0

100

200

300

400

500

Major Currents

Phase 4

If = Pacemaker current, relatively nonselective. IK1 = Inward rectifier, outward K+ current

Phase 0

ICa = Inward Ca2+ current

Phase 3

IK = Delayed rectifier, outward K+ current

EK -94 mV

Time (ms)

160

ECa +150 mV

120 80

K+

K+ Na+

ENa +70 mV

+45

40

Ca2+

A SA node action potential

0

Repolarize

-40 -80

-85

-120 0

100

200

300

400

500

EK -94 mV

Time (ms)

Membrane potential (mV)

Em (mV)

Depolarize

B Ventricular muscle cell

40 20 0 ICa

-40 -60

Phase 0

IK

Phase 4

If

IK1 If 150

0

Time (ms) B Ion currents of SA node action potential 4

Currents across membrane (μA/μF)

SA node action potential, the kinetics of this depolarization are modulated by voltage-gated Na channels that are also expressed in the node. There are gradients of expression of If channels and of the more selective voltage-gated Na and Ca2 channels within the SA node, such that cells at the border of the node express relatively more voltage-gated Na channels and cells in the center of the node express relatively more If and voltage-gated Ca2 channels. The expression of voltage-gated Na channels in the SA node is partly responsible for the effect of certain antiarrhythmics on the automaticity of SA nodal cells (see below). Unlike SA nodal cells, ventricular myocytes do not depolarize spontaneously under physiologic conditions. As a result, the membrane potential of the resting ventricular

Phase 3

1

FIGURE 23-1.

SA node and ventricular muscle cell action potentials. The resting membrane potential of a sinoatrial (SA) node cell is approximately 55 mV, while that of a ventricular muscle cell is 85 mV. The shaded areas represent the approximate depolarization required to trigger an action potential in each cell type. Together, the cardiac action potentials last for approximately half a second. SA node cells (A) depolarize to a peak of 10 mV, and ventricular muscle cells (B) depolarize to a peak of 45 mV. Note that the ventricular action potential has a much longer plateau phase. This long plateau ensures that ventricular myocytes have adequate time to contract before the onset of the next action potential. The Nernst equilibrium potentials of the major ions (ECa, ENa, EK) are shown as dashed horizontal lines. Em, membrane potential.

IK

-20

2

IK

IK1

IK1

0 -2

If

If ICa

-4 -6

(Outward currents are +; inward currents are -) 0

150

Time (ms)

FIGURE 23-2. SA node action potential and ion currents. A. SA nodal cells are depolarized slowly by the pacemaker current (If ) (phase 4), which consists of an inward flow of sodium (mostly) and calcium ions. Depolarization to the threshold potential opens highly selective voltage-gated calcium channels, which drive the membrane potential toward ECa (phase 0). As the calcium channels close and potassium channels open (phase 3 ), the membrane potential repolarizes. B. The flux of each ion species correlates roughly with each phase of the action potential. Positive currents indicate an outward flow of ions (blue and purple ), while negative currents are inward (gray and black ).

PR

QT R

5 mm = 0.5 mV T

P Q

S ST

QRS 5 mm = 0.2 second

406 Principles of Cardiovascular Pharmacology

Determination of Firing Rate The specialized conduction system of the heart consists of the SA node, AV node, bundle of His, and Purkinje system. These different populations of cells have different intrinsic rates of firing. Three factors determine the firing rate. First, as the rate of spontaneous depolarization in phase 4 increases, the rate of firing increases because the threshold potential (the minimum potential necessary to trigger an action potential) is reached more quickly at the end of phase 4. Second, if the threshold potential becomes more negative, the rate of firing increases because the threshold potential is reached more quickly at the end of phase 4. Third, if the maximum diastolic potential (the resting membrane potential) becomes more positive, the rate of firing increases because less time is needed to repolarize the membrane fully at the end of phase 3. Because the various populations of pacemaker cells possess different intrinsic rates of firing, the pacemaker population with the fastest firing rate sets the heart rate. The SA node possesses the fastest intrinsic firing rate—60–100 times per minute—and is the native pacemaker of the heart. The cells of the atrioventricular (AV) node and bundle of His fire intrinsically between 50 and 60 times per minute, and the cells of the Purkinje system have the slowest intrinsic firing rate—30–40 times per minute. The cells of the AV node, bundle of His, and Purkinje system are termed latent pacemakers, because their intrinsic rhythm is overridden by the faster SA-node automaticity. In a mechanism termed overdrive suppression, the SA node suppresses the intrinsic rhythm of the other pacemaker populations and entrains them to fire at the SA nodal firing rate.

 PATHOPHYSIOLOGY OF ELECTRICAL DYSFUNCTION Causes of electrical dysfunction in the heart can be divided into defects in impulse formation and defects in impulse conduction. In the former case, SA-node automaticity is interrupted or altered, leading to missed beats or ectopic beats, respectively. In the latter case, impulse conduction is altered (for example, in the case of re-entrant rhythms), and sustained arrhythmias can result.

Defects in Impulse Formation (SA Node) As the native pacemaker of the heart, the SA node has a pivotal role in normal impulse formation. Electrical events that alter SA nodal function or disturb overdrive suppression can lead to impaired impulse formation. Two mechanisms commonly associated with defective impulse formation are altered automaticity and triggered activity. Altered Automaticity Some mechanisms that alter automaticity of the SA node are physiologic. In particular, the autonomic nervous system often modulates automaticity of the SA node as part of a physiologic response. In sympathetic stimulation during exercise, an increased concentration of catecholamines leads to greater 1-adrenergic receptor activation. Activation of 1 receptors causes the opening of a greater number of pacemaker channels (If channels); a larger pacemaker current is then conducted through these channels; and faster phase 4 depolarization results. Sympathetic stimulation also causes the opening of a greater

number of Ca2 channels, and thereby shifts the threshold to more negative potentials. Both of these mechanisms increase heart rate. The parasympathetic vagus nerve affects the SA node by a number of mechanisms that oppose the sympathetic regulation of heart rate. Vagus nerve release of acetylcholine initiates an intracellular signaling cascade that: (1) reduces the pacemaker current by decreasing pacemaker channel opening; (2) shifts the threshold to more positive potentials by reducing Ca2 channel opening; and (3) makes the maximum diastolic potential (analogous to the resting membrane potential in these spontaneously firing cells) more negative by increasing K channel opening. The SA node, atria, and AV node are highly innervated and are thus more sensitive than the ventricular conducting system to the effects of vagal stimulation. In pathologic conditions, automaticity can be altered when latent pacemaker cells take over the SA node’s role as the pacemaker of the heart. When the SA nodal firing rate becomes pathologically slow or when conduction of the SA impulse is impaired, an escape beat may occur as a latent pacemaker initiates an impulse. A series of escape beats, known as an escape rhythm, may result from prolonged SA nodal dysfunction. On the other hand, an ectopic beat occurs when latent pacemaker cells develop an intrinsic rate of firing that is faster than the SA nodal rate, in some cases despite the presence of a normally functioning SA node. A series of ectopic beats, termed an ectopic rhythm, can result from ischemia, electrolyte abnormalities, or heightened sympathetic tone. Direct tissue damage (such as can occur after a myocardial infarction) also results in altered automaticity. Tissue injury can cause structural disruption of the cell membrane. Disrupted membranes are unable to maintain ion gradients, which are critical for maintaining appropriate membrane potentials. If the resting membrane potential becomes sufficiently positive (more positive than 60 mV), nonpacemaker cells may begin to depolarize spontaneously. Another mechanism by which tissue damage leads to altered automaticity is through the loss of gap junction connectivity. Direct electrical connectivity is important for the effective delivery of overdrive suppression from the SA node to the rest of the cardiac myocytes. When connectivity is disrupted due to tissue injury, overdrive suppression is not efficiently relayed, and the unsuppressed cells can initiate their own rhythm. This abnormal rhythm can lead to cardiac arrhythmia. Triggered Activity Afterdepolarizations occur when a normal action potential triggers extra abnormal depolarizations. That is, the first (normal) action potential triggers additional oscillations of membrane potential, which may lead to arrhythmia. There are two types of afterdepolarizations—early afterdepolarizations and delayed afterdepolarizations. If the afterdepolarization occurs during the inciting action potential, it is termed an early afterdepolarization (Fig. 23-5). Conditions that prolong the action potential (e.g., drugs that prolong the QT interval, such as procainamide and ibutilide) tend to trigger early afterdepolarizations. Specifically, an early afterdepolarization can occur during the plateau phase (phase 2) or the rapid repolarization phase (phase 3). During the plateau phase, because most of the Na channels are inactivated, an inward Ca2 current is responsible for the early afterdepolarization. On the other hand, during the rapid repolarization phase, partially recovered Na channels can conduct an inward Na current

CHAPTER 23 / Pharmacology of Cardiac Rhythm 407

Defects in Impulse Conduction

Membrane potential (mV)

50

Early afterdepolarization

Repetitive afterdepolarization

0 Na+ channels recover from inactivation

-50

Triggered arrhythmia

-100 0

0.2

0.6

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Time (sec)

FIGURE 23-5.

Early afterdepolarization. Early afterdepolarizations generally occur during the repolarizing phase of the action potential, although they can also occur during the plateau phase. Repetitive afterdepolarizations can trigger an arrhythmia.

that contributes to the early afterdepolarization. If an early afterdepolarization is sustained, it can lead to a type of ventricular arrhythmia termed torsades de pointes. Torsades de pointes, French for “twisting of the points,” is characterized by QRS complexes of varying amplitudes as they “twist” along the baseline; this rhythm is a medical emergency that can lead to death if not treated emergently with antiarrhythmics and/or defibrillation. In contrast to early afterdepolarizations, delayed afterdepolarizations occur shortly after the completion of repolarization (Fig. 23-6). The mechanism of delayed afterdepolarizations is not well understood; it has been proposed that high intracellular Ca2 concentrations lead to an inward Na current, which, in turn, triggers the delayed afterdepolarization.

The second type of electrical disturbance of the heart involves defects in impulse conduction. Normal cardiac function requires unobstructed and timely propagation of an electrical impulse through the cardiac myocytes. In pathologic conditions, altered impulse conduction can result from one or a combination of three mechanisms: re-entry, conduction block, and accessory tract pathways. Re-entry Normal cardiac conduction is initiated at the SA node and propagated to the AV node, bundle of His, Purkinje system, and myocardium in an orderly fashion. The cellular refractory period ensures that stimulated regions of the myocardium depolarize only once during propagation of an impulse. Figure 23-7A depicts normal impulse conduction, in which an impulse arriving at point a travels synchronously down two parallel pathways, 1 and 2. Re-entry of an electrical impulse occurs when a selfsustaining electrical circuit stimulates an area of the myocardium repeatedly and rapidly. Two conditions must be present Cardiac action potential A Normal conduction

a 1

Non-excitable area 2

Cardiac action potential B Re-entrant circuit

Unidirectional conduction block

50

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A delayed afterdepolarization that reached threshold voltage

Re-entrant conduction

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a

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1 0

depolarized cells)

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FIGURE 23-6. Delayed afterdepolarization. Delayed afterdepolarizations occur shortly after repolarization. Although the mechanism has not been firmly elucidated, it appears that intracellular Ca2 accumulation activates the Na/Ca2 exchanger, and the resulting electrogenic influx of 3 Na for each extruded Ca2 depolarizes the cell.

FIGURE 23-7. Normal and re-entrant electrical pathways. A. In normal impulse conduction, an impulse traveling down a pathway arrives at point a, where it is able to travel down two alternate pathways, 1 and 2. In the absence of re-entry, the impulses continue on and depolarize different areas of the ventricle. B. A re-entrant circuit can develop if one of the branch pathways is pathologically disrupted. When the impulse arrives at point a, it can travel only down pathway 1 because pathway 2 is blocked unidirectionally (i.e., the effective refractory period of the cells in pathway 2 is prolonged to such an extent that anterograde conduction is prohibited). The impulse conducts through pathway 1 and proceeds to point b. At this point, the cells in pathway 2 are no longer refractory, and the impulse conducts in a retrograde fashion up pathway 2 toward point a. When the retrograde impulse arrives at point a, it can initiate re-entry. Re-entry can result in a sustained pattern of rapid depolarizations that trigger tachyarrhythmias. This mechanism can occur over small or large regions of the heart.

408 Principles of Cardiovascular Pharmacology

for a re-entrant electrical circuit to occur: (1) unidirectional block (anterograde conduction is prohibited, but retrograde conduction is permitted); and (2) slowed retrograde conduction velocity. Figure 23-7B shows a re-entrant electrical circuit. As the impulse arrives at point a, it can travel only down pathway 1 (the left branch), because pathway 2 (the right branch) is blocked unidirectionally in the anterograde direction. The impulse conducts through pathway 1 and travels to point b. At this junction, the impulse travels in a retrograde fashion up pathway 2 toward point a. The conduction time from point b to point a is slowed because of cell damage or the presence of cells that are still in the refractory state. By the time the impulse reaches point a, the cells in pathway 1 have had adequate time to repolarize, and these cells are stimulated to continue conducting the action potential toward point b. In this manner, tachyarrhythmias result from the combination of unidirectional block and decreased conduction velocity in the abnormal pathway. Conduction Block Conduction block occurs when an impulse fails to propagate because of the presence of an area of inexcitable cardiac tissue. This area of inexcitable tissue could consist of normal tissue that is still refractory, or it could represent tissue that has been damaged by trauma, ischemia, or scarring. In either case, the myocardium is unable to conduct an impulse. Because conduction block removes overdrive suppression by the SA node, the cardiac myocytes are free to beat at their intrinsically slower frequency. For this reason, conduction block can be manifested clinically as bradycardia. Accessory Tract Pathways During the normal cardiac cycle, the SA node initiates an impulse that travels quickly through the atrial myocardium and arrives at the AV node. Impulse conduction then slows through the AV node, allowing sufficient time for filling of the ventricles with blood before ventricular contraction is

AV node

Bundle of His

SA node

Purkinje fibers

Bypass tract (Bundle of Kent)

FIGURE 23-8.

Bundle of Kent. The bundle of Kent is an accessory electrical pathway that conducts impulses directly from the atria to the ventricles, bypassing the AV node. Impulse conduction through this accessory tract is more rapid than conduction through the AV node, setting up the conditions for re-entrant tachyarrhythmias.

initiated. After the impulse travels through the AV node, it again propagates quickly throughout the ventricles to trigger ventricular contraction. Some individuals possess accessory electrical pathways that bypass the AV node. One common accessory pathway is the bundle of Kent, a band of myocardium that conducts impulses directly from the atria to the ventricles, bypassing the AV node (Fig. 23-8). In these individuals, an impulse originating in the SA node is conducted through the bundle of Kent to the ventricles more rapidly than the same impulse would be conducted through the AV node. Because the bundle of Kent is an accessory pathway, the ventricular tissue receives impulses from both the normal conduction pathway and the accessory pathway. As a result, electrocardiograms from these individuals typically exhibit a wider-than-normal QRS complex and an earlier-than-normal ventricular upstroke. More importantly, because the two conduction tracts have different conduction velocities, the presence of an accessory tract can set up the conditions for a re-entrant loop, and thereby predispose the individual to tachyarrhythmias.

 PHARMACOLOGIC CLASSES AND AGENTS Ion currents across the plasma membrane induce changes in the membrane potential of cells. Changes in the membrane potential of cardiac pacemaker cells underlie the timely contraction of cardiac myocytes. Defects in impulse formation and altered impulse conduction can lead to disturbances in cardiac rhythm. Antiarrhythmic agents are used to restore normal cardiac rhythm by targeting proarrhythmic regions of the heart.

General Mechanisms of Action of Antiarrhythmic Agents Although there are many different antiarrhythmic agents, there are surprisingly few mechanisms of antiarrhythmic action. In general, drugs that affect cardiac rhythm act by altering: (1) the maximum diastolic potential in pacemaker cells (and/ or the resting membrane potential in ventricular cells); (2) the rate of phase 4 depolarization; (3) the threshold potential; or (4) the action potential duration. The specific effect of a particular channel blocker follows directly from the role of the current carried by that channel in the cardiac action potential. For example, Na and Ca2 channel blockers typically alter the threshold potential, while K channel blockers tend to prolong action potential duration. These drugs generally block the pore from inside the cell; they can access their sites of action by either traversing the pore of the channel or diffusing across the lipid bilayer within which the channel is embedded. State-dependent ion channel block is an important concept in antiarrhythmic drug action. Ion channels are capable of switching among various conformational states, and changes in the permeability of the membrane to a particular ion are mediated by conformational changes in the channels that pass that ion. Antiarrhythmic drugs often have different affinities for different conformational states of the ion channel; that is, these drugs bind to one conformation of the channel with higher affinity than they do to other conformations of the channel. This type of binding is referred to as “state-dependent.” Na channel blockers serve as an excellent example to illustrate the concept of state-dependent ion channel block. The Na channel undergoes three major state changes

410 Principles of Cardiovascular Pharmacology

(Fig. 23-9). The block of Na channels leaves fewer channels available to open in response to membrane depolarization, thereby raising the threshold for action potential firing and slowing the rate of depolarization. Both of these effects extend the duration of phase 4, and thereby decrease heart rate. Furthermore, the shift in threshold potential means that, in patients with implanted defibrillators who are treated with Na channel blockers, a higher voltage is needed to defibrillate the heart. Therefore, it is important to take into account the effect of Na channel blockers when choosing appropriate settings for implanted defibrillators. In addition to decreasing SA-node automaticity, Na channel blockers act on ventricular myocytes to decrease reentry. This is achieved mainly by decreasing the upstroke velocity of phase 0 and, for some Na channel blockers, by prolonging repolarization (Fig. 23-10). By decreasing

A

Membrane potential (mV)

60

Class IA Antiarrhythmics

30

0 -30

Increased threshold Normal threshold

-60 Decreased slope of phase 4 depolarization

-90 0

100

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B 60

Membrane potential (mV)

phase 0 upstroke velocity, Na channel blockers decrease the conduction velocity through cardiac tissue. Ideally, conduction velocity is reduced to such an extent that the propagating wavefront is extinguished before it is able to restimulate myocytes in a re-entrant pathway. However, if conduction velocity is not sufficiently decreased, and the impulse is not extinguished, then the slowed impulse can support reentry as it reaches cells that are no longer refractory (see above), and thereby precipitate an arrhythmia. In addition to decreasing phase 0 upstroke velocity, class IA Na channel blockers prolong repolarization. Prolonged repolarization increases the effective refractory period, so that cells in a reentrant circuit cannot be depolarized by the re-entrant action potential. In summary, Na channel blockers decrease the likelihood of re-entry, and thereby prevent arrhythmia, by: (1) decreasing conduction velocity, and (2) increasing the refractory period of ventricular myocytes. Although the three subclasses of class I antiarrhythmics (class IA, IB, and IC) have similar effects on the action potential in the SA node, there are important differences in their effects on the ventricular action potential.

30

0 -30 Normal threshold

-60 Addition of ACh or adenosine

Decreased slope of phase 4 depolarization

-90 Hyperpolarization

0

100

200

300

400

500

600

700

Time (ms)

FIGURE 23-9. Effects of class I antiarrhythmics and natural agonists on the SA-node action potential. A. The normal SA-node action potential is shown as a solid curve. Class I antiarrhythmics (Na channel blockers) alter SA-node automaticity by affecting two aspects of the SA nodal action potential: (1) the threshold is shifted to more positive potentials; and (2) the slope of phase 4 depolarization is decreased. B. Acetylcholine and adenosine slow the SA nodal firing rate by opening K channels that hyperpolarize the cell and decrease the slope of phase 4 depolarization.

Class IA antiarrhythmics exert a moderate block on Na channels and prolong the repolarization of both SA nodal cells and ventricular myocytes. By blocking Na channels, these agents decrease the phase 0 upstroke velocity, which decreases conduction velocity through the myocardium. Class IA antiarrhythmics also block K channels, and thereby reduce the outward K current responsible for repolarization of the membrane. This prolongation of repolarization increases the effective refractory period of the cells. Together, the decreased conduction velocity and increased effective refractory period decrease re-entry. Quinidine is often considered the prototypical drug among the class IA antiarrhythmics, but it is becoming less frequently used due to its adverse effects. In addition to the pharmacologic actions described above for all class IA antiarrhythmics, quinidine exerts an anticholinergic (vagolytic) effect, most likely by blocking the K channels that are opened upon vagal stimulation of M2 muscarinic receptors in the AV node (see Fig. 23-9B, Fig. 9-1). The anticholinergic effect is significant clinically because it can increase conduction velocity through the AV node. Increased AV nodal conduction can have potentially detrimental effects in patients with atrial flutter. Such patients manifest an average atrial firing rate of 280–300 beats per minute. Because some of these impulses reach the AV node while it is still refractory, not all of the impulses are transmitted to the ventricles. Therefore, the atria fire much faster than the ventricles—there is typically a 2:1 or 4:1 ratio of atrial to ventricular firing rates. When quinidine is administered to patients with atrial flutter, the atrial firing rate decreases because of quinidine’s pharmacologic action in slowing conduction velocity through the myocardium. At the same time, however, AV nodal conduction velocity increases because of the vagolytic effects of the drug. The increase in AV nodal conduction velocity abolishes the 2:1 or 4:1 ratio of atrial to ventricular firing rates, and a 1:1 ratio of atrial to ventricular firing rates is often established. For example, with an atrial flutter rate of 300 and 2:1 “A–V block,” the ventricles are driven at a rate of 150, which most individuals can tolerate. If the flutter rate is slowed to 200 and A–V conduction is enhanced to 1:1, however, the

CHAPTER 23 / Pharmacology of Cardiac Rhythm 411

Membrane potential (mV)

Mild Na+ channel block

Moderate Na+ channel block

Class IA

No change in repolarization

Shortened repolarization

Prolonged repolarization

Marked Na+ channel block

Class IB

Class IC

Time

Effects of class IA, IB, and IC antiarrhythmics on the ventricular action potential. Class I antiarrhythmics (Na channel blockers) act on ventricular myocytes to decrease re-entry. All subclasses of the class I antiarrhythmics block the Na channel to some degree: class IA agents exhibit moderate Na channel block, class IB agents rapidly bind to (block) and dissociate from (unblock) Na channels, and class IC agents produce marked Na channel block. Class IA, IB, and IC agents also differ in the degree to which they affect the duration of the ventricular action potential.

FIGURE 23-10.

ventricles are driven at a rate of 200, which is usually too fast for effective ventricular pumping. For this reason, an agent that slows AV nodal conduction—such as a -adrenergic antagonist or verapamil (a Ca channel blocker)—should be used in conjunction with quinidine to prevent an excessively rapid ventricular response in patients with atrial flutter. The most common adverse effects of quinidine are diarrhea, nausea, headache, and dizziness. These effects make it difficult for patients to tolerate chronic therapy with quinidine. Quinidine is contraindicated in patients with QT prolongation and in patients who are taking medications that predispose to QT prolongation, because of the increased risk of torsades de pointes. Relative contraindications to quinidine use include sick sinus syndrome, bundle branch block, myasthenia gravis (because of quinidine’s anticholinergic action), and liver failure. Quinidine is administered orally and metabolized by cytochrome P450 enzymes in the liver. Quinidine increases plasma levels of digoxin (an inotropic agent), most likely by competing for the P450 enzymes that are responsible for digoxin metabolism. Because digoxin has a narrow therapeutic index (see Chapter 24, Pharmacology of Cardiac Contractility), quinidine-induced digoxin toxicity occurs in a significant fraction of patients. The plasma potassium level must be carefully monitored in patients treated with quinidine, because hypokalemia decreases quinidine efficacy, exacerbates QT prolongation, and, most importantly, predisposes to torsades de pointes. It is hypothesized that torsades de pointes is the mechanism most likely responsible for quinidine-induced syncope. Because of quinidine’s numerous adverse effects and contraindications, this drug has largely been replaced by class III agents—such as ibutilide and amiodarone—for the pharmacologic conversion of atrial flutter or atrial fibrillation to normal sinus rhythm. Procainamide is a class IA antiarrhythmic agent that is effective in the treatment of many types of supraventricular and ventricular arrhythmias. Procainamide is often used in the pharmacologic conversion of new-onset atrial fibrillation to normal sinus rhythm, although with less efficacy than intravenous ibutilide. Procainamide can be used safely to decrease the likelihood of re-entrant arrhythmias in the setting of acute

myocardial infarction, even in the presence of decreased cardiac output. Procainamide can also be administered by slow intravenous infusion to treat acute ventricular tachycardia. Unlike quinidine, procainamide has few anticholinergic effects and does not alter plasma levels of digoxin. Procainamide can cause peripheral vasodilation via inhibition of neurotransmission at sympathetic ganglia. With chronic therapy, almost all patients develop a lupus-like syndrome and positive antinuclear antibodies; the precise mechanism of this reaction is not known, but it remits if the drug is discontinued. Procainamide is acetylated in the liver to N-acetyl-procainamide (NAPA); this active metabolite produces the pure class III antiarrhythmic effects of prolonging the refractory period and lengthening the QT interval. NAPA does not appear to cause the lupus-like adverse effects of procainamide. Disopyramide is similar to quinidine in its electrophysiologic and antiarrhythmic effects; the difference between the two drugs lies in their adverse effects. Disopyramide causes fewer gastrointestinal problems but has even more profound anticholinergic effects than quinidine, producing such adverse effects as urinary retention and dry mouth. The profound anticholinergic effects of disopyramide appear to be related to the drug’s action as an antagonist at muscarinic acetylcholine receptors. Disopyramide is contraindicated in patients with obstructive uropathy or glaucoma. Disopyramide is also contraindicated in patients with conduction block between the atria and ventricles and in patients with sinus-node dysfunction. Disopyramide has the prominent but unexplained effect that it depresses cardiac contractility, which has led to its use in the treatment of hypertrophic obstructive cardiomyopathy and neurocardiogenic syncope. Because of its negative inotropic effects, disopyramide is absolutely contraindicated in patients with decompensated heart failure. Oral disopyramide is approved only for the treatment of life-threatening ventricular arrhythmias; oral or intravenous disopyramide is sometimes used to convert supraventricular tachycardia to normal sinus rhythm. The current trend in the treatment of life-threatening arrhythmias, however, is away from class I antiarrhythmic agents and toward class III agents and electrical devices.

412 Principles of Cardiovascular Pharmacology

Class IB Antiarrhythmics

Class IB antiarrhythmics include lidocaine, mexiletine, and phenytoin. Lidocaine is the prototypical class IB agent. These drugs alter the ventricular action potential by blocking Na channels and sometimes by shortening repolarization; the latter effect may be mediated by the drugs’ ability to block the few Na channels that inactivate late during phase 2 of the cardiac action potential (Fig. 23-10). In comparison to class IA antiarrhythmics, which preferentially bind to open Na channels, class IB drugs bind to both open and inactivated Na channels. Therefore, the more time Na channels spend in the open or inactivated state, the more blockade the class IB antiarrhythmics can exert. The major distinguishing characteristic of the class IB antiarrhythmics is their fast dissociation from Na channels. Because Na channels recover quickly from class IB blockade, these drugs are most effective in blocking depolarized or rapidly driven tissues, where there is a higher likelihood of the Na channels being in the open or inactivated state. Thus, class IB antiarrhythmics exhibit use-dependent block in diseased myocardium, where the cells have a tendency to fire more frequently; these antiarrhythmics have relatively little effect on normal cardiac tissue. Myocardial ischemia provides an example of the therapeutic utility of the use-dependent block exerted by class IB antiarrhythmics. The increase in extracellular H concentration in ischemic tissue activates membrane pumps that cause an increase in the extracellular K concentration. This increase in extracellular K shifts EK to a more depolarized (more positive) value; for example, EK may shift from 94 mV to 85 mV. The altered electrochemical K gradient provides a smaller driving force for K ions to flow out of cells, and depolarization of the membrane leads to a higher likelihood of action potential firing. Because ischemic cardiac myocytes tend to fire more frequently, the Na channels spend more time in the open or inactivated state, serving as a better target for blockade by class IB antiarrhythmics. Lidocaine is commonly used to treat ventricular arrhythmias in emergency situations. This drug is not effective in treating supraventricular arrhythmias. In hemodynamically stable patients, lidocaine is reserved for treatment of ventricular tachyarrhythmias or frequent premature ventricular contractions (PVCs) that are bothersome or hemodynamically significant. Lidocaine has a short plasma half-life (approximately 20 minutes), and it is metabolically de-ethylated in the liver. Its metabolism is governed by two factors, liver blood flow and liver cytochrome P450 activity. For patients whose liver blood flow is decreased by old age or heart failure, or whose P450 enzymes are acutely inhibited, for example, by cimetidine (see Chapter 4, Drug Metabolism), a lower dose of lidocaine should be considered. For patients whose P450 enzymes are induced by drugs such as barbiturates, phenytoin, or rifampin, the dose of lidocaine should be increased. Because lidocaine shortens repolarization, possibly by blocking the few Na channels that inactivate late during phase 2 of the cardiac action potential, it does not prolong the QT interval. Therefore, the drug is safe for use in patients with long QT syndrome. However, because lidocaine also blocks Na channels in the central nervous system (CNS), it can produce CNS adverse effects such as confusion, dizziness, and seizures. In addition to its use as an acute intravenous therapy for ventricular arrhythmias, lidocaine is used as a local anesthetic (see Chapter 11).

Mexiletine, an analogue of lidocaine, is available in oral formulation. While the efficacy of mexiletine is similar to that of quinidine, mexiletine does not prolong the QT interval and it lacks vagolytic effects. In addition, little hemodynamic depression has been reported with the use of mexiletine. The primary indication for mexiletine is life-threatening ventricular arrhythmia. In practice, however, mexiletine is often used as an adjunct to other antiarrhythmic agents. For example, mexiletine is used in combination with amiodarone in patients with implantable cardioverter-defibrillators (ICDs) and in patients with recurrent ventricular tachycardia. Mexiletine is also used in combination with quinidine or sotalol to increase antiarrhythmic efficacy while reducing adverse effects. There are no data supporting reduced mortality with the use of mexiletine or any of the other class IB antiarrhythmic agents. Major adverse effects of mexiletine include dose-related nausea and tremor, which can be ameliorated when the medication is taken with food. Mexiletine undergoes hepatic metabolism, and its plasma levels may be altered by inducers of hepatic P450 enzymes such as phenytoin and rifampin. While phenytoin is usually considered an antiepileptic medication, its effects on the myocardium also allow it to be classified as a class IB antiarrhythmic agent. The pharmacologic properties of phenytoin are discussed in detail in Chapter 15, Pharmacology of Abnormal Electrical Neurotransmission in the Central Nervous System. Although the use of phenytoin as an antiarrhythmic agent is limited, it has been found to be effective in ventricular tachycardia of young children. Specifically, phenytoin has been used in the treatment of congenital prolonged QT syndrome when therapy with -adrenergic antagonists alone has failed; it is also used to treat ventricular tachycardia after congenital heart surgery. Phenytoin maintains AV conduction in digoxin-toxic arrhythmias, and it is especially useful in the rare patient who has concurrent epilepsy and cardiac arrhythmia. Phenytoin is an inducer of hepatic enzymes including P450 3A4, and thus affects plasma levels of other antiarrhythmic agents such as mexiletine, lidocaine, and quinidine. Class IC Antiarrhythmics

Class IC antiarrhythmics are the most potent Na channel blockers, and they have little or no effect on action potential duration (Fig. 23-10). By markedly decreasing the rate of phase 0 upstroke of ventricular cells, these drugs suppress premature ventricular contractions. Class IC antiarrhythmics also prevent paroxysmal supraventricular tachycardia and atrial fibrillation. However, these drugs have marked depressive effects on cardiac function and, thus, must be used with discretion. In addition, the CAST (Cardiac Arrhythmia Suppression Trial) and other studies have brought attention to the proarrhythmic effects of these agents. Flecainide is the prototypical class IC drug; other members of this class include encainide, moricizine, and propafenone. Flecainide illustrates the principle that antiarrhythmic agents can also cause arrhythmia. When flecainide is administered to patients with preexisting ventricular tachyarrhythmias and to those with a history of myocardial infarction, it can worsen the arrhythmia even at normal doses. Currently, flecainide is approved for use only in lifethreatening situations; for example, when paroxysmal supraventricular or ventricular arrhythmia is unresponsive to other measures. Flecainide is eliminated very slowly from the body; it has a plasma half-life of 12–30 hours. Because

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of its marked blockade of Na channels and its suppressive effects on cardiac function, flecainide use is associated with adverse effects that include sinus-node dysfunction, a marked decrease in conduction velocity, and conduction block. Class II Antiarrhythmic Agents: ␤-Adrenergic Antagonists Class II antiarrhythmic agents are -adrenergic antagonists (also called -blockers). These agents act by inhibiting sympathetic input to the pacing regions of the heart. (-Adrenergic antagonists are more extensively discussed in Chapter 10, Adrenergic Pharmacology.) Although the heart is capable of beating on its own without innervation from the autonomic nervous system, both sympathetic and parasympathetic fibers innervate the SA node and the AV node, and thereby alter the rate of automaticity. Sympathetic stimulation releases norepinephrine, which binds to 1-adrenergic receptors in the nodal tissues. (1-Adrenergic receptors are the adrenergic subtype preferentially expressed in cardiac tissue.) Activation of 1-adrenergic receptors in the SA node triggers an increase in the pacemaker current (If), which increases the rate of phase 4 depolarization and, consequently, leads to more frequent firing of the node. Stimulation of 1-adrenergic receptors in the AV node increases Ca2 and K currents, thereby increasing the conduction velocity and decreasing the refractory period of the node. 1-Antagonists block the sympathetic stimulation of 1adrenergic receptors in the SA and AV nodes (Fig. 23-11). The AV node is more sensitive than the SA node to the effects of 1-antagonists. 1-Antagonists affect the action potentials of SA and AV nodal cells by: (1) decreasing the rate of phase 4 depolarization; and (2) prolonging repolarization. Decreasing the rate of phase 4 depolarization results in decreased automaticity, and this, in turn, reduces myocardial oxygen demand. Prolonged repolarization at the AV node increases the effective refractory period, which decreases the incidence of re-entry.

Membrane potential (mV)

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Time (ms)

FIGURE 23-11. Effects of class II antiarrhythmics on pacemaker cell action potentials. Class II antiarrhythmics (-antagonists) reverse the tonic sympathetic stimulation of cardiac 1-adrenergic receptors. By blocking the adrenergic effects on the SA and AV nodal action potentials, these agents decrease the slope of phase 4 depolarization (especially important at the SA node) and prolong repolarization (especially important at the AV node). These agents are useful in the treatment of supraventricular and ventricular arrhythmias that are precipitated by sympathetic stimulation.

1-Antagonists are the most frequently used agents in the treatment of supraventricular and ventricular arrhythmias precipitated by sympathetic stimulation. 1-Adrenergic antagonists have been shown to reduce mortality after myocardial infarction, even in patients with relative contraindications to this therapy such as severe diabetes mellitus or asthma. Because of their wide spectrum of clinical application and established safety record, -adrenergic antagonists are the most useful antiarrhythmic agents currently available. There are several generations of -antagonists, each characterized by slightly different pharmacologic properties. First-generation -antagonists, such as propranolol, are nonselective -adrenergic antagonists that antagonize both 1-adrenergic and 2-adrenergic receptors. They are widely used to treat tachyarrhythmias caused by catecholamine stimulation during exercise or emotional stress. Because propranolol does not prolong repolarization in ventricular tissue, it can be used in patients with long QT syndrome. Second-generation agents, including atenolol, metoprolol, acebutolol, and bisoprolol, are relatively selective for 1-adrenergic receptors when administered in low doses. Third-generation -antagonists cause vasodilation in addition to 1-receptor antagonism. Labetalol and carvedilol induce vasodilation by antagonizing -adrenergic receptormediated vasoconstriction; pindolol is a partial agonist at the 2-adrenergic receptor; and nebivolol stimulates endothelial production of nitric oxide. The different generations of -antagonists produce varying degrees of adverse effects. Three general mechanisms are responsible for the adverse effects of -blockers. First, antagonism at 2-adrenergic receptors causes smooth muscle spasm, leading to bronchospasm, cold extremities, and impotence. These effects are more commonly caused by the nonselective first-generation -antagonists. Second, exaggeration of the therapeutic effects of 1-receptor antagonism can lead to excessive negative inotropic effects, heart block, and bradycardia. Third, drug penetration into the CNS can produce insomnia and depression. Class III Antiarrhythmic Agents: Inhibitors of Repolarization Class III antiarrhythmic agents block K channels. Two types of currents determine the duration of the plateau phase of the cardiac action potential: inward, depolarizing Ca2 currents and outward, hyperpolarizing K currents. During a normal action potential, the hyperpolarizing K currents eventually dominate, returning the membrane potential to more hyperpolarized values. Larger hyperpolarizing K currents shorten plateau duration, returning the membrane potential to its resting value more rapidly, while smaller hyperpolarizing K currents lengthen plateau duration and delay return of the membrane potential to its resting value. When K channels are blocked, a smaller hyperpolarizing  K current is generated. Therefore, K channel blockers cause a longer plateau and prolong repolarization (Fig. 23-12). The ability of K channel blockers to lengthen plateau duration is responsible for both their pharmacologic uses and their adverse effects. On the beneficial side, prolongation of the plateau duration increases the effective refractory period, which, in turn, decreases the incidence of re-entry. On the toxic side, prolongation of the plateau duration increases the likelihood of developing early afterdepolarizations and torsades de pointes. With the exception of amiodarone, K channel blockers also exhibit the undesirable property of “reverse use-dependency”: action

Membrane potential (mV)

50 Block of repolarizing K+ channels Balance of Ca2+ (depolarizing) and K+ (hyperpolarizing) currents

0

-50

Prolonged repolarization

-100 0

0.2

0.4

Time (sec)

0.6

0.8

Membrane potential (mV)

60 Slow rise of action potential

30

0 -30 Threshold

-60

-90 0

100

200

300

400

Time (ms)

500

600

700

CHAPTER 23 / Pharmacology of Cardiac Rhythm 417

models are used for the majority of ion channel research; comparatively little is known about the clinical pharmacology of ion channels expressed in humans. With the mouse and human genomes now completely sequenced, researchers will be able to investigate the possibility that newly identified gene products can serve as selective targets for new therapeutic agents. The identification of ion channel gene expression in the various tissues of the human heart (SA node, AV node, atrial conduction pathways, endocardium, ventricular conduction pathways, etc.), both during development and in response to injury, may provide new targets that are not now known. Many of the genes are likely to encode channels that form heteromultimers, and there are likely to be many genetic variants within the population. This enormous complexity will likely represent a boon to drug development because it will allow more tailored strategies to be employed. For example, current research in atrial fibrillation has focused on the development of antiarrhythmics selective for ion channels that are expressed selectively in the atria. In parallel, the development of implantable computers, stimulators, and defibrillators

will constitute an alternative strategy to prevent or terminate arrhythmias.

Suggested Reading Ackerman MJ, Clapham DE. Ion channels—basic science and clinical disease. N Engl J Med 1997;336:1575–1586. (Broad review of ion channels.) Delacretaz E. Clinical practice. Supraventricular tachycardia. N Engl J Med 2006;354:1039–1051. (Discussion of the clinical uses of antiarrhythmic agents in treating supraventricular tachycardia.) Hohnloser SH, Crijns HJ, van Eickels M, et al. Effect of dronedarone on cardiovascular events in patients with atrial fibrillation. N Engl J Med 2009;360:668–678. (Trial of dronedarone suggesting safety in patients with atrial fibrillation.) McBride BF. The emerging role of antiarrhythmic compounds with atrial selectivity in the management of atrial fibrillation. J Clin Pharmacol 2009;49:258–267. (Future directions in drug development for treatment of atrial fibrillation.) Nash DT, Nash SBD. Ranolazine for chronic stable angina. Lancet 2008; 372:1335–1341. (Recent review of ranolazine.) Rudy Y, Silva JR. Computational biology in the study of cardiac ion channels and cell electrophysiology. Quarterly Rev Biophys 2006;39:57–116. (Summarizes the known cardiac ion channels in models of cardiac action potentials.)

24 Pharmacology of Cardiac Contractility Ehrin J. Armstrong and Thomas P. Rocco

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 422-423 PHYSIOLOGY OF CARDIAC CONTRACTION . . . . . . . . . . . . . 422 Myocyte Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 Myocyte Contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 Regulation of Contractility . . . . . . . . . . . . . . . . . . . . . . . . 424 The Sodium Pump and Sodium–Calcium Exchange . . . . . . . . . . . . . . . . . . . . 424 Calcium Storage and Release . . . . . . . . . . . . . . . . . . . 425 Adrenergic Receptor Signaling and Calcium Cycling . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Sensitivity of Contractile Proteins to Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Cellular Pathophysiology of Contractile Dysfunction . . . . . 427

PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 429 Cardiac Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 Digoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 Digitoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 ␤-Adrenergic Receptor Agonists . . . . . . . . . . . . . . . . . . . 431 Dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 Dobutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Epinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Norepinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Isoproterenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Phosphodiesterase (PDE) Inhibitors . . . . . . . . . . . . . . . . . 432 Calcium-Sensitizing Agents . . . . . . . . . . . . . . . . . . . . . . . 433 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 433 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

 INTRODUCTION

as agents that increase the contractile force of cardiac myocytes. Since the advent of digitalis, elucidation of the cellular mechanism of cardiac contraction has facilitated the development of other inotropic agents. After reviewing the physiology of cardiac contraction and the cellular pathophysiology of contractile dysfunction, this chapter describes four classes of positive inotropic drugs that are either approved for use or under investigation in clinical trials. An integrated discussion of therapeutic strategies for HF can be found in Chapter 25, Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure.

In 1785, Dr. William Withering described the cardiovascular benefits of a preparation from the foxglove plant called digitalis. He used this preparation to treat patients suffering from “dropsy,” a condition in which accumulation of extravascular fluid leads to dyspnea (difficulty breathing) and peripheral edema. These symptoms are now recognized as characteristic manifestations of heart failure (HF), a clinical syndrome most commonly caused by systolic dysfunction of the left ventricle (LV). In this condition, the LV is unable to maintain adequate stroke volume despite normal filling volumes, and the LV end-diastolic volume increases in an effort to preserve stroke output. However, beyond a certain end-diastolic volume, LV diastolic pressures begin to increase, often precipitously. This increase in LV diastolic pressure results in increased left atrial and pulmonary capillary pressures, which, in turn, lead to interstitial and alveolar pulmonary edema and to increased right heart and pulmonary artery pressures. The elevated right heart pressures result in systemic venous hypertension and peripheral edema. Dr. Withering’s use of digitalis presaged the current use of digoxin, a member of the cardiac glycoside family of drugs, to treat conditions in which myocardial contractility is impaired. Cardiac glycosides are positive inotropes, defined 422

 PHYSIOLOGY OF CARDIAC CONTRACTION The heart is responsible for receiving deoxygenated blood from the periphery, propelling this blood through the pulmonary circulation (where the hemoglobin is reoxygenated), and ultimately distributing the oxygenated blood to peripheral tissues. To accomplish the latter task, the left ventricle (LV) must develop sufficient tension to overcome the impedance to ejection that resides in the peripheral circulation. The relationship between the tension generated during the systolic phase of the cardiac cycle and the extent of LV filling during diastole is referred to as the contractile state of the myocardium. Together with preload (intraventricular blood volume), afterload (the resistance against which the left ventricle ejects), and

424 Principles of Cardiovascular Pharmacology T-tubule

Sarcotubular network Terminal cisterna

Ca2+

T-tubule

Ca2+

Sarcolemma Mitochondrion Myofibrils Ca2+

Sarcoplasmic reticulum

Sarcomere

I band

A band

Z line

Z line

A band Actin

Z line Myosin

FIGURE 24-1. Cardiac myocyte structure. Each cardiac myocyte contains myofibrils and mitochondria surrounded by a specialized plasma membrane termed the sarcolemma. Invaginations of the sarcolemma, called T-tubules, provide conduits for Ca2⫹ influx. Within the cell, an extensive sarcoplasmic reticulum stores Ca2⫹ for use in contraction. Extracellular Ca2⫹ enters through the sarcolemma and its T-tubules during phase 2 of the action potential. This trigger Ca2⫹ binds to channels on the sarcoplasmic reticulum membrane, causing release into the cytosol of a large pool of so-called activation Ca2⫹. Increased cytosolic Ca2⫹ initiates myofibril contraction. The sarcomere is the functional unit of the myofibril. Each sarcomere consists of interdigitating bands of actin and myosin. These bands form distinctive structures under the electron microscope. The A bands correspond to regions of overlapping actin and myosin. The Z lines demarcate the borders of each sarcomere. The I bands span neighboring sarcomeres and correspond to regions of actin without overlapping myosin. During cardiac myocyte contraction, the I bands become shorter (i.e., the Z lines approach one another), but the A bands maintain a constant length. stretch (length) of the muscle exposes additional sites for calcium binding and for actin–myosin interaction; increased stretch also effects greater release of calcium from the SR. These cellular events provide the mechanistic explanation for the Frank–Starling law: an increase in the end-diastolic volume of the left ventricle leads to an increase in ventricular stroke volume during systole. Chapter 25 describes the organlevel implications of the Frank–Starling law in more detail.

Regulation of Contractility Three major control mechanisms regulate calcium cycling and myocardial contractility in cardiac myocytes. At the sarcolemma, calcium flux is mediated by interactions between the sodium pump and sodium–calcium exchanger. At the sarcoplasmic reticulum, calcium channels and pumps regulate the extent of calcium release and reuptake. Neurohumoral influences, especially the ␤-adrenergic signaling pathway, further modulate calcium cycling through these channels and transporters. The Sodium Pump and Sodium–Calcium Exchange In the sarcolemma, the three key proteins involved in calcium regulation are the Na⫹/K⫹-ATPase, hereafter referred to as the sodium pump, the sodium–calcium exchanger,

and the calcium–ATPase or calcium pump (Fig. 24-3). The activity of the sodium pump is crucial to maintain both the resting membrane potential and the concentration gradients of sodium and potassium across the sarcolemma ([Na⫹]out ⫽ 145 mM, [Na⫹]in ⫽ 15 mM, [K⫹]out ⫽ 5 mM, [K⫹]in ⫽ 150 mM). Sodium-pump activity is closely linked to the intracellular calcium concentration via the sodium–calcium exchanger; this antiporter exchanges sodium and calcium in both directions across the sarcolemma. Changes in the concentration of either sodium or calcium ions inside or outside the cell affect the direction and magnitude of sodium–calcium exchange. Under normal conditions, the low intracellular sodium concentration favors sodium influx and calcium efflux. Some drugs take advantage of the functional coupling between the sodium pump and the sodium– calcium exchanger to exert their effect as positive inotropes. Digoxin, discussed in the introductory case and described in detail below, is the prototype of an inotropic agent that acts by inhibiting the sodium pump. A sarcolemmal calcium pump also helps to maintain calcium homeostasis by actively extruding calcium from the cytoplasm after cardiac contraction. A high concentration of ATP favors calcium removal (relaxation), both directly via the calcium pump and indirectly via the sodium pump.

Tropomyosin Actin filament TN-I

TN-T

TN-C

Troponin complex 1 ATP hydrolysis

ADP

ATP

P

Myosin Ca2+

Relaxed

Relaxed, energized

4

2 Dissociation of actin and myosin

Formation of active complex Ca2+

ATP

Ca2+

Ca2+

3

ADP P

Product dissociation Rigor complex

Active complex ADP + P

426 Principles of Cardiovascular Pharmacology A

Sarcolemma Ca2+ 2 Ca2+ - induced Ca2+ release

1

T-tubule

Ca2+

Ca2+

2+2+ Ca Ca

Ca2+

3 Myofibril contraction free Ca2+

by regulating calcium reuptake into the SR: unphosphorylated phospholamban slows relaxation, while phosphorylated phospholamban accelerates relaxation. Adrenergic Receptor Signaling and Calcium Cycling ␤1-Adrenoceptor stimulation supports cardiac performance in several ways. First, ␤-receptor agonists increase ␤-adrenoceptormediated increases in Ca2⫹ entry during systole; increased Ca2⫹ entry increases fractional shortening of cardiac muscle during contraction. This positive inotropic effect results in a greater stroke volume for any given end-diastolic volume. ␤-Agonists also have a positive chronotropic effect, increasing heart rate in a relatively linear dose-dependent manner. The net effect of these inotropic and chronotropic effects is to increase cardiac output: CO ⴝ HR ⴛ SV

Equation 24–1

where CO is cardiac output, HR is heart rate, and SV is stroke volume. A third, but less widely appreciated, mechanism by B

4 Na+/Ca2+ exchange Ca2+

5 Na+/K+ ATPase

3Na+

3Na+

ATP

Ca2+

3Na+

β1-receptor

β1-agonist

Na+/K+ ATPase

NCX

Cardiac myocyte

2K+

Adenylyl cyclase

Ca2+

ADP

3Na+

2K+

6 Cytoplasm

Phospholamban

Ca2+ PKA

SERCA

Sarcoplasmic reticulum

β

ADP

Ca2+

P

γ

αs

P

Ca2+

GTP

ATP

Amrinone

ATP

Phosphodiesterase

Ca2+ AMP

cAMP

PKA

PKA

inactive

active

Phospholamban

Regulation of cardiac myocyte Ca2⫹ flux. A. During contraction: 1. Extracellular Ca2⫹ enters the cardiac myocyte through Ca2⫹ channels in the sarcolemma. 2. This trigger Ca2⫹ induces release of Ca2⫹ from the sarcoplasmic reticulum into the cytosol (so-called Ca2⫹-induced Ca2⫹ release). 3. The increased cytosolic Ca2⫹ facilitates myofibril contraction. B. During relaxation: 4. The Na⫹/ Ca2⫹ exchanger (NCX) extrudes Ca2⫹ from the cytosol, using the Na⫹ gradient as a driving force. 5. The Na⫹/K⫹ ATPase (sodium pump) maintains the Na⫹ gradient, thus keeping the cardiac myocyte hyperpolarized. The sodium pump is tonically inhibited by phospholemman; phosphorylation of phospholemman by protein kinase A (PKA) disinhibits the pump, thereby increasing sodium extrusion and indirectly enhancing Na⫹/Ca2⫹ exchange (not shown). 6. The sarcoendoplasmic reticulum Ca2⫹ ATPase (SERCA) in the sarcoplasmic reticulum membrane is tonically inhibited by phospholamban. Phosphorylation of phospholamban by PKA disinhibits the Ca2⫹ ATPase, allowing sequestration of cytosolic Ca2⫹ in the sarcoplasmic reticulum. A sarcolemmal Ca2⫹ ATPase (calcium pump) also helps to maintain calcium homeostasis by actively extruding calcium from the cytoplasm (not shown).

FIGURE 24-3.

A third mediator of SERCA activity is phospholamban, an SR membrane protein that inhibits SERCA. High levels of intracellular cAMP stimulate protein kinase A to phosphorylate phospholamban, which reverses its inhibition of SERCA (Fig. 24-3). Phospholamban thus controls the rate of relaxation

ADP

Ca2+

P

ATP

Ca2+ Sarcoplasmic reticulum

Regulation of cardiac contractility by ␤-adrenergic receptors. ␤-Adrenergic receptors increase cardiac myocyte contractility but also enhance relaxation. Binding of an endogenous or exogenous agonist to ␤1-adrenergic receptors on the surface of cardiac myocytes causes G␣ proteins to activate adenylyl cyclase, which in turn catalyzes the conversion of ATP to cAMP. cAMP activates multiple protein kinases, including protein kinase A (PKA). PKA phosphorylates and activates sarcolemmal Ca2⫹ channels and thereby increases cardiac myocyte contractility. PKA also phosphorylates phospholamban. The SERCA pump becomes disinhibited and pumps Ca2⫹ into the sarcoplasmic reticulum; the increased rate of Ca2⫹ sequestration enhances cardiac myocyte relaxation. Finally, PKA phosphorylates phospholemman, thereby disinhibiting the sarcolemmal sodium pump and enhancing sarcolemmal Na⫹/Ca2⫹ exchange (not shown ). cAMP is converted to AMP by phosphodiesterase, resulting in termination of ␤1adrenergic receptor-mediated actions. The phosphodiesterase is inhibited by amrinone (also known as inamrinone), a drug that can be used in the treatment of heart failure.

FIGURE 24-4.

428 Principles of Cardiovascular Pharmacology Failing myocardium

Normal myocardium A Calcium homeostasis Ca2+

3Na+

Ca2+

Ca2+

3Na+

Ca2+

NCX

Ca2+

NCX

Ca2+

3Na+

Ca2+ Phospholamban ADP

Ca2+

3Na+

Ca2+ Phospholamban

P

ATP SERCA

SERCA

Ca2+ Sarcoplasmic reticulum

Sarcoplasmic reticulum

B Contractile filaments ATP

Myosin

ATP

ADP

Actin

Myosin

ADP

Actin

P

TN-I

TN-I

TN-T

TN-C

TN-T

TN-C

C Adenylyl cyclase signaling pathway β-AR β1-agonist

β-AR Adenylyl cyclase

β

γ

Adenylyl cyclase

αs

αs P P

GTP

P P

ATP

cAMP

GTP ATP

cAMP

β-arrestin PKA

PKA

PKA

PKA

inactive

active

inactive

active

Cellular mechanisms of contractile pathophysiology. In the failing myocardium, there are derangements in Ca2⫹ homeostasis, the contractile elements, and the adenylyl cyclase signaling pathway. In each panel (A, B, and C), normal myocardium is shown on the left and failing myocardium on the right. A. In the normal myocardium, Ca2⫹ homeostasis is tightly controlled by Ca2⫹ channels, including the Na⫹/Ca2⫹ exchanger (NCX) and the Ca2⫹ ATPase (SERCA). Operation of these pathways allows the myocardium to relax during diastole. In the failing myocardium, diastolic Ca2⫹ remains elevated because phospholamban is not phosphorylated and therefore tonically inhibits SERCA. Also, the expression of NCX increases (large arrows), so that cytosolic Ca2⫹ is extruded from the cardiac myocyte rather than stored in the sarcoplasmic reticulum. B. In the normal myocardium, phosphorylation of troponin-I (TN-I) exposes the actin–myosin interaction site, and myosin effectively hydrolyzes ATP during each contraction cycle. In the failing myocardium, there is decreased phosphorylation of TN-I, resulting in less efficient actin–myosin cross-linking. Myosin does not hydrolyze ATP as efficiently (dashed arrow), further reducing the effectiveness of each contraction cycle. There is also increased expression of the fetal isoform of TN-T in the failing myocardium; the significance of this alteration is uncertain. C. In the normal myocardium, ␤-agonists stimulate cAMP formation and subsequent activation of protein kinase A (PKA). In the failing myocardium, ␤-arrestin binds to and inhibits the activity of ␤-adrenergic receptors (␤-AR), leading to decreased stimulation of adenylyl cyclase (dashed arrows). Expression of the inhibitory G␣ isoform G␣i is also induced in the failing myocardium (not shown ).

FIGURE 24-5.

2

Sarcolemma

Ca2+

1

Ca2+ extrusion 3Na+

3Na+

Ca2+

Na+/K+ -ATPase 3Na+

Ca2+ stores Ca2+

2K+ Digoxin

Na+/Ca2+ exchanger

3

Na+ extrusion

4

ADP P

stored Ca2+

ATP

Ca2+

3Na+

released Ca2+

2K+

Myofibril contraction

CHAPTER 24 / Pharmacology of Cardiac C ontractility 433

milrinone increase contractility and enhance the rate and extent of diastolic relaxation. PDE3 inhibitors also have important vasoactive effects in the peripheral circulation. These peripheral actions occur through cAMP-mediated effects on intracellular calcium handling in vascular smooth muscle and result in decreased arterial and venous tone. In the systemic arterial circulation, vasodilation leads to a decrease in systemic vascular resistance (decreased afterload); in the systemic venous circulation, an increase in venous capacitance results in a decrease in venous return to the heart (decreased preload). The combination of positive inotropy and mixed arterial and venous dilation has led to the designation of PDE inhibitors as “ino-dilators.” Similar to ␤-agonists, PDE inhibitors have found clinical utility in short-term support of the severely failing circulation. Widespread application of inamrinone has been limited by the adverse effect of clinically significant thrombocytopenia in about 10% of patients. Oral formulations of PDE3 inhibitors have been developed. Unfortunately, long-term use of these agents has been limited by data demonstrating increased mortality.

Calcium-Sensitizing Agents Calcium-sensitizing drugs, such as levosimendan, are a novel class of positive inotropes that are under investigation as possible therapeutic agents. Calcium sensitizers, which have the same “ino-dilator” actions as PDE inhibitors, augment myocardial contractility by enhancing the sensitivity of troponin C to calcium. This potentiating effect increases the extent of actin–myosin interactions at any given concentration of intracellular calcium, without a substantial increase in myocardial oxygen consumption. In the peripheral circulation, levosimendan activates ATP-sensitive K⫹ channels, leading to peripheral vasodilation. Preliminary clinical trial data suggest that levosimendan improves cardiac hemodynamics in severe systolic HF and may reduce short-term mortality. Levosimendan is available in some European countries but is not currently approved for use in the United States.

 CONCLUSION AND FUTURE DIRECTIONS Knowledge of the cellular and molecular bases for myocardial contraction has provided several pharmacologic strategies designed to increase myocardial contractility in patients with heart failure attributable to left ventricular systolic dysfunction. By inhibiting the sodium pump, digoxin raises intracellular calcium levels and thereby increases contractile force. This drug is the only oral inotropic agent in wide clinical use today. Although digoxin has no demonstrable impact on the mortality of patients with heart failure, it helps alleviate symptoms and improves functional capacity. Digoxin also slows AV nodal conduction, an effect that is useful in treating patients with atrial fibrillation and rapid ventricular response rates. The ␤-adrenergic receptor agonists— including the endogenous amines dopamine, norepinephrine,

and epinephrine and the synthetic agents dobutamine and isoproterenol—act through G protein-mediated elevation of intracellular cAMP to enhance both myocardial contractility and diastolic relaxation. The latter effect allows the left ventricle to fill adequately during diastole, despite the increase in heart rate that is stimulated by these agents. ␤-Agonists are administered intravenously, and they provide short-term hemodynamic support to patients with cardiogenic circulatory failure. The longer-term utility of these agents has been limited both by the lack of an oral formulation with acceptable bioavailability and by the adverse-effect profile of these drugs. PDE inhibitors, including inamrinone and milrinone, act as positive inotropes and as mixed arterial and venous dilators by increasing the levels of cyclic AMP in the heart and vascular smooth muscle. The increased mortality associated with longer-term use of these agents has similarly restricted their role to the short-term management of severe HF. New classes of pharmacologic agents are under investigation for their ability to augment myocardial contractility. These agents are directed at a variety of biochemical targets, including the efficiency of actin–myosin interactions (e.g., cardiac myosin activators) and the synthesis of contractile proteins (e.g., cardiac neuregulins). These approaches may improve cardiac contractility without increasing myocardial oxygen demand or significantly altering calcium signaling. Alternative strategies attempt to preserve myocardial contractility by inhibiting the effects of proinflammatory cytokines associated with HF, but recent trials of these agents, such as endothelin receptor antagonists, have met with limited success. Finally, gene therapy methods are being investigated to increase contractility, including the delivery of genes with cardiac-specific promoters that alter the production of contractile proteins, channels, and regulators in the heart. At the present time, the most promising candidates for gene therapy include the SR calcium pump, phospholamban, and cardiac troponin I.

Suggested Reading Endoh M. Cardiac calcium signaling and calcium sensitizers. Circ J 2008;72:1915–1925. (Physiology of excitation–contraction coupling and pharmacology of investigational agents for treatment of heart failure.) Gheorghiade M, Adams KF, Colucci WS. Digoxin in the management of cardiovascular disorders. Circulation 2004;109:2959–2964. (Reviews the clinical pharmacology of digoxin.) Libby P, ed. Braunwald’s heart disease: a textbook of cardiovascular medicine. 8th ed. Philadelphia: WB Saunders; 2008. (Encyclopedic reference that includes a good survey of pharmacologic agents, trials, and new approaches.) Lilly LS, ed. Pathophysiology of heart disease. 4th ed. Baltimore: Lippincott Williams & Wilkins; 2008. [Excellent introduction to cardiovascular medicine: Chapters 1 (Basic Cardiac Structure and Function), 9 (Heart Failure), and 17 (Cardiovascular Drugs) relate to the physiology, pathophysiology, and pharmacology of contractile function.] Peterson JW, Felker GM. Inotropes in the treatment of acute heart failure. Crit Care Med 2008;36:S106–S111. (Evidence underlying the use of inotropes, with emphasis on future directions.) Teerlink JR. A novel approach to improve cardiac performance: cardiac myosin activators. Heart Fail Rev 2009;14:289–298. (One of the possible future approaches to treatment of acute heart failure.)

25 Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure Ehrin J. Armstrong, April W. Armstrong, and Thomas P. Rocco

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 CASE, PART I: HYPERTENSION . . . . . . . . . . . . . . . . . . . . . . 438 PATHOPHYSIOLOGY OF HYPERTENSION . . . . . . . . . . . . . . . 438 Cardiac Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439 Vascular Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439 Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440 Neuroendocrine Function . . . . . . . . . . . . . . . . . . . . . . . . . 440 CLINICAL MANAGEMENT OF HYPERTENSION . . . . . . . . . . . 440 Reduction of Intravascular Volume . . . . . . . . . . . . . . . . . . 441 Diuretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Down-Regulation of Sympathetic Tone . . . . . . . . . . . . . . . 442 ␤-Adrenoceptor Antagonists . . . . . . . . . . . . . . . . . . . . 442 ␣-Adrenoceptor Antagonists . . . . . . . . . . . . . . . . . . . . 443 Central Sympatholytics . . . . . . . . . . . . . . . . . . . . . . . . 443 Modulation of Vascular Smooth Muscle Tone . . . . . . . . . . 443 Ca 2⫹ Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . 443 K ⫹ Channel Openers . . . . . . . . . . . . . . . . . . . . . . . . . 443 Modulation of the Renin-Angiotensin–Aldosterone System . . 444 Renin Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 Angiotensin Converting Enzyme Inhibitors . . . . . . . . . . 444 AT1 Antagonists (Angiotensin Receptor Blockers). . . . . 444 Monotherapy and Stepped Care . . . . . . . . . . . . . . . . . . . . 444 Possible Demographic Factors . . . . . . . . . . . . . . . . . . . . . 445 Hypertensive Crisis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445 CASE, PART II: ISCHEMIC HEART DISEASE . . . . . . . . . . . . . 446 PATHOPHYSIOLOGY OF ISCHEMIC HEART DISEASE . . . . . . 446 Chronic Coronary Artery Disease . . . . . . . . . . . . . . . . . . . 446 Coronary Flow Reduction . . . . . . . . . . . . . . . . . . . . . . 447 Endothelial Dysfunction. . . . . . . . . . . . . . . . . . . . . . . . 448 Acute Coronary Syndromes . . . . . . . . . . . . . . . . . . . . . . . 448 CLINICAL MANAGEMENT OF ISCHEMIC HEART DISEASE . . 449 Chronic Coronary Artery Disease . . . . . . . . . . . . . . . . . . . 450 ␤-Adrenoceptor Antagonists . . . . . . . . . . . . . . . . . . . . 450 Ca 2⫹ Channel Blockers . . . . . . . . . . . . . . . . . . . . . . . . 450 Nitrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451

Lipid-Lowering Agents . . . . . . . . . . . . . . . . . . . . . . . . 451 Metabolic Modulators . . . . . . . . . . . . . . . . . . . . . . . . . 452 Unstable Angina and Non-ST Elevation Myocardial Infarction . 452 Antianginal Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Heparin and Aspirin. . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Glycoprotein IIb–IIIa Antagonists . . . . . . . . . . . . . . . . . 453 ADP Receptor Antagonists. . . . . . . . . . . . . . . . . . . . . . 453 Direct Thrombin Inhibitors . . . . . . . . . . . . . . . . . . . . . . 453 ST Elevation Myocardial Infarction . . . . . . . . . . . . . . . . . . 453 Thrombolytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Primary Percutaneous Intervention . . . . . . . . . . . . . . . 454 Postmyocardial Infarction Management . . . . . . . . . . . . . . 454 CASE, PART III: HEART FAILURE . . . . . . . . . . . . . . . . . . . . . 455 PATHOPHYSIOLOGY OF HEART FAILURE . . . . . . . . . . . . . . . 455 Etiologies of Contractile Dysfunction . . . . . . . . . . . . . . . . 455 Cardiac Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 Frank–Starling Mechanism . . . . . . . . . . . . . . . . . . . . . 457 Cardiac Remodeling and Hypertrophy . . . . . . . . . . . . . 457 Neurohumoral Activation . . . . . . . . . . . . . . . . . . . . . . . 458 CLINICAL MANAGEMENT OF HEART FAILURE . . . . . . . . . . . 458 Preload Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Diuretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Aquaretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460 Aldosterone Receptor Antagonists . . . . . . . . . . . . . . . . 460 Venodilators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 Afterload Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 ACE Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 ␤-Adrenoceptor Antagonists . . . . . . . . . . . . . . . . . . . . 461 Vasodilators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 Inotropic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 Cardiac Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 Sympathomimetic Amines . . . . . . . . . . . . . . . . . . . . . 462 Phosphodiesterase Inhibitors . . . . . . . . . . . . . . . . . . . 462 Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 462 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 437

BP CO

HR

SVR

SV

Direct innervation

PSNS SNS Catecholamines

Circulating regulators Catecholamines ATII

Contractility

Preload

Local regulators NO Prostacyclin Endothelin ATII O2 H+ Adenosine

SNS Catecholamines

Venous tone

Intravascular volume

SNS Catecholamines

Thirst

Na+/H2O retention SNS Aldosterone ADH Natriuretic peptides

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 441 CCB Direct arterial vasodilators

BP

Hypertension Pharmacologic interventions

CO

SVR BP

HR

SV

Contractility

Direct innervation

Circulating regulators

Local regulators

Renin inhibitors

Endothelin antagonists Sodium nitroprusside Renin inhibitors ACE inhibitors AT1 antagonists

Preload

Venous tone

Renal perfusion

Sympathetic ouflow

Renin release

Tachycardia, contractility

Na+/H2O retention

Diuretics Renin inhibitors ACE inhibitors AT1 antagonists

Intravascular volume Vasoconstriction

Renin inhibitors

Baroreceptor reflex

Na+/H2O retention Diuretics Renin inhibitors ACE inhibitors AT1 antagonists

FIGURE 25-2.

Pharmacologic effects of commonly used antihypertensive agents. Antihypertensive agents modulate blood pressure by interfering with the determinants of blood pressure. Many of these antihypertensive drugs have multiple actions. For example, renin–angiotensin system blockers, such as ACE inhibitors and AT1 antagonists, alter the levels of local regulators and circulating regulators, and affect renal Na⫹ retention and venous tone. BP, blood pressure; CO, cardiac output; SVR, systemic vascular resistance; HR, heart rate; SV, stroke volume; CCB, Ca2⫹ channel blockers; ACE, angiotensin converting enzyme.

dose adjustments and/or the use of more than one agent to achieve long-term blood pressure control (Fig. 25-3).

BP

FIGURE 25-3. Compensatory homeostatic responses to antihypertensive treatment. When blood pressure is lowered by pharmacologic interventions, homeostatic responses are activated to increase blood pressure. These homeostatic responses can be divided broadly into baroreceptor reflexes and renal perfusion reflexes. Baroreceptor reflexes originating in the aortic arch and carotid sinus increase sympathetic outflow, leading to tachycardia, increased contractility, and vasoconstriction; these effects all increase blood pressure. Sympatholytics, such as ␤-antagonists, blunt the tachycardia and contractility responses by interrupting the sympathetic nervous system. ␣1-Antagonists inhibit vasoconstriction but have minimal effects on tachycardia or contractility. Decreased renal perfusion causes increased release of renin from juxtaglomerular cells of the kidney. Renin then cleaves angiotensinogen to angiotensin I, which, in turn, is activated to the potent vasoconstrictor angiotensin II (not shown). Angiotensin II increases adrenal secretion of aldosterone, which acts on principal cells of the collecting duct to increase Na⫹ (and, therefore, water) reabsorption. The increased Na⫹ reabsorption increases intravascular volume, and thereby results in increased blood pressure. Diuretics interrupt this homeostatic response by decreasing Na⫹ reabsorption from the nephron; renin inhibitors prevent the generation of angiotensin I; angiotensin converting enzyme (ACE) inhibitors interrupt the formation of angiotensin II; and AT1 antagonists prevent the target-organ signaling of angiotensin II.

Reduction of Intravascular Volume Diuretics Although diuretics have long been a cornerstone of antihypertensive therapy, the mechanism of action of diuretics in hypertension is incompletely understood. As discussed in Chapter 20, Pharmacology of Volume Regulation, diuretics decrease intravascular volume by increasing renal excretion of Na⫹ and H2O. However, volume depletion alone is unlikely to fully explain the antihypertensive effect of diuretics. Thiazide diuretics (e.g., hydrochlorothiazide) are the natriuretic drugs most commonly prescribed for the treatment of hypertension (Table 25-3). The pharmacokinetic and pharmacodynamic characteristics of the thiazides make them especially useful agents in the treatment of chronic hypertension. Thiazides have high oral availability and long duration of action. The initial antihypertensive effect seems to be mediated by decreasing intravascular volume. Therefore, thiazides are particularly effective in patients with volume-based hypertension, such as patients with primary renal disease and African-American patients. Thiazides induce an initial decrease in intravascular volume that decreases blood pressure by lowering cardiac output. However, the decrease in cardiac output stimulates the renin–angiotensin system, which leads to volume retention and attenuation of the effect

of the thiazide on volume status. It is hypothesized that a vasodilatory effect of the thiazides complements the compensated volume depletion, leading to a sustained decrease in blood pressure. This hypothesis is supported by the observation that the maximal antihypertensive effect of the thiazides is frequently achieved at doses lower than those needed to achieve a maximal diuretic effect. Therefore, thiazides achieve their blood pressure effect by influencing both cardiac output and systemic vascular resistance. The Joint National Commission (JNC) “Stepped-Care” algorithm suggests thiazide diuretics as the first-line agents of choice for the majority of patients, unless there is a specific indication for another antihypertensive drug (such as an ACE inhibitor in a patient with diabetes). This recommendation arises from the results of a large-scale trial, which found favorable outcomes and decreased cost associated with thiazide therapy. The practice at present is to initiate thiazide therapy at low doses (e.g., 12.5–25 mg/day); this recommendation represents a significant reduction in dose when compared with earlier iterations of the JNC guidelines. Loop diuretics (e.g., furosemide) are infrequently prescribed for the treatment of mild or moderate hypertension. These agents typically have a relatively short duration of

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 443

the long-term treatment of hypertension. One potential advantage of this drug is that the decrease in blood pressure achieved by reduction of systemic vascular resistance (via antagonism of ␣1-receptors) is not associated with the reflex increase in heart rate or cardiac output (because cardiac ␤1-receptors are also antagonized) that can occur when pure vasodilator drugs are used as monotherapy. In recent years, ␤-adrenoceptor antagonists have been used less frequently in the initial treatment of hypertension, due to clinical data suggesting that they may not be as efficacious as diuretics or inhibitors of the renin-angiotensin–aldosterone system. However, these agents are still important in the treatment of hypertension when there are other clinical indications for a ␤-adrenoceptor antagonist, such as coronary artery disease or heart failure. ␤-receptor antagonists are generally efficacious in the treatment of hypertension in younger patients. ␣-Adrenoceptor Antagonists ␣1-Adrenergic antagonists (e.g., prazosin, terazosin, doxazosin) are also used in the treatment of high blood pressure. ␣1-Adrenergic antagonists inhibit peripheral vasomotor tone, reducing vasoconstriction and decreasing systemic vascular resistance. The absence of adverse effects on the serum lipid profile during long-term treatment with ␣1-adrenergic antagonists is often cited as a distinctive advantage of these agents relative to other antihypertensive medications. However, the long-term benefit of this advantage, if any, remains to be determined in randomized clinical trials. Furthermore, in a large trial comparing different antihypertensives, there was an increased incidence of heart failure in the group randomized to doxazosin. Nonselective ␣-adrenergic antagonists (e.g., phenoxybenzamine, phentolamine) are not employed in the longterm treatment of hypertension, because excessive compensatory responses can result from their long-term use. For example, antagonism of central ␣2-adrenergic receptors disinhibits sympathetic outflow, resulting in unopposed reflex tachycardia. However, these agents are indicated for the medical treatment of pheochromocytoma. Central Sympatholytics The ␣2-adrenergic agonists methyldopa, clonidine, and guanabenz reduce sympathetic outflow from the medulla, leading to decreases in heart rate, contractility, and vasomotor tone. These drugs are available in oral formulations (clonidine is also available as a transdermal patch), and were widely used in the past despite their unfavorable adverseeffect profile. The availability of multiple alternative agents, as well as the current trend toward the use of multidrug regimens at submaximal doses, have substantially diminished the clinical role of ␣2-agonists in the treatment of hypertension. Ganglionic blockers (e.g., trimethaphan, hexamethonium) inhibit nicotinic cholinergic activity at sympathetic ganglia. These agents are extremely effective at lowering blood pressure. However, the severe adverse effects of combined parasympathetic and sympathetic blockade (e.g., constipation, blurred vision, sexual dysfunction, and orthostatic hypotension) have made ganglionic blockers of historic interest only. Some sympatholytic agents (e.g., reserpine, guanethidine) are taken up into the terminals of postganglionic adrenergic neurons, where they induce long-term depletion of neurotransmitter from norepinephrine-containing synaptic vesicles (see Chapter 10). These agents lower blood pressure by decreasing the activity of the sympathetic nervous system. However,

reserpine and guanethidine have little role in the contemporary treatment of hypertension because of their significant adverseeffect profiles, which include severe depression (reserpine) and orthostatic hypotension and sexual dysfunction (guanethidine).

Modulation of Vascular Smooth Muscle Tone As discussed in Chapter 21, vascular tone is dependent on the degree of vascular smooth muscle contraction. Vasodilators reduce systemic vascular resistance by acting on arteriolar smooth muscle and/or the vascular endothelium. The major mechanisms of action of the arterial vasodilators include blockade of Ca2⫹ channels and opening of metabotropic K⫹ channels. Ca 2 Channel Blockers Ca2⫹ channel blockers (e.g., verapamil, diltiazem, nifedipine, amlodipine) are oral agents that are widely used in the long-term treatment of hypertension. Calcium channel blockers (CCBs) have a variety of hemodynamic effects, reflecting the multiple sites at which calcium is involved in the electrical and mechanical events of the cardiac cycle and in vascular regulation. These agents can act as arterial vasodilators, negative inotropes, and/or negative chronotropes. The dihydropyridine agents nifedipine and amlodipine act primarily as vasodilators. In contrast, the nondihydropyridine drugs verapamil and diltiazem act principally as negative inotropes and chronotropes, thereby decreasing myocardial contractility, heart rate, and impulse conduction. Thus, CCBs can lower blood pressure through reduction of both systemic vascular resistance and cardiac output. CCBs are often used in combination with other cardioactive drugs, either as components of a multidrug antihypertensive regimen or for combined antihypertensive and antianginal treatment in patients with ischemic heart disease (IHD). Given the distinctive pharmacodynamic effects of the different CCBs, the potential adverse effects of CCB therapy (including adverse interactions with other cardiovascular therapies) are agent-specific. The nondihydropyridine agents should be used with caution in patients who have impaired left ventricular (LV) systolic function, as these agents can exacerbate systolic heart failure (see below). These agents should also be used with caution in patients with conduction system disease, as these drugs can potentiate functional abnormalities of the sinoatrial (SA) and atrioventricular (AV) nodes. Both of these cautions are particularly relevant in patients receiving concomitant ␤-antagonist therapy. K  Channel Openers Minoxidil and hydralazine are orally available arterial vasodilators that are occasionally used in the long-term treatment of hypertension. Minoxidil is a metabotropic K⫹ channel opener that hyperpolarizes vascular smooth muscle cells and thereby attenuates the cellular response to depolarizing stimuli. Hydralazine is a less powerful vasodilator with an uncertain mechanism of action. Both minoxidil and hydralazine can cause compensatory retention of Na⫹ and H2O as well as reflex tachycardia; these adverse effects are more frequent and more severe with minoxidil than with hydralazine. Concomitant use of a ␤-antagonist and a diuretic can mitigate these adverse effects. The use of hydralazine is limited by the frequent occurrence of tolerance and tachyphylaxis to the drug. In addition, increases in the total daily dose of hydralazine can be associated with a drug-induced lupus syndrome. Given the more favorable safety profile of the Ca2⫹ channel blockers, the use of minoxidil is now largely restricted to

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 447

Ischemic heart disease

This ability to modulate blood flow is referred to as the coronary flow reserve: CFR  maximal CBF/resting CBF

Chronic coronary artery disease (stable angina)

Un

st ab le

an

gi na N m o yo nca ST rd e ia l e l in va fa tio rc n tio n m yo S ca T rd e ia lev l in a fa tion rc tio n

Acute coronary syndromes

FIGURE 25-4. Classification of ischemic heart disease. Ischemic heart disease is divided into two broad categories: chronic coronary artery disease and acute coronary syndromes. Stable angina is the prototypical manifestation of chronic coronary artery disease. Acute coronary syndromes constitute a series (not necessarily a linear progression) of clinical presentations, including unstable angina, non-ST elevation myocardial infarction, and ST elevation myocardial infarction.

where CFR is coronary flow reserve and CBF is coronary blood flow. In healthy individuals, the maximal CBF is approximately five-fold greater than the resting CBF. Because of this wide safety margin, the resting CBF does not decrease until an epicardial stenosis exceeds 80–90% of the original arterial diameter. Changes in maximal CBF can be observed more readily with exercise, as maximal CBF begins to decrease during exercise when an epicardial stenosis exceeds 50–70% of the original arterial diameter. In patients with chronic CAD, the decrease in CFR is directly related to the severity of epicardial artery stenosis; the coronary

A Normal Endothelial cell

Patent lumen Normal endothelial function Platelet aggregation inhibited

Lumen

demand, leading to functional cardiac abnormalities (poor contraction of the ischemic portion of the myocardium) as well as clinical symptoms of CAD. The basic physiology of myocardial oxygen supply and demand is discussed in Chapter 21. Imbalances in myocardial oxygen supply and demand occur mainly as a result of coronary flow reduction and endothelial dysfunction. Coronary Flow Reduction The coronary vasculature is composed of two types of vessels: large, proximal epicardial vessels, and small, distal endocardial vessels. The epicardial vessels are the more frequent sites of atheroma formation; in disease states, total coronary artery blood flow is limited by the extent of epicardial vessel stenosis. In comparison, endocardial vessels regulate intrinsic coronary vascular resistance in response to local metabolic changes. When myocardial oxygen demand is increased, endocardial vessels dilate in response to local metabolic factors, resulting in a regional increase in myocardial blood flow and thereby providing increased oxygen to these metabolically active tissues. Angina pectoris (Fig. 25-5) is the principal clinical manifestation of chronic CAD. This symptom is characterized by precordial pressure-like discomfort resulting from myocardial ischemia. Most patients with chronic CAD experience stable angina, a clinical syndrome in which ischemic chest pain occurs at characteristic and reproducible workloads (e.g., walking up a flight of stairs). Pathologically, chronic CAD is associated with subintimal deposition of atheroma in the epicardial coronary arteries. In general, atherosclerotic plaques in patients with chronic stable angina are characterized by an overlying fibrous cap that is thick and resistant to disruption. The immediate cause of angina pectoris is an imbalance between myocardial oxygen supply and demand. Under normal physiologic conditions, coronary blood flow is modulated carefully to ensure adequate tissue perfusion in response to varying levels of myocardial oxygen demand.

B Stable angina Plaque

Lumen narrowed by plaque Inappropriate vasoconstriction

C Unstable angina Ruptured plaque

Platelet Plaque ruptured Platelet aggregation Thrombus formation Unopposed vasoconstriction Thrombus

D Variant angina

No overt plaques Intense vasospasm

FIGURE 25-5. Pathophysiology of anginal syndromes. A. Normal coronary arteries are widely patent, the endothelium functions normally, and platelet aggregation is inhibited. B. In stable angina, atherosclerotic plaque and inappropriate vasoconstriction (caused by endothelial damage) reduce the vessel-lumen diameter, and hence decrease coronary blood flow. C. In unstable angina, rupture of the plaque triggers platelet aggregation, thrombus formation, and vasoconstriction. Depending on the anatomic site of plaque rupture, this process can progress to non-Q wave (non-ST elevation) or Q wave (ST elevation) myocardial infarction. D. In variant angina, atherosclerotic plaques are absent, and ischemia is caused by intense vasospasm.

448 Principles of Cardiovascular Pharmacology

Relative coronary blood flow

5

4 Maximal coronary flow 3

2

Resting coronary flow

1

0 0

20

40

60

80

100

Percent occlusion of coronary artery

FIGURE 25-6. Effect of coronary artery occlusion on resting and maximal coronary blood flow. The dotted line depicts resting coronary blood flow, and the solid line represents maximal blood flow when there is full dilation of distal coronary arteries. Comparison of these two lines shows that maximal coronary blood flow is compromised when the lesion occludes more than about 50% of the arterial lumen, whereas resting coronary blood flow is relatively unaffected until the lesion exceeds about 80% of the arterial diameter. The y-axis represents coronary artery blood flow relative to the flow in a resting coronary artery with 0% occlusion.

flow reserve may be further impaired as a consequence of endothelial dysfunction (discussed below), resulting in a further reduction in CBF. During periods in which myocardial oxygen demand exceeds myocardial oxygen delivery, demand-related ischemia occurs, and the patient experiences angina pectoris. The degree of epicardial artery stenosis and the degree of compensatory endocardial artery dilation determine the hemodynamic consequence of an atherosclerotic plaque (Fig. 25-6). If the endocardial arteries are normal, an epicardial stenosis that narrows the diameter of the arterial lumen by less than 50% does not significantly reduce maximal coronary blood flow. However, if the stenosis narrows the arterial lumen diameter by more than 80%, then the endocardial vessels must dilate to provide adequate perfusion to the myocardium, even at rest. The need for endocardial vessels to dilate at rest attenuates coronary flow reserve, because the endocardial vessels cannot then dilate further during exercise. This reduction in coronary flow reserve leads to inadequate myocardial blood flow during hyperemic stress. Myocardial ischemia can occur at rest when the epicardial artery stenosis exceeds 90% of the lumen diameter: under these conditions, endocardial vessels cannot maintain adequate myocardial perfusion even at maximal dilation. Endothelial Dysfunction Endothelial dysfunction is a general term for pathologic endothelial cell regulation. Clinically, endothelial dysfunction is manifested by abnormal vascular tone and prothrombotic properties.

Abnormal vascular tone is a result of dysregulated endothelial control of smooth muscle contraction: arterial beds with endothelial dysfunction cannot dilate in response to hyperemic stimuli. For example, when mental stress or physical exertion triggers activation of the sympathetic nervous system (SNS), two opposing forces act on the coronary vascular endothelium: catecholamine-mediated vasoconstriction and nitric oxide (NO)-mediated vasodilation. Normally, endothelial release of NO is stimulated by the shear stress on the coronary vascular endothelium that results from increased blood flow. Eventually, the vasodilator effects of NO predominate over the vasoconstrictor effects of SNS activation, and the overall effect is coronary vasodilation. However, when the vascular endothelium is damaged, the production of endothelial vasodilators is decreased and catecholamine-mediated vasoconstriction predominates. Because the endothelium also plays a crucial role in regulating platelet activation and the coagulation cascade, endothelial dysfunction can promote blood coagulation (thrombosis) at the site of endothelial injury. Endothelial-derived NO and prostacyclin exert significant antiplatelet effects, and molecules on the surface of healthy endothelial cells have significant anticoagulant properties (see Chapter 22, Pharmacology of Hemostasis and Thrombosis). Endothelial damage decreases the ability of the endothelium to utilize these endogenous antiplatelet and anticoagulant mechanisms, leading to a local predominance of procoagulant factors and increasing the likelihood of platelet and coagulation factor activation.

Acute Coronary Syndromes Acute coronary syndromes (ACS) are most often caused by the fissuring or rupture of atherosclerotic plaques. These socalled unstable or vulnerable plaques are characterized by thin fibrous caps that are prone to rupture. Plaque rupture results in the exposure of procoagulant factors, such as subendothelial collagen (Fig. 25-7), that activate platelets and the coagulation cascade. Under physiologic circumstances, hemostasis at a site of vascular injury is self-limited by endogenous anticoagulant mechanisms (see Chapter 22). However, the dysfunctional endothelium overlying the atherosclerotic plaque cannot elaborate sufficient anticoagulant factors to control the extent of clot formation. Dysregulated coagulation can then result in intraluminal thrombus formation, which leads to myocardial ischemia and potentially to irreversible myocardial injury. The three subtypes of acute coronary syndromes are unstable angina, non-ST elevation MI, and ST elevation MI. In unstable angina, patients experience either acceleration in the frequency or severity of chest pain, new-onset anginal pain, or characteristic anginal chest pain that abruptly occurs at rest. Enzymatic evidence of tissue infarction (e.g., elevated troponin levels) is absent in unstable angina, but patients are at high risk for MI because of the presence of an active prothrombotic surface at the site of plaque rupture. Non-ST elevation myocardial infarction occurs when an unstable plaque abruptly ruptures and significantly compromises (but does not completely occlude) the lumen of an epicardial coronary artery. Because the artery is partially occluded and there is a persistent prothrombotic surface at the site of plaque rupture, patients with non-ST elevation MI are at high risk for recurrence of ischemia. The pathophysiology and clinical management of unstable angina and non-ST

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 449

A

B

C

D

E

FIGURE 25-7. Pathogenesis of acute coronary syndromes. A. A normal coronary artery has an intact endothelium surrounded by smooth muscle cells. B. Endothelial cell activation or injury recruits monocytes and T lymphocytes to the site of injury, leading to development of a fatty streak. C. Continued oxidative stress within a fatty streak leads to development of an atherosclerotic plaque. D. Macrophage apoptosis and continued cholesterol deposition cause further plaque organization, and may induce the expression of additional inflammatory proteins and matrix metalloproteinases. At this stage, the cap of the fibroatheroma remains intact. E. Continued inflammation within an atherosclerotic plaque leads to thinning of the fibrous cap and, eventually, to plaque erosion or rupture. Exposure of plaque constituents to the bloodstream activates platelets and the coagulation cascade, with resulting coronary artery occlusion.

elevation MI are very similar, and these two syndromes are often referred to by the combined acronym unstable angina/ non-ST elevation MI (UA/NSTEMI). If the intraluminal thrombus completely occludes the epicardial coronary artery at the site of plaque rupture, then blood flow ceases downstream from the locus of obstruction. Persistent, total epicardial artery occlusion provides the substrate for acute myocardial injury (ST elevation myocardial infarction; STEMI) that will progress inexorably to transmural infarction unless perfusion is reestablished. This clinical syndrome can also present as out-of-hospital sudden cardiac death (⬃30% of patients); in these cases, death is usually caused by ischemia-induced electrical instability of the myocardium. In the absence of fatal electrical instability, ST elevation MI typically presents with unremitting chest pain that is often accompanied by dyspnea and ischemic left heart failure. Mortality in STEMI is significantly reduced by prompt relief of the complete epicardial obstruction. Therefore, the principal management goal in STEMI is expeditious reperfusion of the occluded artery. The extent of myocardial necrosis following ischemic injury depends on the mass of myocardium supplied by the occluded artery, the amount of time over which the artery is totally occluded, and the degree of collateral circulation. Regions of the myocardium that are supplied directly and

exclusively by the occluded artery sustain extensive ischemic injury. Cell death occurs in a “wavefront” that progresses both spatially and temporally from the subendocardial region to the epicardial surface of the myocardium. As a result, the extent of “transmurality” of an MI bears a direct relationship to the duration of coronary artery occlusion. Adjacent to the region of transmural necrosis, a border zone of myocardium receives nutrients and oxygen from collateral vessels; this collateral perfusion can maintain the viability of border-zone cells for some period of time. However, in the absence of reperfusion of the occluded (infarct-causing) artery, lethal cardiomyocyte injury eventually occurs in these border zones as well.

 CLINICAL MANAGEMENT OF ISCHEMIC HEART DISEASE As noted above, both the pathophysiology and the clinical management of ischemic heart disease are different in patients with chronic coronary artery disease compared to patients with acute coronary syndromes. Chronic CAD results from an imbalance between myocardial oxygen supply and demand, and the treatment of chronic CAD focuses on modulating this balance, usually by reduction of oxygen demand. In comparison, treatment of ACS relies on reestablishing and

450 Principles of Cardiovascular Pharmacology

maintaining the patency of the occluded epicardial coronary artery as rapidly as possible. All patients with CAD, irrespective of clinical presentation, also require modification of underlying risk factors, including aggressive lipid-lowering therapy and blood pressure control.

Chronic Coronary Artery Disease The treatment goal in chronic CAD is to restore the balance between myocardial oxygen supply (coronary artery blood flow) and myocardial oxygen demand (myocardial oxygen consumption). Pharmacologic therapies concentrate on the reduction of myocardial oxygen demand, which is governed by heart rate, contractility, and ventricular wall stress (see Chapter 21). Antianginal drugs can be categorized on the basis of their impact on these parameters. ␤-Adrenoceptor Antagonists Activation of ␤1-adrenergic receptors by the sympathoadrenal system leads to an increase in heart rate, contractility, and conduction through the AV node. It follows that antagonists acting at ␤1-adrenergic receptors decrease sinus rate, reduce inotropic state, and slow AV nodal conduction. ␤1-Adrenoceptor antagonists (also referred to as “␤blockers”) are the cornerstone of medical treatment regimens in patients with chronic stable angina. ␤-Antagonists reduce myocardial oxygen demand by decreasing heart rate and contractility, and the drug-induced decrease in heart rate may also increase myocardial perfusion via prolongation of the diastolic filling time. When used in chronic angina, ␤-antagonists decrease both the resting heart rate and the peak heart rate achieved during exercise and delay the time to onset of angina. Dosing regimens for ␤-antagonists are drug-specific, reflecting the characteristic pharmacokinetics of each individual agent. As a general rule, the dose of drug is calibrated to maintain the resting heart rate at approximately 50 beats/min and to maintain the peak heart rate during exertion at approximately 110 to 120 beats/min. ␤-Antagonists are frequently co-administered with organic nitrates in patients with stable angina. This combination is often more effective than either agent used alone. ␤-Antagonists are also frequently combined with CCBs— typically, with agents of the dihydropyridine class (see below). (In early clinical trials, short-acting formulations of the dihydropyridine CCB nifedipine were associated with reflex tachycardia when administered as monotherapy; this tachycardia was attenuated when nifedipine was coadministered with a ␤-antagonist. In current practice, the availability of long-acting dihydropyridine agents has effectively diminished this adverse effect.) Although ␤-antagonists are generally well tolerated in patients with stable angina, certain clinical scenarios require caution. Combining ␤-antagonists with CCBs of the nondihydropyridine classes (e.g., diltiazem or verapamil) can result in synergistic suppression of SA-node automaticity (leading to extreme sinus bradycardia) and/or AV-node conduction (leading to high-grade AV conduction block). Likewise, because of their depressant effects on nodal tissues, ␤-antagonists may exacerbate preexisting bradycardia and/or high-grade AV block. However, given the clear and consistent mortality benefit associated with ␤-antagonists in secondary prevention trials, it is currently standard clinical practice to implant a permanent transvenous pacing device if such rhythm abnormalities are the major contraindication

to the use of ␤-antagonists, and then to administer the ␤antagonist. (Secondary prevention trials test the efficacy of pharmacologic interventions to reduce adverse cardiovascular events in patients with known CAD.) ␤-Antagonists are now also used in patients with clinically stable heart failure (see below). It must be emphasized that the survival benefit demonstrated in HF treatment trials occurred when these agents were initiated during periods of clinical stability. ␤-Antagonists must not be administered to patients with decompensated HF. When used in an attempt to treat the rare patient with pure vasospastic or variant angina (i.e., angina in the absence of epicardial artery obstruction; see Fig. 25-5), ␤-antagonists can induce coronary vasospasm as a consequence of unopposed ␣-receptor–mediated vasoconstriction. ␤-Antagonists can also exacerbate bronchospasm in patients with asthma and chronic airway obstruction. However, in such patients, the decision to exclude ␤-antagonists should be based on objective documentation of exacerbation of airflow obstruction during ␤-antagonist therapy. Peripheral vascular disease is another relative contraindication to ␤-antagonist therapy; the concern in this circumstance is the potential for antagonism of the ␤2-adrenergic receptors that mediate dilation of peripheral vessels. In clinical practice, however, this concern is rarely justified. Furthermore, patients with peripheral arterial disease have an extremely high risk of concomitant CAD and are therefore likely to benefit significantly from ␤-antagonist therapy. Common adverse effects of ␤-antagonists include fatigue, lethargy, insomnia, and impotence. Although the precise mechanism of fatigue is unclear, decreased exercise capacity is directly related to drug-induced blunting of the physiologic tachycardia of exercise. The impotence reported by 1% of patients treated with ␤-antagonists is due to inhibition of ␤2-adrenoceptor–mediated peripheral vasodilation. Ca2 Channel Blockers Calcium channel blockers (CCBs) decrease the influx of calcium through voltage-gated L-type calcium channels in the plasma membrane. The resulting decrease in intracellular calcium concentration leads to reduced contraction of both cardiac myocytes and vascular smooth muscle cells (see Chapter 21). Calcium channel blockers decrease myocardial oxygen demand and may also increase myocardial oxygen supply. Calcium channel blockers decrease myocardial oxygen demand by decreasing systemic vascular resistance and by decreasing cardiac contractility. In the periphery, calcium entry into vascular smooth muscle cells is required for contraction of the cells and is therefore a central determinant of resting vasomotor tone. By blocking calcium entry, CCBs cause relaxation of vascular smooth muscle and thereby reduce systemic vascular resistance. Calcium channel blockers can theoretically increase myocardial oxygen supply by blocking calcium-mediated increases in coronary vasomotor tone; the resulting dilation of epicardial vessels and arteriolar resistance vessels would, in theory, increase coronary blood flow. However, the contribution of this coronary vasodilator mechanism to the clinical effects of the CCBs is controversial, because regional metabolic abnormalities that result from myocardial ischemia should effect a maximal vasodilator response in the absence of pharmacologic modulation. The different classes of calcium channel blockers have distinctive inotropic effects on cardiac myocytes. Compared to

452 Principles of Cardiovascular Pharmacology

Metabolic Modulators Some patients with stable angina continue to experience frequent angina despite maximal attempts at medical management and revascularization. In these cases, metabolic modulators that increase the efficiency of ATP utilization may be clinically useful. In this class of drugs, ranolazine is approved for the second-line treatment of refractory angina despite otherwise maximal therapy. Clinical trials of ranolazine in stable angina have demonstrated improved effort tolerance and decreased frequency of anginal symptoms relative to placebo. Other metabolic modulators remain an active area of investigation and drug development.

latter circumstance, management strategies appropriate for unstable angina take precedence over those for stable CAD.) It is estimated that, in the absence of treatment, patients with UA have a 15–20% risk of progression to acute MI over a period of 4–6 weeks. Aggressive treatment can reduce this risk by more than 50%. Patients with UA have no overt evidence of myocardial damage, whereas patients with NSTEMI have elevated biomarkers that reflect cardiomyocte necrosis. Untreated UA may progress to NSTEMI, or NSTEMI may be the initial result of plaque rupture with extensive inflammation and coagulation at the rupture site. Goals of treatment in UA/NSTEMI are to relieve ischemic symptoms and to prevent additional thrombus formation at the site of plaque rupture. UA/NSTEMI is typically treated with aspirin, heparin, and ␤-antagonists. Other antiplatelet agents (GPIIb–IIIa antagonists and ADP receptor antagonists) and/or direct thrombin inhibitors (bivalirudin) are indicated in high-risk patients to prevent additional thrombus formation (Fig. 25-8). Although conventional antianginal

Unstable Angina and Non-ST Elevation Myocardial Infarction Unstable angina (UA) and non-ST elevation myocardial infarction (NSTEMI) may occur as the first presentation of CAD or in patients with a history of stable CAD. (In the

Ischemic heart disease

Chronic coronary artery disease (stable angina)

Acute coronary syndromes

Aspirin -Antagonists Nitrates Ca2+ channel blockers ACE inhibitors Ranolazine

Aspirin -Antagonists Nitrates

No ST elevation on ECG: Unstable angina or non-ST elevation myocardial infarction

ST elevation on ECG: ST elevation myocardial infarction

Thrombolysis

Add: Heparin GPIIb-IIIa antagonist ADP receptor antagonist or Bivalirudin ADP receptor antagonist

Add: Thrombolytic agent Heparin ADP receptor antagonist

Angioplasty

Add: Heparin GPIIb-IIIa antagonist ADP receptor antagonist or Bivalirudin ADP receptor antagonist

Post-myocardial infarction management

Possible addition of: Statin ACE inhibitor Aldosterone receptor antagonist Continue : Aspirin ADP receptor antagonist

FIGURE 25-8. Pharmacologic management of acute coronary syndromes. All patients with chronic coronary artery disease are given aspirin unless a life-threatening contraindication is present. ␤-Antagonists, nitrates, calcium channel blockers, ACE inhibitors, and ranolazine are primarily used to reduce myocardial oxygen demand. All patients with symptoms that raise concerns about a possible acute coronary syndrome are given aspirin and, if tolerated, a ␤-antagonist. Sublingual or intravenous nitrates can also be given to relieve chest discomfort and minimize ischemia. Electrocardiographic (ECG) findings of ST elevation should prompt emergency measures to open the occluded artery, either with a thrombolytic agent (thrombolysis) or mechanical revascularization (angioplasty). Additional adjunctive pharmacologic therapies for ST elevation myocardial infarction may include aspirin, ␤-antagonists, nitrates, heparin, ADP receptor antagonists, and GPIIb–IIIa antagonists or bivalirudin. For patients with acute coronary syndrome but no ST elevation on the electrocardiogram, laboratory assays of myocyte damage (e.g., troponin I or troponin T) determine whether the patient is classified as experiencing unstable angina or non-ST elevation myocardial infarction. In either case, management generally includes administration of aspirin, ␤-antagonists, nitrates, ADP receptor antagonists, and bivalirudin or heparin with GPIIb–IIIa antagonists. For all patients with acute coronary syndrome, postmyocardial infarction management should include modification of risk factors; possible addition of lipid-lowering agents (statins), ACE inhibitors, and aldosterone receptor antagonists; and continuation of aspirin and ADP receptor antagonists.

456 Principles of Cardiovascular Pharmacology

Ventricular emptying AV closes

ESP

LV Pressure (mm Hg)

Additional causes of systolic HF include chronic abnormalities of the loading conditions imposed on the heart, such as systemic arterial hypertension (pressure loading) and valvular heart disease (volume loading from mitral regurgitation or aortic insufficiency; pressure loading from aortic stenosis). The contractile performance of the myocardium is initially preserved in disease states associated with abnormal loading conditions, but cardiomyocyte injury and whole-organ contractile dysfunction supervene if the abnormal loading conditions are not corrected. The latter phase of cardiac pump dysfunction has been referred to as cardiomyopathy of chronic overload. Systolic dysfunction can also result from diverse conditions in which the proximate pathologic abnormality is cardiomyocyte injury or dysfunction. These conditions are referred to as dilated cardiomyopathies, because the heart characteristically remodels to produce LV chamber dilation (with or without wall thinning) in states of primary myocyte dysfunction. Symptomatic HF can also occur in patients with normal or near-normal LV systolic function (i.e., preserved LV ejection fraction). In such cases, the symptoms of left HF are caused by abnormalities of LV relaxation and/or filling (diastolic heart failure). Impaired relaxation results in an elevation of LV diastolic pressure at any given filling volume. This elevation of LV diastolic pressure causes elevation of left atrial and pulmonary capillary pressures, leading to transudation of fluid into the pulmonary interstitium (as well as secondary, or passive, elevation of pulmonary artery and right heart pressures). The most common acute cause of isolated diastolic HF is acute myocardial ischemia. In the setting of acute reversible ischemia (i.e., ischemia not associated with MI), LV diastolic pressures increase as a consequence of incomplete LV relaxation. (Recall from the discussion in Chapter 24, Pharmacology of Cardiac Contractility, that both contraction and relaxation of cardiomyocytes depend on adequate levels of intracellular ATP.) Both systolic and diastolic HF can be understood by considering the determinants of cardiac performance and the pathophysiologic conditions that affect these parameters. Although diastolic dysfunction is now appreciated as a common cause of clinical heart failure, the balance of this section will deal principally with heart failure due to systolic dysfunction. Each of the major factors affecting stroke volume— preload, afterload, and contractility—can be described by its effect on cardiac function curves. Figure 25-9 illustrates a normal LV pressure-volume loop. In the normal cycle, LV volume increases when the mitral valve opens during diastole. Isovolumetric contraction begins when LV pressure exceeds left atrial pressure and the mitral valve closes; during this segment of the cardiac cycle, intraventricular pressure increases while intracavitary volume remains constant. Ejection begins when the impedance to LV ejection is exceeded and the aortic valve opens; ejected blood is then transmitted to the systemic circulation by the elastic properties of the aorta. The aortic valve closes when LV pressure falls below aortic pressure; at this point, intraventricular pressure decreases rapidly (isovolumetric relaxation), up to (and perhaps beyond) the point at which the mitral valve opens, and the cycle is repeated. As illustrated in Figure 25-10A, the forward stroke volume ejected by the LV depends on the degree of LV filling during diastole, or preload. This fundamental relationship between preload and stroke volume is the Frank–Starling law; it derives from the relationship between muscle length

AV opens

Stroke volume

Relaxation

EDP

Contraction

MV closes MV opens

Ventricular filling

ESV

EDV

LV Volume (ml)

FIGURE 25-9.

Normal left ventricular pressure-volume loop. Mitral valve (MV ) opening allows the left ventricular (LV ) volume to increase as the chamber fills with blood during diastole. When ventricular pressure exceeds left atrial pressure, the mitral valve closes. During the isovolumetric phase of systolic contraction, the left ventricle generates a high pressure, which eventually forces open the aortic valve (AV ). Ejection of the stroke volume ensues, and the aortic valve closes when aortic pressure exceeds LV pressure. Isovolumetric relaxation returns the ventricle to its lowest pressure state, and the cycle is repeated. Stroke volume (i.e., the volume of blood ejected with each contraction cycle) is the difference between end-diastolic volume (EDV ) and end-systolic volume (ESV ). EDP, enddiastolic pressure; ESP, end-systolic pressure.

and degree of muscle shortening, as described in Chapter 24. In brief, increased diastolic volume increases myocardial fiber length. As a result, a higher fraction of the actin filament length is exposed in each sarcomere and is thereby available for myosin cross-bridge formation when the cardiomyocyte is depolarized. Impedance to LV ejection, or afterload, is the second determinant of stroke volume (Fig. 25-10B). As impedance to ejection (afterload) increases, the stroke output of the ventricle falls. This characteristic of the intact heart derives from the fact that increasing the resistance against which cardiac muscle must contract leads to a decrease in the extent of shortening (i.e., to reduced stroke volume). Because the sensitivity of stroke volume to outflow resistance is accentuated in the failing ventricle, agents that decrease afterload are able to increase LV stroke volume in patients with systolic HF (see below). A third determinant of cardiac performance is contractility, also described in Chapter 24. The contractile state of the LV is described by the end-systolic pressure-volume relationship (ESPVR, Fig. 25-10C). The ESPVR is, in effect, a variant of the Frank–Starling law. While the Frank–Starling law defines the relationship between LV diastolic volume (or preload) and LV stroke volume (or cardiac output), the ESPVR describes the relationship between diastolic filling volume and LV tension development during isovolumetric contraction. As shown in Figure 25-10C, an increase in the contractile state of the LV, reflected by an upward shift of the ESPVR, results in a greater degree of tension development for any given end-diastolic volume. In the presence of a fixed afterload, increased contractility results in a greater degree of muscle shortening and an increase in LV stroke volume.

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 457

A

B

C ESPVR-2

LV Pressure (mm Hg)

ESPVR

ESPVR-1

3

ESV 2 1 1

2

3

2

1

EDV

LV Volume (ml)

FIGURE 25-10. Determinants of cardiac output. Changes in preload, afterload, and myocardial contractility alter the pressure-volume relationship of the cardiac cycle. A. Increases in preload (lines 1, 2, 3) result in greater stretch of ventricular myocytes, development of greater ventricular end-diastolic pressure, and ejection of greater stroke volume (the Frank–Starling mechanism). Note that the end-systolic volume (ESV ) is the same in each case, because the contractility of the heart has not changed. B. Increases in afterload (points 1, 2, 3) create greater impedance to left ventricular output and result in proportionately decreased stroke volume (the difference between end-diastolic volume [EDV ] and ESV ). The end-systolic pressure is linearly related to ESV; this linear relationship is called the end-systolic pressurevolume relationship (ESPVR ). C. Increases in myocardial contractility (lines 1, 2), as occurs after administration of a positive inotrope, shift the ESPVR up and to the left, resulting in increased stroke volume.

A final determinant of cardiac pump performance is heart rate. However, if LV contractile performance is preserved, then impairment of cardiac output occurs as a consequence of abnormal heart rate only at extreme rates outside the physiologic range. Heart rate can be an important determinant of cardiac output in patients with systolic contractile dysfunction.

Cardiac Compensation As the ability of the myocardium to maintain normal forward output fails, compensatory mechanisms are activated to preserve circulatory function. The Frank–Starling mechanism increases stroke volume in direct response to increased preload. This recruitment of preload reserve is the first response of the system to hemodynamic stress. Hemodynamic stress that cannot be fully compensated by the Frank–Starling mechanism stimulates signaling systems that initiate structural changes at the cellular level, a process referred to as remodeling of the myocardium. Although the underlying stimuli for remodeling remain an active area of investigation, it has been noted that the specific pattern of remodeling is determined by the nature of the applied stress. If the Frank– Starling mechanism and remodeling mechanisms are unable to reestablish adequate forward cardiac output, neurohumoral systems are then activated. These systems modulate intravascular volume and vasomotor tone to maintain oxygen delivery to critical organs. Although each of these compensatory mechanisms contributes to the maintenance of circulatory function, each may also contribute to the development and progression of pump dysfunction and circulatory failure, as described below. Frank–Starling Mechanism In the intact heart, increased preload leads to increased stroke volume via the Frank–Starling mechanism. This mechanism remains operative in the failing heart; importantly, though, the relationship between end-diastolic volume and stroke volume is altered. In patients with systolic dysfunction, the

relationship between end-diastolic volume and stroke volume is characterized by a flatter plateau (Fig. 25-11). Therefore, unlike normal individuals who are operating on the ascending limb of the Starling curve, where volume expansion can be a useful strategy for increasing stroke volume, the majority of patients with heart failure operate with elevated intravascular volume. This increased intravascular volume reflects the end result of neurohumoral activation (i.e., the sympathoadrenal axis and the renin-angiotensin–aldosterone system; see below). Thus, the treatment of cardiogenic circulatory failure rarely involves volume expansion. It also merits emphasis that preload expansion can result in significant LV dilation, thereby increasing LV systolic and diastolic wall stress and exacerbating pulmonary congestion. Cardiac Remodeling and Hypertrophy In the setting of increased myocardial wall stress, cardiac hypertrophy develops in order to maintain ventricular systolic performance. Because LV ejection fraction is inversely proportional to wall stress, adaptations that decrease systolic wall stress increase LV ejection fraction. Laplace’s law states that wall stress (␴) is directly proportional to the pressure (P) and radius (R) of a chamber, and inversely proportional to wall thickness (h):   P  R/2h

Equation 25-1

In cases of chronic pressure overload, such as aortic stenosis or systemic hypertension, the LV develops a concentric pattern of hypertrophy as contractile proteins and new sarcomeres are added in parallel to the existing myofilaments. Concentric hypertrophy simultaneously increases wall thickness (h) and decreases cavity size (R), resulting in a net reduction in systolic wall stress and thereby preserving systolic performance. The disadvantage of concentric remodeling derives from the decrease in LV compliance that occurs as a consequence of this pattern of hypertrophy. In a ventricle with reduced compliance, diastolic pressure in the

458 Principles of Cardiovascular Pharmacology Symptoms of high end-diastolic pressure

Normal

D A

E

C

HF with positive inotrope Untreated HF

Normal

Cardiac output

Cardiac output

Symptoms of high end-diastolic pressure

HF with ACE inhibitor F

Afterload reduction

C G

Untreated HF

Preload reduction

B

Ventricular end-diastolic pressure

Symptoms of low cardiac output

Ventricular end-diastolic pressure

Symptoms of low cardiac output

FIGURE 25-11. The Frank–Starling relationship in heart failure (HF). Left panel: The normal Frank–Starling relationship shows a steep increase in cardiac output with increasing ventricular end-diastolic pressure (preload). Point A describes the end-diastolic pressure and cardiac output of a normal heart under resting conditions. With contractile dysfunction (untreated HF), cardiac output falls ( B ) and the Frank–Starling curve flattens, so that increasing preload translates to only a modest increase in cardiac output ( C ). This increase in cardiac output is accompanied by symptoms of high end-diastolic pressure, such as dyspnea. Treatment with a positive inotrope, such as digitalis, shifts the Frank–Starling curve upward, and cardiac output increases ( D ). The improvement in myocardial contractility supports a sufficient reduction in preload that the venous congestion is relieved ( E ). Right panel: Two of the principal pharmacologic treatments of HF are afterload reduction (e.g., ACE inhibitors) and preload reduction (e.g., diuretics). Afterload reduction ( F ) increases cardiac output at any given preload, and thereby elevates the Frank–Starling relationship. Preload reduction ( G ) alleviates congestive symptoms by decreasing ventricular end-diastolic pressure along the same Frank–Starling curve.

chamber is increased at any given filling volume. This in turn leads to elevation of LA and pulmonary capillary pressures, thereby predisposing to congestive symptoms. In conditions of chronic volume overload, such as mitral or aortic regurgitation, the LV develops an eccentric pattern of hypertrophy as contractile proteins and new sarcomeres are added in series to the existing myofilaments. Eccentric hypertrophy helps to maintain cardiac performance via modulation of diastolic wall stress. In contrast to the situation that occurs after concentric remodeling, eccentric hypertrophy is associated with increased LV compliance. The increase in compliance allows LV end-diastolic volume to increase without a significant elevation in left ventricular and left atrial diastolic pressures. This attenuation of the rise in chamber pressure allows the system to maintain forward cardiac output by a volume-driven increase in total stroke volume. During the compensated phase of eccentric hypertrophy, LV wall thickness increases in approximate proportion to the increase in chamber radius. Neurohumoral Activation Failure of the heart to provide adequate forward output activates several neurohumoral systems, often with deleterious consequences (Fig. 25-12). Decreased arterial pressure activates the baroreceptor reflex, stimulating release of catecholamines; in turn, the catecholamines produce tachycardia (via ␤1-receptors) and vasoconstriction (via peripheral ␣1-receptors). Stimulation of ␤1-receptors on renal juxtaglomerular (JG) cells promotes the release of renin. JG cells also release renin in response to the decreased renal perfusion that accompanies decreased cardiac output. Renin cleaves circulating angiotensinogen to angiotensin I, which is subsequently converted by angiotensin converting enzyme (ACE) to angiotensin II (AT II). AT II acts through AT1 receptors to

increase arterial vasomotor tone. AT II also activates several physiologic mechanisms that increase intravascular volume, including aldosterone release from the adrenal glands (thus promoting salt and water retention), vasopressin (ADH) release from the posterior pituitary gland, and thirst center activation in the hypothalamus. In addition, AT II appears to be an important mediator of vascular and myocardial hypertrophy. The tachycardia and increased intravascular volume that accompany activation of these neurohumoral mechanisms help to maintain forward cardiac output, and the systemic vasoconstriction provides a mechanism by which central regulatory centers can override local autoregulation of blood flow. Together, these mechanisms allow the cardiovascular system to maintain perfusion of critical organs in the setting of reduced cardiac output. However, sympathetic stimulation of the heart also increases myocardial oxygen demand by increasing both afterload (arteriolar constriction) and preload (retention of sodium and water). Continued sympathetic stimulation eventually results in down-regulation of ␤-adrenergic receptors, further impairing the ability of the system to maintain forward output. The central aim of the current pharmacologic management of HF is to modulate the action of these neurohumoral effectors (Fig. 25-13).

 CLINICAL MANAGEMENT OF HEART FAILURE The pharmacologic treatment of HF has expanded dramatically over the past three decades. Numerous large-scale clinical trials have demonstrated that the new, “load-active” therapies are associated with statistically significant reductions in morbidity and mortality in patients with HF. In addition, improvements in the detection and treatment of hypertension

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 459

Increased renin

β

Compromised cardiac function

Compromised cardiac function

Decreased arterial blood pressure

Decreased arterial blood pressure

Baroreceptor reflex

Baroreceptor reflex

Increased sympathetic outflow

α Increased aldosterone

Increased ATII

Vasoconstriction

Increased renin

Increased sympathetic outflow

Increased ATII

Vasoconstriction

ACE inhibitors Increased aldosterone

Vasodilators Spironolactone Diuretics

Increased afterload

Intravascular volume expansion

Increased afterload

Na+ retention

Na+ retention

Increased preload

Increased myocardial O2 demand

Worsening heart failure

FIGURE 25-12.

Neurohumoral effects of heart failure. Compromised cardiac function leads to decreased arterial blood pressure, which activates baroreceptors that increase sympathetic outflow. ␣-Adrenergic sympathetic outflow () causes vasoconstriction, an effect that increases afterload. The increased afterload creates a greater pressure against which the heart must contract, and thereby increases myocardial O2 demand. ␤-Adrenergic sympathetic outflow () increases juxtaglomerular cell release of renin. Renin cleaves angiotensinogen to angiotensin I, and angiotensin I is then converted to the active hormone angiotensin II (AT II). AT II has a direct vasoconstrictor action; it also increases aldosterone synthesis and secretion. Aldosterone increases collecting duct Na⫹ reabsorption, leading to intravascular volume expansion and increased preload. Together, the increased afterload and preload increase myocardial O2 demand. In the already compromised heart, these increased stresses can lead to worsening heart failure.

and the management of complex multivessel CAD have dramatically altered the clinical course of patients with contractile dysfunction. It is helpful to organize the treatment strategies for contractile dysfunction in patients who exhibit or are at risk to develop symptomatic heart failure according to the following physiologic goals: preload reduction, afterload reduction, and contractility enhancement (increased inotropy). Table 25-6 provides a summary of the hemodynamic effects and mechanisms of action of the drug classes that are commonly used to treat heart failure.

Preload Reduction Diuretics Diuretics have long been cornerstones of the pharmacologic management of patients with left ventricular failure and remain integral components of the treatment of patients with congestive symptoms and/or intravascular volume overload. However, despite the efficacy of these agents at reducing congestive symptoms, there is no evidence of a mortality benefit from treatment with either loop diuretics or thiazide diuretics.

Intravascular volume expansion

Increased preload

Venodilators

Increased myocardial O2 demand

Worsening heart failure

FIGURE 25-13. Pharmacologic modulation of the neurohumoral effects of heart failure. Many therapeutic agents used in the management of heart failure modulate the neurohumoral systems that are activated by compromised cardiac function. The renin-angiotensin–aldosterone system can be inhibited by (1) ␤adrenergic antagonists, which inhibit renin release by the juxtaglomerular cells of the kidney; (2) ACE inhibitors, which prevent the conversion of angiotensin I to the active hormone angiotensin II; and (3) spironolactone, which competitively antagonizes aldosterone binding to the mineralocorticoid receptor. Diuretics promote Na⫹ excretion, and thereby counteract the Na⫹ retention stimulated by activation of the renin-angiotensin–aldosterone system. Venodilators counteract the effect of intravascular volume expansion by increasing peripheral venous capacitance and thereby decreasing preload. Direct arterial vasodilators alleviate the ␣-adrenergic receptor-mediated and angiotensin II receptor-mediated vasoconstriction induced by increased sympathetic outflow. Cardiac glycosides, ␤-adrenergic agonists, and cardiac phosphodiesterase inhibitors are also used in HF to increase myocardial contractility (not shown).

The natriuretic agents most commonly used in HF are the loop diuretics furosemide and bumetanide. These drugs inhibit the Na⫹-K⫹-2Cl– co-transporter (NKCC2) in the thick ascending limb of Henle, resulting in increased excretion of sodium, potassium, and water. Thiazide diuretics such as hydrochlorothiazide are also used to treat congestive symptoms, particularly in patients with hypertensive heart disease and LV systolic dysfunction. Thiazides inhibit sodium and chloride reabsorption via the Na⫹-Cl⫺ co-transporter (NCC) in the distal convoluted tubule. Thiazides are less efficacious natriuretic agents than loop diuretics and are often ineffective as monotherapy for congestive symptoms in patients with chronic kidney disease. Thiazides are sometimes co-administered with loop diuretics in patients with reduced GFR and refractory volume overload, and in selected patients with HF in whom treatment with loop diuretics alone does not achieve adequate diuresis. (Refer to Chapter 20 for an extended discussion of diuretics.) In the introductory case, the decrease in

CHAPTER 25 / Integrative Cardiovascular Pharmacology: Hypertension, Ischemic Heart Disease, and Heart Failure 463

Ischemic Heart Disease Abrams J. Chronic stable angina. N Engl J Med 2005;352:2524–2533. (Clinical pharmacology of chronic coronary artery disease treatments.) American Heart Association 2005 guidelines for cardiopulmonary resuscitation and emergency cardiac care. Part 8: stabilization of the patient with acute coronary syndromes. Circulation 2005;IV(Suppl):89–110. (Emergency management of acute coronary syndromes.) Anderson JL, Adams CD, Antman EM, et al. ACC/AHA 2007 guidelines for the management of patients with unstable angina and non-ST elevation myocardial infarction. Summary article: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol 2007;50:652–726. (Current guidelines for evaluating and treating patients with unstable angina and non-ST elevation myocardial infarction.) Armstrong EJ, Morrow DA, Sabatine MS. Inflammatory biomarkers in acute coronary syndromes. Part I: introduction and cytokines. Part II: acute-phase reactants and biomarkers of endothelial cell activation. Part III: biomarkers of oxidative stress and angiogenic growth factors. Part IV: matrix metalloproteinases and biomarkers of platelet activation. Circulation 2006;113:72–75, 152–155, 289–292, 382–385. (Four-part series reviewing pathophysiology and clinical evidence concerning the role of inflammatory mediators in acute coronary syndromes.) Cannon CP, Braunwald E, McCabe CH, et al. Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med

2004;350:1495–1504. (Trial demonstrating clinical benefit for aggressive statin therapy after acute coronary syndrome.) Libby P. The molecular mechanisms of the thrombotic complications of atherosclerosis. J Int Med 2008;263:517–527. (Molecular basis of coronary artery atherosclerosis.)

Heart Failure ACCF/AHA 2009 focused update: guidelines for the diagnosis and management of heart failure in adults. J Am Coll Cardiol 2009;53:e1–e90. (Consensus guidelines for management of heart failure.) Jessup M, Brozena S. Heart failure. N Engl J Med 2003;348:2007–2018. (Clinical approach to heart failure.) Opie LH. Cellular basis for therapeutic choices in heart failure. Circulation 2004;110:2559–2561. (Molecular basis of heart failure therapeutics.) Stevenson LW. Clinical use of inotropic agents for heart failure: looking backward or forward. Part I: inotropic infusions during hospitalization. Part II: chronic inotropic therapy. Circulation 2003;108:367–372, 492–497. (Two-part series examining use of inotropic agents in heart failure.) Taylor AL, Ziesche S, Yancy C, et al. Combination of isosorbide dinitrate and hydralazine in blacks with heart failure. N Engl J Med 2004;351:2049–2057. (Trial showing mortality benefit in self-identified black patients.)

IV Principles of Endocrine Pharmacology

26 Pharmacology of the Hypothalamus and Pituitary Gland Anand Vaidya and Ursula B. Kaiser

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 465-466 HYPOTHALAMIC AND PITUITARY PHYSIOLOGY . . . . . . . . . 465 Relationship Between the Hypothalamus and Pituitary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Feedback Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468 PHYSIOLOGY, PATHOPHYSIOLOGY, AND PHARMACOLOGY OF INDIVIDUAL AXES . . . . . . . . . . . . . . . 468 Anterior Pituitary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . 468 Hypothalamic-Pituitary–Growth Hormone Axis . . . . . . 468

Hypothalamic-Pituitary–Prolactin Axis . . . . . . . . . . . . . 471 Hypothalamic-Pituitary–Thyroid Axis . . . . . . . . . . . . . . 472 Hypothalamic-Pituitary–Adrenal Axis. . . . . . . . . . . . . . 472 Hypothalamic-Pituitary–Gonadal Axis . . . . . . . . . . . . . 473 Posterior Pituitary Gland. . . . . . . . . . . . . . . . . . . . . . . . . . 474 Antidiuretic Hormone (ADH) . . . . . . . . . . . . . . . . . . . . 474 Oxytocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 476 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

 INTRODUCTION

(adenohypophysis) is derived from ectodermal tissue. The posterior pituitary (neurohypophysis) is a neural structure derived from the ventral surface of the diencephalon. The prefixes adeno- and neuro- denote the oral ectodermal and neural ectodermal origin of the anterior and posterior pituitary gland components, respectively. An intermediate lobe also exists in most mammals but is vestigial in humans. Although the anterior and posterior pituitary glands derive from different embryologic origins, the hypothalamus controls the activity of both lobes. The connection between hypothalamus and pituitary gland is one of the most important points of interaction between the nervous and endocrine systems. The hypothalamus acts as a neuroendocrine transducer by integrating neural signals from the brain and converting those signals into chemical messages (largely peptides) that regulate the secretion of pituitary hormones. In turn, the pituitary hormones alter the activities of peripheral endocrine organs. Hypothalamic control of the anterior pituitary gland occurs via hypothalamic secretion of hormones into the hypothalamic–pituitary portal vascular system (Fig. 26-1). The initial capillary bed of this portal system is formed from branches of the superior hypophyseal artery that fan around the axon terminals of hypothalamic neurons. Endothelial fenestrations in this capillary bed allow hypothalamic factors to be released into the bloodstream. These capillaries then coalesce into short veins that extend to the anterior pituitary gland. Upon arriving at the anterior pituitary, the veins

The hypothalamus and pituitary gland function cooperatively as master regulators of the endocrine system. Together, hormones secreted by the hypothalamus and pituitary gland control important homeostatic and metabolic functions, including reproduction, growth, lactation, thyroid and adrenal gland physiology, and water homeostasis. This chapter introduces the physiology and regulation of hypothalamic and pituitary hormones through a discussion of feedback regulation and the various axes of hormonal regulation. The pharmacologic utility of hypothalamic and pituitary factors is then discussed, with emphasis on the regulation of specific endocrine pathways. Three concepts are of special importance in this chapter: (1) hypothalamic control of pituitary hormone release; (2) negative feedback inhibition; and (3) endocrine axes. A thorough understanding of these pathways and their mechanisms provides the foundation for understanding the use of pharmacotherapy to modulate the hypothalamic–pituitary axes.

 HYPOTHALAMIC AND PITUITARY PHYSIOLOGY Relationship Between the Hypothalamus and Pituitary Gland From a developmental perspective, the pituitary gland consists of two closely associated organs. The anterior pituitary

465

Hypothalamic area

Optic chiasm Superior hypophyseal artery Hypothalamicpituitary portal system

Paraventricular and supraoptic nuclei

Inferior hypophyseal artery

To systemic circulation

Pituitary gland: Anterior lobe Posterior lobe

To systemic circulation

Hypothalamus CRH

Pituitary gland

ACTH

Cortisol

Adrenal gland

CHAPTER 26 / Pharmacology of the Hypothalamus and Pituitary Gland 469

gastric fundal cells during the fasting state, linking growth with nutritional status and energy balance. Nonpeptide, orally active ghrelin mimetics are currently under clinical investigation as GH secretagogues, and antagonists are being studied for appetite control. Several environmental factors inhibit GH release, including hyperglycemia, sleep deprivation, and poor nutritional status. The most significant endogenous biological factors that inhibit GH secretion are somatostatin, IGF-1, and GH. A Normal axis

Pathophysiology and Pharmacology of Growth Hormone Deficiency

Failure to secrete growth hormone or to enhance IGF-1 secretion during puberty results in growth retardation (Fig. 26-3A–D). GH deficiency most commonly results from defective hypothalamic release of GHRH (tertiary deficiency, Fig. 26-3D) or from pituitary insufficiency (secondary deficiency, Fig. 26-3C). Importantly, however, failure of IGF-1 secretion in response to GH (Laron B Growth hormone insensitivity

Ghrelin GHRH Somatostatin

Ghrelin GHRH Somatostatin

GH

GH

IGF-1

IGF-1

C Secondary deficiency Ghrelin GHRH Somatostatin

GH

IGF-1

D Tertiary deficiency Ghrelin GHRH Somatostatin

GH

IGF-1

E Growth hormone excess Ghrelin GHRH Somatostatin

GH

IGF-1

FIGURE 26-3. Hypothalamic-pituitary–growth hormone axis in health and disease. A. In the normal hypothalamic-pituitary–growth hormone axis, hypothalamic secretion of growth hormone-releasing hormone (GHRH) or ghrelin stimulates release of growth hormone (GH), while somatostatin inhibits release of GH. Secreted GH then stimulates the liver to synthesize and secrete insulin-like growth factor 1 (IGF-1), which promotes systemic growth. IGF-1 also inhibits GH release from the anterior pituitary gland. B. In GH insensitivity, the anterior pituitary gland secretes GH, but the liver is unresponsive to stimulation by GH. As a result, IGF-1 secretion is reduced (indicated by dashed lines). The decreased feedback inhibition of GH release results in higher plasma levels of GH (thick line). C. In secondary deficiency, the pathology lies in an unresponsive anterior pituitary gland, which secretes reduced amounts of GH. Because GH levels are low, the liver is not stimulated to produce IGF-1. D. In tertiary deficiency, the hypothalamus fails to secrete GHRH appropriately (dashed line); the role of ghrelin in this condition is unknown. Lack of sufficient GHRH results in lack of adequate stimulation of GH secretion by the anterior pituitary gland and, therefore, diminished production of IGF-1. E. In GH excess, GH is most commonly hypersecreted from an anterior pituitary adenoma. Elevated, and unregulated, GH levels result in increased hepatic production of IGF-1, and thus in systemic trophic effects. Because GH secretion occurs via an autonomous adenoma in the pituitary, negative feedback by IGF-1 is usually less effective.

TRH

Dopamine

PRL

Breasts/ Mammary tissue

Estrogen

Lactation

472 Principles of Endocrine Pharmacology

frequently used to treat prolactinomas have not shown a significant link to valvular heart disease to date. Hypothalamic-Pituitary–Thyroid Axis The hypothalamus secretes TRH, which stimulates thyrotrophs in the anterior pituitary gland to produce and secrete TSH. In turn, TSH promotes biosynthesis and secretion of thyroid hormone by the thyroid gland. Thyroid hormone regulates overall body energy homeostasis. Thyroid hormone negatively controls hypothalamic and pituitary release of TRH and TSH, respectively (see Fig. 27-4). Because thyroid hormone replacement is an effective therapy for hypothyroidism, TRH and TSH are used mainly for diagnosis of disease etiology. If hypothyroidism is caused by an unresponsive thyroid gland (primary deficiency), serum TSH levels will be high because of decreased negative feedback from thyroid hormone. For this reason, serum TSH is the main test used in screening for primary thyroid disease. TRH administration would produce an exaggerated increase in TSH, although this test is no longer used regularly in clinical practice. Conversely, if hypothyroidism is caused by a defect in pituitary TSH production (secondary deficiency), the TSH level will not be high despite the presence of low thyroid hormone levels. In this scenario, if TRH were to be administered, the normally expected rise in TSH would be absent or significantly reduced. Recombinant TSH (thyrotropin) is commonly used during radioactive iodine treatment of thyroid cancer. Thyrotropin is administered before radioactive iodine therapy to maximize uptake of radiolabeled 131I isotope into thyroid tissue in patients with thyroid cancer. This approach enables the administration of smaller quantities of radioisotope, maintaining maximum radiation exposure specifically to thyroid tissue with less radiation exposure to other tissues. Other aspects of thyroid gland pharmacology are discussed in Chapter 27, Pharmacology of the Thyroid Gland. Hypothalamic-Pituitary–Adrenal Axis Neurons from the paraventricular nucleus of the hypothalamus synthesize and secrete corticotropin-releasing hormone (CRH). CRH binds to cell-surface receptors on corticotrophs of the anterior pituitary gland and stimulates corticotrophs to synthesize and release adrenocorticotropinreleasing hormone (ACTH). ACTH is synthesized as part of proopiomelanocortin (POMC), a precursor polypeptide that is cleaved into multiple effector molecules. In addition to ACTH, cleavage of POMC yields melanocyte-stimulating hormone (MSH), lipotropin, and ␤-endorphin. MSH has effects on skin pigmentation. Because of the structural similarities between ACTH and MSH, high concentrations of ACTH can bind to and activate MSH receptors. This becomes important in primary hypoadrenalism, where increased ACTH levels result in enhanced skin pigmentation. ACTH stimulates the synthesis and secretion of adrenocortical steroid hormones, including glucocorticoids, androgens, and mineralocorticoids (Fig. 26-5A). ACTH is required for secretion of glucocorticoids and adrenal androgens. Mineralocorticoid production is also regulated by potassium balance and volume status, and ACTH has a relatively minor role in regulating mineralocorticoids. ACTH also has a trophic effect on the zona fasciculata and zona reticularis of the adrenal cortex (see Fig. 28-1); excessive ACTH secretion causes adrenal hyperplasia, while ACTH

A Normal axis

B Primary adrenal tumor/ Cushing's syndrome

CRH

ACTH

ACTH

Cortisol

Cortisol

C Pituitary adenoma/ Cushing's disease

ACTH

D Ectopic ACTH-secreting tumor

ACTH

Cortisol

Cortisol

Tumor

ACTH

FIGURE 26-5. Hypothalamic-pituitary–adrenal axis in health and disease. A. In the normal hypothalamic-pituitary–adrenal axis, hypothalamic secretion of corticotropin-releasing hormone (CRH) stimulates release of adrenocorticotropic hormone (ACTH). ACTH, in turn, stimulates synthesis and secretion of cortisol by the adrenal cortex. Cortisol inhibits further release of CRH and ACTH. B. A primary adrenal tumor causes Cushing’s syndrome by autonomously producing cortisol (thick line), independent of regulation by ACTH. The excessive cortisol production suppresses ACTH production (dashed line). C. An ACTH-producing pituitary adenoma causes Cushing’s disease by autonomously secreting excessive levels of ACTH (thick line), which stimulate the adrenal gland to produce increased levels of cortisol (thick line). ACTH secretion by the tumor has a blunted sensitivity to feedback inhibition by cortisol. D. An ectopic ACTH-secreting tumor (such as a small cell carcinoma of the lung) also stimulates the adrenal gland to produce increased levels of cortisol, which suppress pituitary ACTH production. However, circulating ACTH levels remain elevated due to the ectopic-source production of the hormone.

deficiency ultimately causes adrenal atrophy. Among the several steroid products of adrenal biosynthesis, cortisol is arguably the most crucial. In addition to serving as the main feedback inhibitor of pituitary ACTH release, cortisol functions as a “stress hormone” and is involved in vascular tone, electrolyte balance, and glucose homeostasis. Deficiency of cortisol can rapidly lead to critical illness or death, while cortisol excess results in Cushing’s syndrome (Fig. 26-5B).

CHAPTER 26 / Pharmacology of the Hypothalamus and Pituitary Gland 473

A synthetic form of ACTH, known as cosyntropin, can be used to diagnose suspected cases of adrenal insufficiency, and specifically to assist in ascertaining whether the insufficiency is primary or secondary. Administration of cosyntropin to a patient with primary adrenal insufficiency will fail to increase plasma cortisol concentration due to the inherent dysfunction of adrenal biosynthesis. Conversely, administration of cosyntropin to a patient with new-onset secondary adrenal insufficiency will result in a robust increase in plasma cortisol. However, patients with long-standing secondary adrenal insufficiency may have a blunted cortisol response to cosyntropin, owing mainly to progressive adrenal cortical atrophy in the absence of the trophic effects of ACTH. Conditions requiring physiologic replacement of glucocorticoids are usually treated with synthetic analogues of cortisol, rather than ACTH, because use of the target hormone generally allows for more precise physiologic control. Cortisol physiology and pharmacology are discussed in greater detail in Chapter 28, Pharmacology of the Adrenal Cortex. CRH is used as a diagnostic tool in petrosal sinus sampling for ACTH. It can be used to distinguish whether excessive cortisol secretion results from an ACTH-secreting pituitary adenoma or from an ectopic ACTH-secreting tumor (Fig. 26-5). If the hypercortisolism derives from a pituitary corticotroph adenoma (Cushing’s disease), administration of CRH will usually increase blood ACTH levels (Fig. 26-5C). This response is not seen in the case of an ACTH-secreting ectopic tumor, which secretes ACTH at a constant autonomous rate (Fig. 26-5D). Cushing’s syndrome resulting from primary adrenal tumors is often treated with surgical resection; however, several medical therapies also exist. Metyrapone, ketoconazole, and mitotane all have potent inhibitory effects on adrenal steroidogenesis and can be used to reduce cortisol production, while mifepristone antagonizes peripheral cortisol receptor binding (see Chapter 28). Hypothalamic-Pituitary–Gonadal Axis Gonadotrophs are unique among anterior pituitary gland cells because they secrete two glycoprotein hormones—LH and FSH. Together, these hormones are referred to as gonadotropins. LH and FSH are both heterodimers composed of ␣ and ␤ subunits. LH and FSH share the same ␣ subunit with TSH and hCG, but possess unique ␤ subunits. Gonadotrophs regulate the secretion of FSH and LH independently. This axis is diagrammed in Figure 26-6. Gonadotropins control hormone production by the gonads, promoting the synthesis of androgens and estrogens. The effects of estrogen and other reproductive hormones on the anterior pituitary gland are complex. In males, gonadotropins are inhibited via negative feedback by testosterone. In contrast, in females, depending on the rate of change and absolute concentration of estrogen, as well as the stage of the menstrual cycle, estrogen can exert both inhibitory and excitatory effects on gonadotropins. Inhibin is a hormone produced in the gonads that has inhibitory effects primarily on FSH secretion, with little effect on LH secretion. Activin is a paracrine factor that is produced and acts locally both in the pituitary and in the gonads, and functions in the pituitary gland to stimulate primarily FSH secretion (Fig. 26-6). Endocrine control of the reproductive process is discussed in greater detail in Chapter 29, Pharmacology of Reproduction.

GnRH (pulsatile)

GnRH (continuous)

Activin LH and

FSH

Estrogen (+/-) Inhibin (-)

Testosterone (-) Inhibin (-)

Ovaries or testes

FIGURE 26-6.

The hypothalamic-pituitary–gonadal axis. Gonadotropinreleasing hormone (GnRH) is secreted by the hypothalamus in a pulsatile fashion, stimulating gonadotroph cells of the anterior pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH stimulate the ovaries or testes to produce the sex hormones estrogen or testosterone, respectively, which inhibit further release of LH and FSH. Paradoxically, however, the increasing estrogen levels that are secreted from developing follicles during the follicular phase of the menstrual cycle induce a positive-feedback, mid-cycle ovulatory surge of LH and FSH secretion. Inhibin is also produced by the gonads in response to FSH and exerts negative feedback on gonadotrophs to inhibit further release of FSH. Locally produced pituitary activin acts in a paracrine fashion to stimulate FSH secretion. Exogenous pulsatile GnRH can be used to induce ovulation in women with infertility of hypothalamic origin. However, continuous administration of GnRH suppresses the gonadotroph response to endogenous GnRH, and thereby causes decreased production of sex hormones. Analogues of GnRH with increased metabolic stability and prolonged half-lives take advantage of this effect and are used to suppress sex hormone production in clinical conditions such as precocious puberty and prostate cancer.

Native GnRH, which has a short half-life, can be administered in a pulsatile fashion to stimulate patterned gonadotropin release, while GnRH analogues with longer half-lives are used to suppress production of sex hormones by desensitizing the pituitary gland to the stimulating activity of the releasing factor (Fig. 26-6). The main pharmacologic difference among the currently approved GnRH agonists is the method of administration. Leuprolide is the most commonly used GnRH agonist and can be administered as a daily subcutaneous injection or as a monthly depot injection. Osmotic pump implants (see Chapter 54, Drug Delivery Modalities) are also available that deliver leuprolide acetate at a controlled rate for up to 12 months. Long-acting agonists are utilized therapeutically to suppress gonadotropins in several conditions, including endometriosis, uterine fibroids,

474 Principles of Endocrine Pharmacology

precocious puberty, and androgen-dependent prostate cancer. Their main drawback is that gonadotroph suppression does not occur immediately; instead, there is a transient (several days) increase (“flare”) in sex hormone levels, followed by a lasting suppression of hormone synthesis and secretion. FSH is used clinically to stimulate ovulation for in vitro fertilization. Two Food and Drug Administration (FDA)approved formulations are available. Urofollitropin is purified FSH isolated from the urine of postmenopausal women, and follitropin is a recombinant form of FSH. Both agents effectively stimulate ovulation but may cause ovarian hyperstimulation syndrome. Interestingly, a rare form of ovarian hyperstimulation syndrome that occurs during pregnancy (familial gestational ovarian hyperstimulation syndrome) is caused by an inherited mutation in the FSH receptor. This mutation allows human chorionic gonadotropin (hCG), a hormone present in high concentrations during the early stages of pregnancy, to activate the FSH receptor. The resulting overstimulation of the FSH receptor is thought to cause the follicular enlargement and other sequelae characteristic of this syndrome. Whether similar mutations in the FSH receptor could be associated with cases of druginduced ovarian hyperstimulation syndrome is an area of active investigation. The GnRH antagonists cetrorelix and ganirelix are sometimes used in assisted reproduction; they suppress premature surges in LH in the early to mid-follicular phase of the menstrual cycle, resulting in improved rates of implantation and pregnancy (see Chapter 29, Pharmacology of Reproduction). GnRH antagonists also have applications for palliation of metastatic prostate cancer. In this situation, a direct GnRH antagonist has the advantage of avoiding the initial surge in testosterone caused by treatment with GnRH agonists.

Posterior Pituitary Gland In comparison to the numerous hormones of the anterior pituitary gland, the posterior lobe of the pituitary gland (neurohypophysis) secretes only two hormones, antidiuretic hormone (ADH) and oxytocin. ADH is an important regulator of plasma volume and osmolality, while oxytocin has physiologic effects on uterine contraction and lactation. Antidiuretic Hormone (ADH) ADH is a peptide hormone produced by magnocellular cells of the hypothalamus. Cells in this region possess osmoreceptors that sense changes in extracellular osmolality. Increased osmolality stimulates ADH secretion from nerve terminals in the posterior pituitary gland. ADH binds to two types of receptors, V1 and V2. V1 receptors, located in systemic arterioles, mediate vasoconstriction. This property gives ADH its alternative name, vasopressin. V2 receptors, located in the nephron, stimulate the cell surface expression of water channels in order to increase water reabsorption in the collecting duct, as discussed in Chapter 20, Pharmacology of Volume Regulation. These two actions of ADH combine to maintain vascular tone by: (1) increasing blood pressure; and (2) increasing water reabsorption. Disruption of ADH homeostasis results in two important pathophysiologic conditions. Excessive secretion of ADH causes the syndrome of inappropriate ADH (SIADH); deficient secretion of ADH or decreased responsiveness to ADH causes diabetes insipidus. In SIADH, ADH secretion

occurs irrespective of plasma volume status or osmolality. One of the most common causes of SIADH is the ectopic secretion of ADH by small cell carcinoma of the lung, but SIADH may be caused by a medication effect or result from almost any pulmonary process, central nervous system insult, or pituitary surgery. Excessive ADH secretion results in persistent stimulation of V1 and V2 receptors, causing hypertension and excessive water retention. The inappropriate water retention can result in low extracellular sodium concentration. Until recently, if the source of excess ADH could not be removed, the only effective therapy for SIADH was restriction of fluid intake or administration of hypertonic saline. Over the past decade, the discovery and clinical use of vasopressin receptor antagonists has provided more weapons in the arsenal to combat SIADH. Conivaptan and tolvaptan are vasopressin receptor antagonists that have recently been approved by the FDA for SIADH-induced hyponatremia. Tolvaptan is a specific V2 receptor antagonist approved for use in heart failure, while conivaptan is a mixed V1a and V2 receptor antagonist approved for use in euvolemic and hypervolemic hyponatremia. Both are available as oral agents. Demeclocycline (a tetracycline antibiotic; see Chapter 33, Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation) and lithium (see Chapter 14, Pharmacology of Serotonergic and Central Adrenergic Neurotransmission) are two other pharmacologic treatments that can also be used to treat SIADH. Both diabetes insipidus and diabetes mellitus are characterized by symptoms of thirst, polydipsia, and polyuria. Despite their phenotypic similarities, however, the etiologies of diabetes mellitus and diabetes insipidus are unrelated. Diabetes insipidus is a disorder of vasopressin deficiency or resistance, whereas diabetes mellitus is caused by deficient production of insulin or target tissue insensitivity to insulin (see Chapter 30, Pharmacology of the Endocrine Pancreas and Glucose Homeostasis). Diabetes insipidus is characterized by polyuria and polydipsia secondary to an inability to concentrate urine and retain free water at the level of the renal collecting duct. A distinction is made between two types of diabetes insipidus. Neurogenic diabetes insipidus results from an inability of hypothalamic neurons to synthesize or secrete ADH. In this condition, administration of the exogenous ADH analogue, desmopressin, results in stimulation of V2 receptors and a robust concentration of urine and decrease in thirst (Fig. 26-7). Nephrogenic diabetes insipidus results from an inability of renal collecting duct cells to respond to ADH (or, in other words, resistance to ADH). Nephrogenic diabetes insipidus can be caused by a mutation in the V2 receptor, such that ADH is unable to bind the receptor or stimulate receptor signaling, or by medication-induced resistance; lithium is one such medication. In nephrogenic diabetes insipidus, administration of desmopressin results in little or no change in urine concentration or thirst because of receptor insensitivity to ADH and its analogues. Patients with nephrogenic diabetes insipidus can be treated with diuretics such as amiloride or hydrochlorothiazide. The proposed mechanism by which these diuretics prevent excessive loss of free water is paradoxical: they induce a volume-contracted state, which promotes enhanced absorption of water in the proximal tubule and thereby decreases delivery of water to the site of ADH resistance, the collecting ducts.

CHAPTER 26 / Pharmacology of the Hypothalamus and Pituitary Gland 475

B

ADH

A

Water

V2-receptor

D

Pituitary gland No change in water channel expression Collecting duct cell ADH

ADH

Water

C Water

Water

V2-receptor

Pituitary gland

Water

Increased water channel expression

Missing or unresponsive V2-receptor

No change in water channel expression

Collecting duct cell

Desmopressin

Water Water

Water

V2-receptor

Increased water channel expression

FIGURE 26-7. Comparison of neurogenic and nephrogenic diabetes insipidus. A. Antidiuretic hormone (ADH), released by nerve terminals in the posterior pituitary gland, stimulates V2 receptors on renal collecting duct cells, and thereby increases expression of water channels in the apical membrane of these cells. Increased water channel expression increases water flux through the cell. B. In neurogenic diabetes insipidus, the posterior pituitary gland is unable to secrete ADH. Consequently, there is no stimulation of renal V2 receptors by ADH, and the collecting duct cells do not increase water channel expression. C. Exogenous administration of desmopressin, an ADH analogue, can replace the deficiency of posterior pituitary gland-derived ADH, and thereby treat neurogenic diabetes insipidus. D. In nephrogenic diabetes insipidus, the V2 receptor is either missing or unresponsive to stimulation by ADH. The lack of functional V2 receptors prevents the cell from responding to ADH with an increase in water channel expression.

Oxytocin Oxytocin is a peptide hormone produced by paraventricular cells of the hypothalamus. Many of the known physiologic roles of oxytocin involve muscular contraction; two such effects are milk release during lactation and uterine contraction. In the milk letdown response, stimuli to the hypothalamus cause oxytocin release into the blood from nerve terminals in the posterior pituitary gland. Oxytocin

causes contraction of myoepithelial cells surrounding the mammary gland alveoli. This is an important physiologic action during breast feeding. In addition, it has long been known that administration of oxytocin causes uterine contraction. Oxytocin release is probably not the physiologic stimulus for initiation of labor during pregnancy; however, oxytocin is used pharmacologically to induce labor artificially.

476 Principles of Endocrine Pharmacology

 CONCLUSION AND FUTURE DIRECTIONS Hormones of the hypothalamus and pituitary gland can be used as pharmacologic agents to modify the respective endocrine axes of each hormone. Recognizing the relationships and effects of primary, secondary, and tertiary disorders of any hypothalamic–pituitary axis is of paramount importance in understanding the appropriate diagnostic and treatment choices. Hypothalamic hormones can be used as diagnostics to determine the causes of underlying endocrine pathology (CRH, GHRH, TRH) or as therapeutics to suppress an axis (GnRH, somatostatin, dopamine). Hormones of the anterior pituitary gland can be given as replacement therapy in cases of deficiency (GH) or used diagnostically (ACTH, TSH). The posterior pituitary gland produces two hormones, ADH and oxytocin, which can be used to treat neurogenic diabetes insipidus and to induce labor, respectively. Future directions in hypothalamic and pituitary gland pharmacology will include: design of new drug delivery systems; synthesis of orally

active, nonpeptide analogues of hormones; and investigations to better understand hormone receptor mechanisms and signaling to assist in the design of new pharmacotherapies.

Acknowledgment We thank Ehrin J. Armstrong and Armen H. Tashjian, Jr. for their valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Hays R. Vasopressin antagonists – progress and promise. N Engl J Med 2006;355:2146–2148. (Perspective on SIADH and the future of vasopressin antagonists.) Melmed S. Acromegaly. N Engl J Med 2006;355:2558–2273. (Review of growth hormone pathophysiology and treatment for acromegaly.) Verhelst J, Abs R. Hyperprolactinemia. Treat Endocrinol 2003;2:23–32. (Review of the pathophysiology and management of hyperprolactinemia.)

27 Pharmacology of the Thyroid Gland Ehrin J. Armstrong, Armen H. Tashjian, Jr., and William W. Chin

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 480-481 THYROID GLAND PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . 480 Synthesis and Secretion of Thyroid Hormones . . . . . . . . . 480 Metabolism of Thyroid Hormones . . . . . . . . . . . . . . . . . . . 482 Effects of Thyroid Hormones on Target Tissues. . . . . . . . . 482 Hypothalamic-Pituitary–Thyroid Axis . . . . . . . . . . . . . . . . 483 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 484 Treatment of Hypothyroidism . . . . . . . . . . . . . . . . . . . . . . 484

Treatment of Hyperthyroidism . . . . . . . . . . . . . . . . . . . . . 485 Inhibitors of Iodide Uptake . . . . . . . . . . . . . . . . . . . . . 485 Inhibitors of Organification and Hormone Release . . . . 485 Inhibitors of Peripheral Thyroid Hormone Metabolism . 486 Other Drugs Affecting Thyroid Hormone Homeostasis. . . . 487 Lithium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 Amiodarone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 487 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

 INTRODUCTION

trachea. The main function of the thyroid gland is to produce the thyroid hormones, T3 and T4. Structurally, the thyroid hormones are built on a backbone of two tyrosine molecules that are iodinated and connected by an ether linkage (Fig. 27-1). An important structural feature of thyroid hormones is the placement of iodines on this backbone. The position and relative orientation of iodines attached to the tyrosine residues determine the specific form of thyroid hormone. 3,5,3ⴕ,5ⴕ-Tetraiodothyronine (thyroxine, T4) has four iodines attached to the tyrosine backbones and is the major form of thyroid hormone secreted by the thyroid gland. 3,5,3ⴕ-Triiodothyronine (T3) has three iodines. Most T3 is produced by peripheral 5⬘ deiodination of T4 (see below). A biologically inactive form of thyroid hormone is 3,3⬘,5⬘-triiodothyronine, also referred to as reverse triiodothyronine (rT3) because the single iodine is on the opposite tyrosine in the backbone relative to T3. In a normal individual, circulating thyroid hormone consists of about 90% T4, 9% T3, and 1% rT3, and most of the hormone is bound to plasma proteins (both specific binding proteins and albumin). Iodide is a trace element that is a crucial component of thyroid hormone structure. Thyroid follicular cells, which synthesize and secrete thyroid hormones, selectively concentrate iodide (I⫺) via a Na⫹/I⫺ symporter located on the basolateral membrane of the cell (Fig. 27-2). This active transport mechanism has the ability to concentrate iodide to intracellular concentrations up to 500 times that of plasma; most individuals have thyroid gland to plasma iodide ratios of approximately 30.

The thyroid gland has diverse and important effects on many aspects of metabolic homeostasis. Follicular thyroid cells constitute the majority of thyroid tissue; these cells produce and secrete the classical thyroid hormones: thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). Thyroid hormones regulate growth, metabolism, and energy expenditure, from oxygen consumption to cardiac contractility. Parafollicular C cells of the thyroid gland secrete calcitonin, a regulator of bone mineral homeostasis. Calcitonin is discussed in Chapter 31, Pharmacology of Bone Mineral Homeostasis. The major diseases of the thyroid gland involve disruption of the normal hypothalamic-pituitary–thyroid axis (see Chapter 26, Pharmacology of the Hypothalamus and Pituitary Gland). Replacement of deficient thyroid hormone is an effective and established therapy for hypothyroidism. Treatment of hyperthyroidism is more complex, with options including antithyroid drugs, radioactive iodide, and surgical excision of abnormal tissue. Understanding the pathways and mechanisms of feedback regulation of thyroid hormone synthesis and thyroid hormone actions serves to explain the rationale for effective drug treatment of thyroid diseases.

 THYROID GLAND PHYSIOLOGY Synthesis and Secretion of Thyroid Hormones The thyroid is an endocrine gland located in the neck inferior to the larynx and spanning the ventral surface of the 480

O H2N

I

OH

3

3'

I

5'

O

5

OH

I

I

Thyroxine (T4) Outer ring deiodination

Inner ring deiodination

O H2N

O

I

OH

3

I

3'

OH

H2N

I

OH

3

I

3'

OH

O

5 I

3,5,3'-Triiodothyronine (T3) (biologically active)

O

5' I

3,3',5'-Triiodothyronine (rT3) (biologically inactive)

482 Principles of Endocrine Pharmacology I-

Na+ Na+/ I- symporter

Extracellular space

TG

I-

Na+

Thyroid peroxidase (organification)

TG-MIT, TG-DIT Thyroid peroxidase (coupling)

Colloid space

T3

T4 TG

T3,T4

Follicular cell

T3

T4 Peripheral conversion T3

FIGURE 27-2.

Thyroid hormone synthesis, storage, and release. Follicular cells of the thyroid gland concentrate iodide (I⫺) from plasma via a basolateral membrane Na⫹/I⫺ symporter. In a reaction (called “organification”) catalyzed by thyroid peroxidase, intracellular iodide reacts covalently with tyrosine residues on thyroglobulin (TG) molecules at the apical membrane. Addition of one I⫺ to tyrosine results in the formation of monoiodinated tyrosine (MIT); addition of two I⫺ to tyrosine results in the formation of diiodinated tyrosine (DIT). MIT and DIT associate covalently on thyroglobulin via a mechanism known as “coupling,” which is also catalyzed by thyroid peroxidase. The derivatized thyroglobulin is stored as colloid within follicles in the thyroid gland. Upon stimulation by TSH, thyroid follicular cells endocytose colloid into lysosomal compartments, where the thyroglobulin is degraded to yield free T4, free T3, and uncoupled MIT and DIT. T3 and T4 are secreted into the plasma, and MIT and DIT are deiodinated intracellularly to yield free iodide for use in new thyroid hormone synthesis (not shown). The thyroid gland secretes more T4 than T3, although T4 is converted to T3 in peripheral tissues.

the follicular cells endocytose colloid. The ingested thyroglobulin enters lysosomes, where proteases digest the thyroglobulin. Proteolytic digestion releases free T3, T4, MIT, and DIT. T3 and T4 are transported across the follicular cell basolateral membrane and into the blood. Free MIT and DIT are rapidly deiodinated within the cell, allowing the iodide to be recycled for new thyroid hormone synthesis. Most endocrine organs concurrently synthesize and release new hormone when activated, rather than storing large quantities of precursor hormone. The thyroid gland is unusual among endocrine glands in that it stores large quantities of thyroid prohormone in the form of thyroglobulin. It is not understood why the thyroid gland maintains this elaborate pathway for hormone synthesis and release, but doing so makes it possible to maintain plasma thyroid hormone at a constant level despite fluctuations in the availability of dietary iodide.

Metabolism of Thyroid Hormones Thyroid hormone circulates mostly bound to plasma proteins, notably thyroid binding globulin (TBG) and transthyretin. Although T4 is the predominant thyroid hormone found in the blood, T3 has four times the physiologic activity of T4 on target tissues. Some serum T4 is inactivated by deamination, decarboxylation, or conjugation and excretion by the liver. Most T4, however, is deiodinated to the more active T3 form in several locations in the body. This deiodination reaction is catalyzed by the enzyme iodothyronine 5ⴕ-deiodinase (Fig. 27-1). There are three subtypes of deiodinase. Type I 5ⴕdeiodinase, expressed in the liver and kidneys, is important for converting T4 to the majority of serum T3. Type II 5ⴕdeiodinase is expressed primarily in the pituitary gland, brain, and brown fat. This enzyme is located intracellularly and converts T4 to T3 locally. Type III 5-deiodinase is responsible largely for conversion of T4 to the biologically inactive rT3. The presence of T4 in the blood provides a buffer, or reservoir, for thyroid hormone effects. Most T4 to T3 conversion occurs in the liver, and many pharmacologic agents that increase hepatic cytochrome P450 enzyme activity also increase T4 to T3 conversion. In addition, T4 has a half-life in the plasma of approximately 6 days, whereas plasma T3 has a half-life of only 1 day. Because T4 has a long plasma halflife, changes in thyroid hormone-regulated functions caused by pharmacologic intervention are generally not observed for a period of 1 to 2 weeks, as seen with Ms. L in the introductory case.

Effects of Thyroid Hormones on Target Tissues Thyroid hormones have effects on virtually every cell of the body. While the majority of the effects of thyroid hormones likely occur at the level of gene transcription, there is growing evidence that these hormones also act at the plasma membrane. Both modes of action are mediated by hormone binding to thyroid hormone receptors (TRs). Free hormone enters the cell by both passive diffusion and active transport, the latter mediated by hormone-specific and nonspecific carriers such as organic anion and monocarboxylate transporters. TRs are proteins containing thyroid hormone-binding, DNA-binding, and dimerization domains. There are two classes of thyroid hormone receptor, termed TR␣ and TR␤. In addition, both TR␣ and TR␤ can be expressed as multiple isoforms. TR monomers can interact in a dimerization reaction to form homodimers, or with another transcription factor, retinoid X receptor (RXR), to form heterodimers. These TR dimers bind to gene promoter regions and are activated by binding of thyroid hormones. Together, the multiple different combinations of TRs and the variability in their tissue distributions create tissue specificity for thyroid hormone effects. In the absence of hormone, thyroid hormone receptor dimers associate with corepressor molecules and constitutively bind to (and thereby inactivate) thyroid hormone-stimulated genes. Binding of thyroid hormone to TR:RXR or TR:TR dimers promotes dissociation of the corepressors and recruitment of coactivators to the DNA. Thus, thyroid hormone binding to TR dimers serves as a molecular switch from inhibition to activation of gene transcription (Fig. 27-3). Thyroid hormone also acts to down-regulate gene expression by a TR-dependent mechanism, the exact nature of which is not fully understood. For example, thyroid hormone is able to down-regulate TSH

CHAPTER 27 / Pharmacology of the Thyroid Gland 483

RxR TR

Decreased transcription

Corepressor

5'

3'

No thyroid hormone

T3 Coactivator

Corepressor

T3

RxR TR

DNA transcription

5'

3'

With thyroid hormone

FIGURE 27-3. Thyroid hormone receptor actions. In the absence of thyroid hormone, the thyroid hormone receptor (TR):retinoid X receptor (RXR) heterodimer associates with a corepressor complex, which binds to promoter regions of DNA and inhibits gene expression. In the presence of thyroid hormone (T3), the corepressor complex dissociates from the TR:RXR heterodimer, coactivators are recruited, and gene transcription occurs. This example demonstrates the action of T3 on a TR:RXR heterodimer, but similar mechanisms are probable for TR:TR homodimers. A useful therapeutic strategy in the future may involve pharmacologic targeting of tissue-specific corepressors or coactivators.

A Normal axis

Thyroid gland

Hypothalamic-Pituitary–Thyroid Axis Thyroid hormone secretion follows a negative regulatory feedback scheme similar to that of the other hypothalamicpituitary–target organ axes (Fig. 27-4). Thyrotropin-releasing

B Graves' disease

TRH

TSH

gene expression, causing negative feedback of thyroid hormone on the hypothalamic-pituitary–thyroid axis (see Chapter 26). Increasing evidence suggests that thyroid hormone also has nongenomic effects on mitochondrial metabolism and that it interacts with plasma membrane receptors to stimulate intracellular signal transduction. Thyroid hormone is important in infancy for growth and development of the nervous system. Congenital deficiency of thyroid hormone results in cretinism, a severe but preventable form of mental retardation. In the adult, thyroid hormone regulates general body metabolism and energy expenditure. Enzymes regulated by thyroid hormone include the Na⫹/K⫹ ATPase and many of the enzymes of intermediary metabolism, both anabolic and catabolic. At high levels of thyroid hormone, this effect can result in futile cycling and a consequent increase in body temperature—this is why Ms. L started turning down the heat in her home. Many of the effects of thyroid hormone resemble the effects of sympathetic neural stimulation, including increased cardiac contractility and heart rate, excitability, nervousness, and diaphoresis (sweating). These symptoms were also seen in Ms. L—she was nervous all the time and was startled by slight provocations. Conversely, low levels of thyroid hormone result in myxedema, a hypometabolic state characterized by lethargy, dry skin, coarse voice, and cold intolerance.

C Hashimoto's thyroiditis

TRH

TSH

Thyroid hormone

Thyroid hormone

Target tissues

Stimulatory autoantibody

TRH

TSH

Thyroid hormone

Thyroid hormone Target tissues

Destructive autoantibody

Thyroid hormone

Thyroid hormone Target tissues

FIGURE 27-4. The hypothalamic-pituitary–thyroid axis in health and disease. A. In the normal axis, thyrotropin-releasing hormone (TRH) stimulates thyrotropes of the anterior pituitary gland to release thyroid-stimulating hormone (TSH). TSH stimulates synthesis and release of thyroid hormone by the thyroid gland. Thyroid hormone, in addition to its effects on target tissues, inhibits further release of TRH and TSH by the hypothalamus and anterior pituitary gland, respectively. B. In Graves’ disease, a stimulatory autoantibody autonomously activates the TSH receptor in the thyroid gland, resulting in sustained stimulation of the thyroid gland, increased plasma thyroid hormone (thick lines), and suppression of TRH and TSH release (dashed lines). C. In Hashimoto’s thyroiditis, a destructive autoantibody attacks the thyroid gland, causing thyroid insufficiency and decreased synthesis and secretion of thyroid hormone (dashed lines). Consequently, feedback inhibition on the hypothalamus and anterior pituitary gland does not occur, and plasma TSH levels rise (thick lines).

Perchlorate Thiocyanate Pertechnetate

Extracellular space

TG

I-

Na+

I-

Na+

Thyroid peroxidase (organification)

Thioamines Iodides (high)

TG-MIT, TG-DIT Thyroid peroxidase (coupling)

Colloid space

T3

T4 TG

Iodides (high)

131I-

T3,T4

Follicular cell T3

T4 Peripheral conversion

Propylthiouracil

T3

CHAPTER 27 / Pharmacology of the Thyroid Gland 487

␤-adrenergic stimulation (e.g., sweating, tremor, tachycardia). It has also been demonstrated that ␤-blockers can reduce peripheral conversion of T4 to T3, but this effect is not thought to be clinically relevant. Because of its rapid onset of action and short elimination half-life (9 minutes), esmolol is a preferred ␤-adrenergic antagonist for the treatment of thyroid storm. Ipodate

Ipodate is a radiocontrast agent formerly used for visualization of the biliary ducts in endoscopic retrograde cholangiopancreatography (ERCP) procedures. In addition to its usefulness as a radiocontrast agent, ipodate significantly inhibits conversion of T4 to T3 by inhibiting the enzyme 5⬘-deiodinase. Although ipodate was sometimes used in the past to treat hyperthyroidism, it is no longer commercially available.

Other Drugs Affecting Thyroid Hormone Homeostasis Lithium Lithium, a drug used in the treatment of bipolar affective disorder (see Chapter 14, Pharmacology of Serotonergic and Central Adrenergic Neurotransmission), can cause hypothyroidism. Lithium is actively concentrated in the thyroid gland, and high levels of lithium have been shown to inhibit thyroid hormone release from thyroid follicular cells. There is some evidence that lithium may inhibit thyroid hormone synthesis as well. The mechanism(s) responsible for these actions is unknown. Amiodarone Amiodarone is an antiarrhythmic drug (see Chapter 23, Pharmacology of Cardiac Rhythm) that has both positive and negative effects on thyroid hormone function. Amiodarone structurally resembles thyroid hormone and, as a result, contains a large concentration of iodine (each 200-mg tablet of amiodarone contains 75 mg of iodine). Metabolism of amiodarone releases this iodine as iodide, resulting in increased plasma concentrations of iodide. The increased plasma iodide is concentrated in the thyroid gland; this can result in hypothyroidism by the Wolff–Chaikoff effect. Amiodarone can also cause hyperthyroidism by two mechanisms. In type I thyrotoxicosis, the excess iodide load provided by amiodarone leads to increased thyroid hormone synthesis and release. In type II thyroiditis, an autoimmune thyroiditis is induced that leads to release of excess thyroid hormone from the colloid. Because of its close structural similarity to thyroid hormone, amiodarone may also act as a homologue of thyroid hormone at the level of the receptor.

In addition, amiodarone competitively inhibits type I 5⬘deiodinase. This results in decreased peripheral conversion of T4 to T3 and increased plasma concentrations of rT3. Corticosteroids Corticosteroids, such as cortisol and glucocorticoid analogues, inhibit the 5⬘-deiodinase enzyme that converts T4 to the metabolically more active T3. Because T4 has less physiologic activity than T3, treatment with corticosteroids reduces net thyroid hormone activity. In addition, the decreased serum T3 results in increased release of TSH. The increased TSH stimulates greater T4 synthesis, until the amount of T4 produced generates a sufficient level of T3 to inhibit the hypothalamus and pituitary gland. Thus, when faced with decreased peripheral conversion of T4 to T3, the thyroid gland releases T4 at a higher rate, and serum T4 and T3 levels reach a new steady state.

 CONCLUSION AND FUTURE DIRECTIONS Thyroid hormone synthesis consists of a complex set of synthesis and degradation steps. This pathway creates numerous points for pharmacologic intervention, from iodide uptake to peripheral conversion of T4 to T3. Thyroid hormone replacement is a safe and effective long-term therapy for thyroid hormone deficiencies. Several effective therapies exist for the management of thyrotoxicosis. Radioactive iodide and thioamines are commonly used for this purpose, leading to selective destruction of the thyroid gland and antagonism of organification and coupling, respectively. Future potential therapies for diseases of the thyroid gland may focus on treating the etiology of autoimmune thyroid diseases, such as Graves’ disease and Hashimoto’s thyroiditis, and better defining the molecular targets of thyroid hormone action.

Suggested Reading Anonymous. Drugs for hypothyroidism and hyperthyroidism. Treat Guidel Med Lett 2006;4:17–24. (Review of therapeutic considerations, including important drug interactions.) Brent GA. Graves’ disease. N Engl J Med 2008;358:2594–2605. (Reviews the clinical approach to Graves’ disease and discusses other causes of hyperthyroidism.) Cooper DS. Antithyroid drugs. N Engl J Med 2005;352:905–917. (An excellent, detailed summary of the clinical uses and adverse effects of methimazole and propylthiouracil.) Davis PJ, Leonard JL, Davis FB. Mechanisms of nongenomic actions of thyroid hormone. Front Neuroendocrinol 2008;29:211–218. (Review of recent developments in thyroid hormone signaling.) Jonklaas J, Davidson B, Bhagat S, Soldin SJ. Triiodothyronine levels in athyreotic individuals during levothyroxine therapy. JAMA 2008;299:769– 777. (Clinical study suggesting that levothyroxine replacement alone is sufficient in individuals without a thyroid gland.)

28 Pharmacology of the Adrenal Cortex Rajesh Garg and Gail K. Adler

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 489-490 OVERVIEW OF THE ADRENAL CORTEX . . . . . . . . . . . . . . . . 489 GLUCOCORTICOIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Physiologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492 Pathophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 Adrenal Insufficiency. . . . . . . . . . . . . . . . . . . . . . . . . . 493 Glucocorticoid Excess . . . . . . . . . . . . . . . . . . . . . . . . . 494 Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . 494 Cortisol and Glucocorticoid Analogues. . . . . . . . . . . . . 494 Inhibitors of Adrenocortical Hormone Synthesis . . . . . . 498 Glucocorticoid Receptor Antagonists . . . . . . . . . . . . . . 499 MINERALOCORTICOIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Physiologic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 Pathophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 Aldosterone Hypofunction . . . . . . . . . . . . . . . . . . . . . . 500 Aldosterone Hyperfunction . . . . . . . . . . . . . . . . . . . . . 500 Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . 500 Mineralocorticoid Receptor Agonists . . . . . . . . . . . . . . 500 Mineralocorticoid Receptor Antagonists . . . . . . . . . . . 500 ADRENAL ANDROGENS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501 Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501 Pathophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501 Pharmacologic Classes and Agents . . . . . . . . . . . . . . . . . 502 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 502 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502

 INTRODUCTION

and there is considerable interest in the use of mineralocorticoid receptor antagonists as therapies for these disorders. Adrenal androgens, although lacking a definitive therapeutic indication at present, are the focus of investigation for use in female sexual dysfunction. Both deficiency and excess of adrenocortical hormones can cause human disease. Deficiency states are treated by replacing the hormones in the form of therapeutic agents, while inhibitors of adrenocortical biosynthetic enzymes can be used to treat hormone excess.

Like the pituitary gland, the adrenal gland consists of two organs fused together during embryologic development. The outer adrenal cortex originates from mesoderm, and the inner adrenal medulla is derived from neural crest cells. The adrenal cortex synthesizes and secretes steroid hormones essential for salt balance, intermediary metabolism, and, in females, androgenic actions. The adrenal medulla synthesizes and secretes the catecholamine epinephrine, which is important, although not essential, for maintaining sympathetic tone. This chapter focuses on the adrenal cortex; because of its importance in neuropharmacology, the adrenal medulla is discussed in Chapter 10, Adrenergic Pharmacology. The therapeutic utility of adrenocortical hormones spans almost every area of medicine. This is largely because of the usefulness of glucocorticoid analogues as efficacious and potent anti-inflammatory agents. Unfortunately, long-term systemic glucocorticoid therapy also produces a number of predictable but undesirable adverse effects. The physiology of mineralocorticoids has been studied in the etiology of hypertension, cardiovascular disease, and renal disease,

 OVERVIEW OF THE ADRENAL CORTEX The adrenal cortex synthesizes three classes of hormones: mineralocorticoids, glucocorticoids, and androgens. Histologically, the adrenal cortex is divided into three zones. Moving from the capsule toward the medulla, these regions are the zona glomerulosa, zona fasciculata, and zona reticularis (Fig. 28-1). The glomerulosa is responsible for mineralocorticoid production and is under the control of angiotensin II, blood potassium concentration, and, to a lesser extent, adrenocorticotropic hormone (ACTH). The 489

Zona glomerulosa Angiotensin II K+

Aldosterone synthase

Aldosterone

11β-hydroxylase 17α-hydroxylase

Cortisol, androgens

ACTH

Zona fasciculata/ reticularis

CHAPTER 28 / Pharmacology of the Adrenal Cortex 491

Feedback inhibition ACTH Anterior pituitary gland

Adrenal gland

Aminoglutethimide Ketoconazole (high)

Cholesterol

Trilostane Pregnenolone

Ketoconazole

17 Progesterone

17-hydroxypregnenolone 17

21 11-deoxycorticosterone Metyrapone

Trilostane 17-hydroxyprogesterone

11

Corticosterone

Dehydroepiandrosterone Trilostane Androstenedione

21 11-deoxycortisol Metyrapone

11

Aldosterone

Cortisol

Testosterone

Mineralocorticoids

Glucocorticoids

Sex steroids

FIGURE 28-2. Hormone synthesis in the adrenal cortex. The hormones of the adrenal cortex are steroids derived from cholesterol. The rate-limiting step in adrenal hormone biosynthesis is the modification of cholesterol to pregnenolone by side-chain cleavage enzyme. From this step, pregnenolone metabolism can be directed toward the formation of aldosterone, cortisol, or androstenedione. The flux of metabolites through each of these pathways depends on the tissue-specific expression of enzymes in the different cell types of the cortex and on the relative activity of the different synthetic enzymes. Note that several enzymes are involved in more than one pathway and that defects in these enzymes can affect the synthesis of more than one hormone. For example, a defect in steroid 21-hydroxylase prevents the synthesis of both aldosterone and cortisol. This overlap of synthetic activities also contributes to the nonselective action of glucocorticoid synthesis inhibitors such as trilostane. Enzymes are shown as numbers: 17, steroid 17␣-hydroxylase; 21, steroid 21-hydroxylase; 11, steroid 11␤-hydroxylase. Aminoglutethimide and high levels of ketoconazole inhibit side-chain cleavage enzyme. Ketoconazole also inhibits 17, 20-lyase. Trilostane inhibits 3␤-hydroxysteroid dehydrogenase. Metyrapone inhibits steroid 11␤-hydroxylase.

overall capacity, whereas albumin has low cortisol affinity but high overall capacity. Only molecules of cortisol that are unbound to protein (the so-called free fraction) are bioavailable, that is, available to diffuse through plasma membranes into cells. Thus, the affinity and capacity of plasma binding proteins regulate the availability of active hormone and, consequently, hormone activity. The liver and kidneys are the primary sites of peripheral cortisol metabolism. Through reduction and subsequent conjugation to glucuronic acid, the liver is responsible for inactivating cortisol in the plasma. The conjugation reaction makes cortisol more water soluble, thus enabling renal excretion. Importantly, the liver and kidneys express different isoforms of the enzyme 11␤-hydroxysteroid dehydrogenase, a regulator of cortisol activity. The two isoforms catalyze opposing reactions. In distal collecting duct cells of the kidney, 11␤hydroxysteroid dehydrogenase type 2 (11␤-HSD 2) converts cortisol to the biologically inactive compound cortisone, which (unlike cortisol) does not bind to the mineralocorticoid receptor (see below, Fig. 28-3B). Expression of 11␤-HSD 2

protects the mineralocorticoid receptor from activation by cortisol in a variety of cell types, including endothelial cells and vascular smooth muscle cells. In contrast, cortisone can be converted back to cortisol (also referred to as hydrocortisone) in the liver by 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD 1, Fig. 28-3A). The interplay between these opposing reactions determines overall glucocorticoid activity. In addition, as discussed below, the activity of these enzymes is important in glucocorticoid pharmacology. Physiologic Actions Like other steroid hormones, unbound cortisol diffuses through the plasma membrane into the cytosol of target cells, where the hormone binds to a cytosolic receptor. There are two types of glucocorticoid receptors: the Type I (mineralocorticoid) and Type II glucocorticoid receptors. The Type I receptor is expressed in the organs of excretion (kidney, colon, salivary glands, sweat glands) and other tissues including the hippocampus, vasculature, heart, adipose tissue, and peripheral blood cells. The Type II receptor has a broader

A

O

O OH

OH OH

O H H

OH

HO H

11β-HSD 1 (liver)

H

H

H

O

O Cortisone

Cortisol

O

B

O

OH

OH

OH

HO H H

OH

O H

11β-HSD 2

H

O

(kidney)

H

H

O Cortisol

Cortisone

(agonist at mineralocorticoid receptor)

(inactive at mineralocorticoid receptor)

CHAPTER 28 / Pharmacology of the Adrenal Cortex 493

Hypothalamus

Thermoregulatory centers

CRH

Immune stimulus

Fever Macrophages

Pituitary gland

Inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α)

ACTH

Cortisol

Adrenal gland

Mediators of inflammation (eicosanoids, serotonin, PAF, bradykinin)

FIGURE 28-4.

The immune–adrenal axis. Cortisol has profound immunosuppressive effects. Cortisol inhibits the action of several mediators of inflammation (eicosanoids, serotonin, platelet activating factor [PAF], bradykinin). Cortisol also inhibits the release of a number of cytokines from macrophages, including IL-1␣, IL1␤, IL-6, and TNF-␣. Because these cytokines in turn promote the hypothalamic release of CRH and thereby increase serum cortisol levels, it is hypothesized that the stress-induced increase in cortisol limits the extent of the inflammatory response.

CRH then travels through the hypothalamic–pituitary portal system and binds to G protein-coupled receptors on the surface of corticotroph cells in the anterior pituitary gland. CRH binding stimulates the corticotrophs to synthesize proopiomelanocortin (POMC), a precursor polypeptide that is cleaved into multiple peptide hormones including ACTH. The neurons in the paraventricular nucleus that respond to stress by synthesizing and secreting CRH can also respond to stress by synthesizing and secreting vasopressin. This vasopressin is released into the hypothalamic–pituitary portal system together with CRH, and it synergizes with CRH to increase the release of ACTH by the anterior pituitary gland. Interestingly, the stress-responsive parvocellular neurons that secrete CRH and vasopressin into the hypothalamic–pituitary portal system are different from the osmolality-responsive magnocellular neurons that synthesize vasopressin and transport this hormone to the posterior pituitary gland (see Chapter 26), even though both types of neurons are located in the paraventricular nucleus of the hypothalamus. Potential crosstalk between the parvocellular and magnocellular systems in the paraventricular nucleus is an area of active investigation. Proteolytic cleavage of POMC yields not only ACTH but also ␥-melanocyte–stimulating hormone (MSH), lipotropin, and ␤-endorphin. MSH binds to receptors on skin melanocytes, promoting melanogenesis and thereby increasing skin pigmentation. Because of the similarities between the ACTH and MSH peptide sequences, high concentrations of ACTH can also bind to and activate MSH receptors. This action becomes apparent in primary hypoadrenalism (see below), in which increased ACTH levels result in increased skin pigmentation. The role of lipotropin in human physiology is uncertain but is thought to involve control of lipolysis. ␤Endorphin is an endogenous opioid that is important for pain modulation and for regulation of reproductive physiology. Because steroid hormones are able to diffuse freely through cell membranes and because the adrenal gland

stores little cortisol, ACTH regulates cortisol production by promoting synthesis of the hormone. ACTH also has a trophic effect on the zona fasciculata and zona reticularis of the adrenal cortex, and hypertrophy of the cortex can occur in response to chronically elevated levels of ACTH. As in other endocrine axes, the hormone (cortisol) produced by the target organ (adrenal cortex) exerts negative feedback regulation at the level of both the hypothalamus and the anterior pituitary gland. High cortisol levels decrease both synthesis and release of CRH and ACTH. Because ACTH has important trophic effects on the adrenal cortex, the absence of ACTH leads to atrophy of the cortisolproducing zona fasciculata and the androgen-producing zona reticularis. However, the aldosterone-producing zona glomerulosa cells continue to function in the absence of ACTH, because angiotensin II and blood potassium continue to stimulate the production of aldosterone.

Pathophysiology Diseases affecting glucocorticoid physiology can be divided into disorders of hormone deficiency and disorders of hormone excess. Addison’s disease is the classic example of adrenocortical insufficiency, while Cushing’s syndrome exemplifies cortisol excess. Adrenal Insufficiency Addison’s disease is an example of a primary adrenal insufficiency in which the adrenal cortex is selectively destroyed, most commonly due to a T cell-mediated autoimmune reaction but alternatively due to infection, infiltration, cancer, or hemorrhage. Destruction of the cortex results in decreased synthesis of all classes of adrenocortical hormones. By comparison, secondary adrenal insufficiency is caused by hypothalamic or pituitary disorders or by prolonged administration of exogenous glucocorticoids. In secondary adrenal insufficiency, the decrease in ACTH levels causes decreased

O OH OH

HO H F O

6

16

H

CHAPTER 28 / Pharmacology of the Adrenal Cortex 495

O

A

O OH HO

OH

HO 11

H

OH

HO

OH OH

11

OH

O 11

H

H H

H

H

H

H

O

H

O

O Cortisol

Methylprednisolone

Prednisolone

O

O

OH

OH OH

HO 11

OH

HO 11

H

F

H

F

H

H

O

O

Fludrocortisone

Dexamethasone

B

O

O

OH

OH

OH

O 11

H

OH

O 11

H H

O

H

H H

O Prednisone

Cortisone

FIGURE 28-6. Glucocorticoid analogues. Panel A shows a number of 11-hydroxy glucocorticoids, while panel B shows two 11-keto congeners. Note that the drugs in A are physiologically active, while the drugs in B are prodrugs that must be activated by 11␤-HSD 1 to become active compounds. The structural class to which a glucocorticoid analogue belongs can be an important consideration in therapeutic decision making. For example, because the skin lacks significant 11␤-HSD 1 activity, only 11-hydroxy glucocorticoids can be used in topical glucocorticoid creams. HSD, hydroxysteroid dehydrogenase.

example, addition of a double bond between carbons 1 and 2 of cortisol creates prednisolone (Fig. 28-6), which has 4–5 times the anti-inflammatory potency of cortisol. Further addition of an ␣-methyl group (where ␣ is defined as the side-group orientation axial to the compound, while ␤ is the equatorial orientation) to carbon 6 of prednisolone creates methylprednisolone, which has an anti-inflammatory potency 5–6 times that of cortisol. Although prednisolone and methylprednisolone have significantly greater glucocorticoid potency than cortisol, their potency at the mineralocorticoid receptor is lower than that of cortisol. In contrast, addition of an ␣-fluorine (F) to carbon 9 of cortisol increases both the glucocorticoid and mineralocorticoid potencies of the resulting compound, known as fludrocortisone (Fig. 28-6). Because of its remarkably enhanced mineralocorticoid activity, fludrocortisone is useful in the treatment of mineralocorticoid deficiency states (see below). Dexamethasone incorporates two of the above changes to the cortisol backbone (1,2 double bond, 9␣ fluorine) as well as the addition of an ␣-methyl group at the 16-carbon position (Fig. 28-6). This compound has more than 18 times the glucocorticoid potency of cortisol, but virtually no mineralocorticoid activity.

A number of other permutations have been made to the cortisol backbone in other synthetic glucocorticoids, but the above discussion highlights the pertinent structural differences among the most common synthetic glucocorticoids. Clinically, it is most important to be aware of the potency of each agent relative to cortisol, especially when considering a change from one analogue to another that has different relative glucocorticoid and mineralocorticoid activities. In general, glucocorticoids used at pharmacologic doses should have minimal mineralocorticoid activity to avoid the consequences of mineralocorticoid excess (i.e., hypokalemia, volume expansion, and hypertension). Table 28-1 summarizes the relative glucocorticoid potencies and mineralocorticoid activities of several common glucocorticoid analogues. Duration of Action

The duration of glucocorticoid action is a complex pharmacokinetic variable that depends on: 1. Fraction of the drug bound to plasma proteins. More than 90% of circulating cortisol is protein-bound, primarily to CBG and, to a lesser degree, to albumin. In contrast, glucocorticoid analogues generally bind to CBG with low affinity. As a result, approximately 2/3 of a typical glucocorticoid

498 Principles of Endocrine Pharmacology

FIGURE 28-7.

O O O O

HO H

O

Cl

H

O Beclomethasone dipropionate

Structures of common inhaled glucocorticoids. Most of the inhaled glucocorticoids are halogenated analogues of cortisol that are highly potent glucocorticoid agonists with little mineralocorticoid activity (halogen atoms are shown in blue). Their high potency allows low doses of the inhaled glucocorticoids to inhibit the local inflammatory response that is a critical component of asthma pathophysiology. In addition, because a number of these compounds are subject to almost complete first-pass metabolism in the liver, the fraction of inhaled glucocorticoid that is inadvertently swallowed (80% of the inhaled dose) becomes inactivated so that it is not systemically bioavailable. The fraction of inhaled glucocorticoid delivered to the lung is eventually absorbed into the systemic circulation.

OH

dosing at many-fold higher local concentrations than could be achieved safely with systemic administration. The glucocorticoid that is administered must be biologically active because the skin has little, if any, of the 11␤-HSD 1 enzyme needed to convert glucocorticoid prodrugs to active compounds. Hydrocortisone, methylprednisolone, and dexamethasone are effective steroids for cutaneous use.

O O HO O

H H

H

O

Depot Glucocorticoids. Depot intramuscular preparations of glucocorticoid analogues last for days to weeks and can be an alternative to daily or alternate-day oral glucocorticoids in the treatment of inflammatory diseases. Although depot formulations reduce the necessity for daily oral administration, these preparations are seldom used because the dose cannot be titrated on a frequent basis. Depot preparations of methylprednisolone suspended in polyethylene glycol are commonly used, however, for intra-articular administration. This approach can be indicated for inflammatory processes restricted to the joints, such as rheumatoid arthritis or gout. Intra-articular glucocorticoid injection is useful in acute attacks of gout that are unresponsive to colchicine or indomethacin. Intra-articular and bursa injections require the use of active glucocorticoid, because joint tissue lacks 11␤HSD 1.

Budesonide

OH O O HO O

H H

H

O F Flunisolide

O O O HO

S

F

H F

H

O F Fluticasone propionate

O OH OH

HO H F

OH H

O Triamcinolone

Pregnancy. The placental–maternal barrier provides another example of selective glucocorticoid targeting. During pregnancy, the placenta metabolically separates the fetus from the mother. Because of this, prednisone can be administered to the mother during pregnancy without fetal side effects. The maternal liver activates the prednisone to prednisolone, but placental 11␤-HSD 2 converts the prednisolone back to inactive prednisone. Because the liver does not function during fetal life, the fetus does not, in turn, activate prednisone. Therefore, use of prednisone in pregnancy does not result in delivery of an active glucocorticoid to the fetus. Glucocorticoids promote lung development in the fetus. If glucocorticoid therapy is indicated to promote fetal lung maturation, dexamethasone is commonly administered to the mother. Dexamethasone is a poor substrate for placental 11␤-HSD 2, and therefore crosses the placenta in active form from the maternal circulation to the fetal circulation, where it stimulates lung maturation. The dose of dexamethasone must be titrated carefully because exposure to excessive glucocorticoid can have deleterious effects on fetal development.

Inhibitors of Adrenocortical Hormone Synthesis Several compounds are available to inhibit hormone biosynthesis by the adrenal cortex. Although these drugs have some specificity for individual adrenal enzymes (Table 28-2), it is

500 Principles of Endocrine Pharmacology

cell surface. The physiologic and pathophysiologic roles of this second signaling mechanism are an active area of investigation. In the kidney, aldosterone increases Na⫹/K⫹ATPase expression in the basolateral membrane of distal nephron cells. Enhanced Na⫹/K⫹ ATPase activity secondarily increases sodium reabsorption and potassium secretion across the lumenal epithelium of the nephron (see Chapter 20). As a result, sodium retention, potassium excretion, and H⫹ excretion are all enhanced by aldosterone. Increased sodium retention is accompanied by increased water retention and, thus, extracellular volume expansion. Excess aldosterone can cause hypokalemic alkalosis and hypertension, while hypoaldosteronism can cause hyperkalemic acidosis and hypotension. The mineralocorticoid receptor is also expressed in cells not involved in sodium reabsorption, including endothelial cells, vascular smooth muscle cells, cardiomyocytes, adipocytes, neurons, and inflammatory cells. Preclinical studies demonstrate a role for the mineralocorticoid receptor in the pathophysiology of vascular injury, atherosclerosis, heart disease, renal disease, and stroke. Activation of the mineralocorticoid receptor increases oxidative stress, promotes inflammation, regulates adipocyte differentiation, and reduces insulin sensitivity. In humans, antagonists of aldosterone action at the mineralocorticoid receptor, such as spironolactone and eplerenone, reduce morbidity and mortality in heart failure, improve vascular function, reduce cardiac hypertrophy, and reduce albuminuria. These beneficial effects of mineralocorticoid receptor blockade appear to be independent of changes in blood pressure. Regulation Three systems regulate aldosterone synthesis: the renin– angiotensin system, blood potassium levels, and ACTH. The renin-angiotensin–aldosterone system is a central regulator of extracellular fluid volume. Decreases in extracellular fluid volume decrease perfusion pressure at the afferent arteriole of the renal glomerulus, which acts as a baroreceptor. This stimulates the juxtaglomerular cells to secrete renin, a protease that cleaves the prohormone angiotensinogen to angiotensin I. Angiotensin I is then converted to angiotensin II by angiotensin converting enzyme, which is expressed at high concentrations by the capillary endothelium of the lungs. Angiotensin II has direct arteriolar pressor effects, and it stimulates aldosterone synthesis by binding to and activating a G protein-coupled receptor in zona glomerulosa cells of the adrenal cortex. Potassium loading increases aldosterone synthesis independent of renin activity. Because aldosterone activity at the distal nephron promotes potassium excretion, this control mechanism serves a homeostatic role in regulating potassium balance. Finally, ACTH acutely stimulates aldosterone synthesis in the zona glomerulosa. Changes in ACTH levels contribute to the circadian regulation of aldosterone and to the aldosterone rises associated with acute stress, such as hypoglycemia. Unlike cortisol, aldosterone does not negatively regulate ACTH secretion.

Pathophysiology Aldosterone Hypofunction Aldosterone hypofunction (hypoaldosteronism) can result from a primary decrease in aldosterone synthesis or action, or from a secondary decrease in aldosterone regulators such

as angiotensin II. Most cases of hypoaldosteronism result from decreased aldosterone synthesis. Defects in the gene coding for steroid 21-hydroxylase, an enzyme necessary for both aldosterone and glucocorticoid synthesis, lead to congenital adrenal hyperplasia (discussed under adrenal androgen pathophysiology) and cause salt wasting as a result of aldosterone deficiency. Addison’s disease, or primary adrenal insufficiency, results in hypoaldosteronism secondary to destruction of the zona glomerulosa. Most cases of Addison’s disease are caused by autoimmune adrenalitis; other causes of adrenal cortex destruction include tuberculosis, metastatic cancer, and hemorrhage. In each case, aldosterone hypofunction can lead to salt wasting, volume depletion, hyperkalemia, and acidosis. Hypoaldosteronism can also result from states of decreased renin production (socalled hyporeninemic hypoaldosteronism, which is common in diabetic renal insufficiency). Both resistance to the action of aldosterone at the level of the mineralocorticoid receptor and inactivating mutations of the aldosterone-regulated epithelial sodium channel (ENaC) in the cortical collecting duct of the nephron result in clinical hypoaldosteronism, despite normal to elevated aldosterone levels in the blood. Aldosterone Hyperfunction Primary hyperaldosteronism results from excess aldosterone production by the adrenal cortex. Bilateral zona glomerulosa adrenal hyperplasia and an aldosterone-producing adenoma are the two most common causes. Increased aldosterone synthesis leads to positive sodium balance, with consequent extracellular volume expansion, suppression of plasma renin activity, potassium wasting and hypokalemia, and hypertension. Independent of its effect on blood pressure, primary hyperaldosteronism also has adverse cardiovascular effects, including endothelial dysfunction, increased intimamedia thickness, vascular stiffness, and increased left ventricular wall thickness. Primary hyperaldosteronism is also a cause of insulin resistance.

Pharmacologic Classes and Agents Mineralocorticoid Receptor Agonists Pathophysiologic conditions leading to hypoaldosteronism necessitate replacement with physiologic doses of a mineralocorticoid. It is not possible to administer aldosterone itself as a therapeutic agent, because the liver converts more than 75% of oral aldosterone to an inactive metabolite during first-pass metabolism. Instead, the cortisol analogue fludrocortisone, which has minimal first-pass hepatic metabolism and a high mineralocorticoid-to-glucocorticoid potency ratio, is used. The adverse effects of fludrocortisone therapy are all related to the ability of this agent to mimic a state of mineralocorticoid excess, including hypertension, hypokalemia, and even cardiac failure. To ensure that an appropriate dose of drug is being administered, it is important to monitor serum potassium and blood pressure levels closely in all patients receiving fludrocortisone. Mineralocorticoid Receptor Antagonists Spironolactone (also discussed in Chapters 20 and 29) is a competitive antagonist at the mineralocorticoid receptor, but the drug also binds to and inhibits the androgen and progesterone receptors. The latter actions, which result in adverse effects such as gynecomastia in males, limit the usefulness

CHAPTER 28 / Pharmacology of the Adrenal Cortex 501

of this agent in some patient subsets. Eplerenone is a mineralocorticoid receptor antagonist that binds selectively to the mineralocorticoid receptor; this selectivity may make eplerenone free of the unwanted adverse effects of spironolactone. Both spironolactone and eplerenone can be used as antihypertensive agents, and both are approved for use in patients with heart failure. Antagonism of the mineralocorticoid receptor can result in significant hyperkalemia. Because many patients with heart failure are prescribed both spironolactone or eplerenone and an angiotensin converting enzyme inhibitor (which also raises blood potassium levels), it is important to monitor potassium levels closely in these patients.

 ADRENAL ANDROGENS Physiology Sex steroids produced by the adrenal cortex, primarily dehydroepiandrosterone (DHEA), have an uncertain role in human physiology. DHEA seems to be a prohormone that is converted to more potent androgens, primarily testosterone,

in the periphery. Adrenocortical androgens are an important source of testosterone in females; these hormones are necessary for the development of female axillary and pubic hair at the time of puberty, when adrenal androgen secretion is activated (adrenarche).

Pathophysiology Congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome are two important diseases related to adrenocortical androgen production. Congenital adrenal hyperplasia is a clinical term denoting a number of inherited enzyme deficiencies in the adrenal cortex. Enzyme defects leading to increased adrenocortical androgen production cause hirsutism and virilization in females. Polycystic ovarian syndrome, discussed in Chapter 29, may be caused by congenital adrenal hyperplasia in a subset of patients. The most common form of congenital adrenal hyperplasia results from a deficiency of steroid 21-hydroxylase. Deficiency of 21-hydroxylase results in the inability of adrenocortical cells to synthesize both aldosterone and cortisol (Fig. 28-8). Because cortisol is the main negative feedback

Feedback inhibition ACTH Anterior pituitary gland

Hyperplastic adrenal cortex

Cholesterol

Pregnenolone

Progesterone

17-hydroxypregnenolone

Dehydroepiandrosterone

17-hydroxyprogesterone

Androstenedione

21 11-deoxycorticosterone

21 Corticosterone

11-deoxycortisol

Aldosterone

Cortisol

Mineralocorticoids

FIGURE 28-8.

Glucocorticoids

Testosterone Sex steroids

Congenital adrenal hyperplasia. Steroid 21-hydroxylase deficiency, the most common cause of congenital adrenal hyperplasia, results in impaired biosynthesis of aldosterone and cortisol (dashed lines). Therefore, steroid hormone synthesis in the adrenal cortex is shunted toward increased production of sex steroids (thick lines). The lack of cortisol production decreases the negative feedback on corticotroph cells of the anterior pituitary gland (dashed line), causing increased ACTH release (thick blue arrow). Increased levels of ACTH induce adrenal hyperplasia and further stimulate the synthesis of sex steroids. This pathway can be interrupted by administering exogenous cortisol. The deficient enzyme is shown as a number: 21, steroid 21-hydroxylase.

502 Principles of Endocrine Pharmacology

regulator of pituitary ACTH release, the decreased cortisol synthesis that results from 21-hydroxylase deficiency disinhibits ACTH release. Increased ACTH restores the level of cortisol in patients with partial enzyme defects, but there is also shunting of precursor compounds into the “unblocked” androgen pathway, resulting in greater production of DHEA and androstenedione. The liver subsequently converts these compounds into testosterone. In severe 21-hydroxylase deficiency, there may be a virilizing effect on the developing female fetus. As a result, female neonates with severe 21hydroxylase deficiency typically have masculinized or ambiguous external genitalia. In the male neonate, however, increased adrenal androgens may have little or no noticeable phenotypic effect. Infants with severe 21-hydroxylase deficiency are commonly diagnosed in infancy during an acute salt-wasting crisis, which results from the inability to synthesize aldosterone and cortisol. Mild 21-hydroxylase deficiency may manifest later in life as hirsutism, acne, and oligomenorrhea in young women after menarche. Treatment of congenital adrenal hyperplasia due to severe enzyme defects requires physiologic replacement doses of glucocorticoids and mineralocorticoids. Treatment of congenital adrenal hyperplasia in patients with mild enzyme defects may include therapy with exogenous glucocorticoid to suppress excessive hypothalamic and pituitary release of CRH and ACTH, and thus to decrease production of adrenal androgens.

reabsorption and fluid retention. Cortisol regulates diverse physiologic processes, including energy homeostasis and inflammatory responses. The physiologic role of adrenal androgens is unknown, but pathophysiologic states causing increased adrenal androgen production have significant masculinizing effects in women. Antagonists of aldosterone are currently used to control high blood pressure. However, accumulating evidence suggests that antagonists specific for the aldosterone receptor may also become important therapies for cardiovascular and renovascular diseases in heart failure and diabetes as well as hypertension. Aldosterone synthase inhibitors are being developed and may be used in the future to reduce aldosterone production. Glucocorticoid pharmacology is an immense field, primarily because glucocorticoids are used to suppress inflammation in a number of disease states. Chronic glucocorticoid use is associated with a multitude of predictable adverse effects, and future research in this area will attempt to minimize the adverse effects of glucocorticoid therapy while maintaining the anti-inflammatory actions. Such efforts could include the development of tissue-selective glucocorticoid agonists and antagonists (analogous to the selective estrogen receptor modulators), as well as further refinement of drug delivery methods. The pharmacology of adrenal androgens needs to be studied more extensively to determine the indications, if any, for DHEA therapy.

Pharmacologic Classes and Agents

Acknowledgments

The androgens synthesized by the adrenal gland can be viewed as prohormones. Because no specific receptors for either DHEA or androstenedione have been described, the activity of these hormones depends on their conversion to testosterone, and subsequently to dihydrotestosterone, in peripheral target tissues. As discussed above, adrenal androgen excess can cause a variety of syndromes in women; the pharmacologic interruption of excessive androgenic activity is discussed in Chapter 29. DHEA is not regulated by the Food and Drug Administration (FDA) and is commonly used as an “over the counter” drug. Population cross-sectional studies have shown a reciprocal relationship between an age-related decline in DHEA levels and the risk of cardiovascular disease and cancer. Replacement therapy with DHEA may be indicated for cases of Addison’s disease in which there is bona fide DHEA deficiency. Exogenous DHEA can be converted to testosterone by the liver. As a result, DHEA is commonly abused for its anabolic effects.

We thank Ehrin J. Armstrong and Robert G. Dluhy for their valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

 CONCLUSION AND FUTURE DIRECTIONS Aldosterone, cortisol, and the adrenal androgens regulate many aspects of basic homeostasis. Aldosterone regulates extracellular fluid volume by promoting sodium

Suggested Reading Barnes PJ. Corticosteroids: the drugs to beat. Eur J Pharmacol 2006;533:2–14. (Review of glucocorticoid pharmacology, with emphasis on inhaled steroids.) Fuller PJ, Young MJ. Mechanisms of mineralocorticoid action. Hypertension 2005;46:1227–1235. (Molecular mechanisms of mineralocorticoid action, including cardiovascular effects.) Nair KS, Rizza RA, O’Brien P, et al. DHEA in elderly women and DHEA or testosterone in elderly men. N Engl J Med 2006;355:1647–1659. (Large clinical trial of DHEA.) Salvatori R. Adrenal insufficiency. JAMA 2005;294:2481–2488. (Pathophysiology and treatment of adrenal insufficiency.) Sapolsky R. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Rev 2000;21:55–89. (Thorough review discussing the numerous roles of glucocorticoids in stress responses.) Stellato C. Post-transcriptional and nongenomic effects of glucocorticoids. Proc Am Thorac Soc 2004;1:255–263. (Details of recent advances in glucocorticoid signaling.) Williams JS, Williams GH. 50th anniversary of aldosterone. J Clin Endocrinol Metab 2003;88:2364–2372. (Historical review of mineralocorticoids.)

29 Pharmacology of Reproduction Ehrin J. Armstrong and Robert L. Barbieri

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 505-506 PHYSIOLOGY OF REPRODUCTIVE HORMONES . . . . . . . . . . 505 Synthesis of Progestins, Androgens, and Estrogens . . . . . 505 Hormone Action and Metabolism . . . . . . . . . . . . . . . . . . . 506 Hypothalamic-Pituitary–Reproduction Axis . . . . . . . . . . . . 508 Integration of Endocrine Control: The Menstrual Cycle . . . 509 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510 Disruption of the HypothalamicPituitary–Reproduction Axis . . . . . . . . . . . . . . . . . . . . . . . 511 Inappropriate Growth of Hormone-Dependent Tissues . . . 511 Decreased Estrogen or Androgen Secretion . . . . . . . . . . . 512 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 512 Inhibitors of Gonadal Hormones . . . . . . . . . . . . . . . . . . . . 512 Synthesis Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . 512 Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . 513

Hormones and Hormone Analogues: Contraception . . . . . 516 Combination Estrogen–Progestin Contraception . . . . . 516 Progestin-Only Contraception . . . . . . . . . . . . . . . . . . . 517 Emergency (Morning-After) Contraception. . . . . . . . . . 517 Male Contraception . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 Hormones and Hormone Analogues: Replacement . . . . . . 518 Estrogens and Progestins . . . . . . . . . . . . . . . . . . . . . . 518 Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 519 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

 INTRODUCTION

is discussed both here and in Chapter 31, Pharmacology of Bone Mineral Homeostasis. Androgen replacement therapy is discussed at the end of this chapter.

This chapter presents endocrine pharmacology relevant to both the male and female reproductive tracts. Although men and women differ in their hormonal profiles, androgens and estrogens are both under the control of the anterior pituitary gland gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and ultimately regulated by hypothalamic release of gonadotropinreleasing hormone (GnRH). Female hormone patterns are temporally more complex and cyclic than male patterns: hormonal control of the menstrual cycle is an illustrative example of how sex hormones are integrated into a complex physiologic system. Understanding the menstrual cycle also provides a basis for understanding the pharmacology of contraception. A number of diseases are treated pharmacologically via modification of reproductive hormone activity; these range from infertility and endometriosis to breast and prostate cancer. Key concepts in this chapter include: (1) the interactions between estrogen and the pituitary gland; (2) the effects of GnRH release frequency on gonadotropin release; (3) the tissue selectivity of estrogen receptor agonists and antagonists; and (4) the various strategies used to antagonize the effects of endogenous sex hormones, from suppression of the hypothalamic-pituitary–reproduction axis to antagonism at the target tissue receptor. Because of its historical role in the prevention of osteoporosis, estrogen replacement therapy

 PHYSIOLOGY OF REPRODUCTIVE HORMONES Synthesis of Progestins, Androgens, and Estrogens The synthesis of progestins, androgens, and estrogens is closely intertwined. All three groups are steroid hormones derived from the metabolism of cholesterol. The synthesis of these hormones is similar to that of adrenal sex hormones, which is discussed in Chapter 28, Pharmacology of the Adrenal Cortex. The terminology “progestins,” “androgens,” and “estrogens” denotes a number of related hormones, rather than a single molecule in each group (Fig. 29-1). The progestins consist of progesterone, a common precursor to testosterone and estrogen synthesis (see also Fig. 28-2), and a number of synthetically altered progesterone derivatives used for therapeutic purposes. Progestins generally exert antiproliferative effects on the female endometrium by promoting the endometrial lining to secrete rather than proliferate (see below). Progesterone is also required for the maintenance of pregnancy. Androgens, all of which have masculinizing properties, 505

CHAPTER 29 / Pharmacology of Reproduction 507

HO

Cholesterol Cholesterol side chain cleavage enzyme O

HO

Pregnenolone 17α-hydroxylase

3β-HSD

O

O OH

HO

O

17-Hydroxypregnenolone

Progesterone

Progestins

17α-hydroxylase 17, 20-lyase

O OH

O

O

17-Hydroxyprogesterone

HO

Dehydroepiandrosterone OH

O

3β-HSD Androgens 17β-HSD O

O

Androstenedione

Testosterone

Aromatase

Aromatase

O

OH

Estrogens HO

HO

Estrone

FIGURE 29-1.

Estradiol

Synthesis of progestins, androgens, and estrogens. Progestins, androgens, and estrogens are steroid hormones derived from cholesterol. The major progestins include progesterone and 17␣-hydroxyprogesterone. The androgens include dehydroepiandrosterone (DHEA), androstenedione, and testosterone. Estrogens include estrone and estradiol. Estrogens are aromatized forms of their conjugate androgens: androstenedione is aromatized to estrone, and testosterone is aromatized to estradiol. Estradiol and estrone are both metabolized to estriol, a weak estrogen (not shown). Some of the precursor–product relationships among the hormones are omitted for clarity (see Fig. 28-2). HSD, hydroxysteroid dehydrogenase.

508 Principles of Endocrine Pharmacology SHBG

Testosterone Plasma Cytoplasm Dihydrotestosterone Androgen receptor 5α-reductase

Finasteride Dutasteride

Nucleus

Promoters Coding sequences Transcription of testosterone-dependent genes

When GnRH is administered continuously, gonadotroph release of LH and FSH is suppressed rather than stimulated. This effect has the important pharmacologic consequence that pulsatile administration of exogenous GnRH stimulates gonadotropin release, whereas continuous GnRH administration inhibits LH and FSH release and thereby blocks target cell function. LH and FSH have analogous but somewhat different effects in males and females. The pertinent target cells in the male are the Leydig and Sertoli cells of the testis, while the thecal and granulosa cells of the ovary mediate gonadotropin function in the female (Fig. 29-4). In each case, a twocell system is coordinated to mediate sex hormone actions. In the male, LH stimulates testicular Leydig cells to increase the synthesis of testosterone, which then diffuses into neighboring Sertoli cells. In the Sertoli cell, FSH stimulation increases the production of androgen binding protein (ABP), which is important for maintaining the high testicular concentrations of testosterone necessary for spermatogenesis. In addition, FSH stimulates the Sertoli cell to produce other proteins necessary for sperm maturation. In the female, LH

FIGURE 29-2.

Intracellular conversion of testosterone to dihydrotestosterone. Testosterone circulates in the plasma bound to sex hormone-binding globulin (SHBG) and albumin (not shown). Free testosterone diffuses through the plasma membrane of cells into the cytosol. In target tissues, the enzyme 5␣reductase converts testosterone to dihydrotestosterone, which has increased androgenic activity relative to testosterone. Dihydrotestosterone binds with high affinity to the androgen receptor, forming a complex that is transported into the nucleus. Homodimers of dihydrotestosterone and androgen receptor initiate transcription of androgen-dependent genes. Finasteride and dutasteride, drugs used in the treatment of benign prostatic hyperplasia and male pattern hair loss, inhibit the enzyme 5␣-reductase.

GnRH (pulsatile)

LH/FSH

thoroughly as those of the estrogen receptor, it is likely that the same complexities exist for these receptors. The recognition that differential binding of modular transcription factors to ERs could alter estrogenic effects will likely prove a burgeoning area of pharmacologic research in the near future, as pharmaceutical researchers continue to develop receptor agonists and antagonists with selective actions in specific tissues. Selective estrogen receptor modulators (SERMs, see below) are the first drugs to take advantage of the tissue selectivity of sex hormone receptor function.

Estrogen or testosterone Estrogen (at ovulation) LH

FSH

Activin

Inhibin

Ovaries or testes

Hypothalamic-Pituitary–Reproduction Axis The hypothalamic-pituitary–reproduction axis regulates sex hormone synthesis. Gonadotropin-releasing hormone (GnRH) resides at the top of this three-tiered hierarchy. The hypothalamus secretes GnRH in pulses (Fig. 29-3). GnRH travels via the hypothalamic–pituitary portal system to stimulate gonadotroph cells of the anterior pituitary gland. Stimulation of gonadotroph cells via a G protein-coupled cell surface receptor increases the synthesis and secretion of LH and FSH, which are jointly referred to as the gonadotropins. Although one cell type produces both LH and FSH, the synthesis and release of these two hormones are controlled independently. Current research suggests that the rate of GnRH secretion may preferentially alter the secretion patterns of LH and FSH. Pulsatile secretion of GnRH is critical for the proper functioning of the hypothalamic-pituitary–reproduction axis.

FIGURE 29-3. The hypothalamic-pituitary–reproduction axis. The hypothalamus secretes gonadotropin-releasing hormone (GnRH) into the hypothalamic– pituitary portal system in a pulsatile pattern. GnRH stimulates gonadotroph cells in the anterior pituitary gland to synthesize and release luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These two hormones, referred to as gonadotropins, promote ovarian and testicular synthesis of estrogen and testosterone, respectively. Estrogen and testosterone inhibit release of GnRH, LH, and FSH. Depending on the time in the menstrual cycle, the concentration of estrogen in the plasma, and the rate at which estrogen concentration increases in the plasma, estrogen can also stimulate pituitary gonadotropin release (e.g., at ovulation). Both the ovaries and testes secrete inhibin, which selectively inhibits FSH secretion, and activin, which selectively promotes FSH secretion.

CHAPTER 29 / Pharmacology of Reproduction 509 LH

FSH

Male

LH-R

FSH-R

ABP Testosterone synthesis

Testosterone Testosterone-ABP

Leydig cell

Sertoli cell

LH

FSH

Female

FSH-R

LH-R

Aromatase Androgen synthesis

Androgen Estrogen

Thecal cell

Granulosa cell

FIGURE 29-4.

Two-cell systems for gonadal hormone action. In the male, the binding of luteinizing hormone (LH) to the LH receptor (LH-R) activates testosterone synthesis in Leydig cells. Testosterone then diffuses into nearby Sertoli cells, where the binding of follicle-stimulating hormone (FSH) to its receptor (FSH-R) increases levels of androgen binding protein (ABP). ABP stabilizes the high concentrations of testosterone that, together with other FSH-induced proteins synthesized in Sertoli cells, promote spermatogenesis in the nearby germinal epithelium (not shown). In the female, LH acts in an analogous manner to promote androgen (androstenedione) synthesis in thecal cells. Androgen then diffuses into nearby granulosa cells, where aromatase converts androstenedione to estrone, which is then reduced to the biologically active estrogen, estradiol. FSH increases aromatase activity in granulosa cells, promoting the conversion of androgen to estrogen. Note that dihydrotestosterone is not a substrate for aromatase.

stimulates the thecal cells to synthesize the androgen androstenedione, which is then aromatized to estrone and estradiol in the granulosa cells under the influence of FSH. Both Sertoli cells and granulosa cells synthesize and secrete the regulatory proteins inhibin A, inhibin B, and activin. Inhibins secreted by the gonad act on the anterior pituitary gland to inhibit the release of FSH, while activin stimulates FSH release. Neither the inhibins nor activin has an effect on anterior pituitary gland LH release (Fig. 29-3). The role of these regulatory proteins in controlling hormone action is still not completely understood. In the male, testosterone is also an important negative regulator of pituitary gland and hypothalamic hormone release. The role of estrogen in

the female is more complex and can involve either positive or negative feedback depending on the prevailing hormonal milieu; this topic is addressed below as part of the menstrual cycle discussion. In the female, the combination of estradiol and progesterone synergistically suppresses GnRH, LH, and FSH secretion by actions at both the hypothalamus and pituitary gland.

Integration of Endocrine Control: The Menstrual Cycle The female menstrual cycle is governed by the cycling of hormones with an approximate periodicity of 28 days (normal range, 24–35 days). This cycle begins at the onset of puberty and continues uninterrupted (with the exception of pregnancy) until menopause (Fig. 29-5). The start of the cycle, cycle day 1, is arbitrarily defined as the first day of menstruation. Ovulation occurs at the midportion (about day 14) of each cycle. The portion of the menstrual cycle before ovulation is often referred to as the follicular or proliferative phase; during this time, the developing ovarian follicle produces most of the gonadal hormones, which stimulate cellular proliferation of the endometrium. Subsequent to ovulation, the corpus luteum produces progesterone, and the endometrium becomes secretory rather than proliferative. The second half of the menstrual cycle is thus often referred to as the luteal or secretory phase, depending on whether the ovary or the endometrium is considered as the frame of reference. At the start of the menstrual cycle, there is low production of estrogen and inhibin A. As a result, the anterior pituitary gland secretes increasing amounts of FSH and LH. These hormones stimulate the maturation of four to six follicles, each of which contains an ovum arrested in the first stage of meiosis. Maturing follicles secrete increasing concentrations of estrogen, inhibin A, and inhibin B. Estrogen causes the follicles to increase the expression of LH and FSH receptors on thecal and granulosa cells, respectively. Receptor up-regulation increases the follicular response to pituitary gland gonadotropins and allows one follicle to secrete increasing quantities of estrogen. The increased plasma estrogen and inhibin levels partially suppress pituitary gland LH and FSH release. In turn, the decreased gonadotropin levels cause other follicles to become atretic, so that usually only one follicle matures. At the same time, increased estrogen levels stimulate the uterine endometrium to proliferate rapidly. As the dominant follicle continues to grow, it secretes high, sustained levels of estrogen. Although the mechanism is still not completely understood, the combination of high estrogen levels and the rapid rate of increase of estrogen levels causes a brief positive feedback effect on gonadotroph release of gonadotropins, stimulating rather than inhibiting release of LH and FSH. The resulting midcycle surge of LH and FSH stimulates the dominant follicle to swell and to increase the activity of its proteolytic enzymes. Approximately 40 hours after the onset of the LH surge, the follicle ruptures and ovulation occurs. The ovum is released into the peritoneal cavity and is then taken up by a fallopian tube, where it begins its route toward the uterus. If the oocyte becomes fertilized in the fallopian tube, it reaches the uterus approximately 4 days after ovulation and implants into the endometrium approximately 5–6 days after ovulation.

Follicular phase

Luteal phase

Ovulation

Ovarian follicle

Growing

Corpus luteum

Mature

Estrogen

Progesterone (+ estrogen)

LH (mlU/ml)

60 40 20 0

FSH (mlU/ml)

20

10

0

Estrogen (pg/ml)

20

10

Progesterone (ng/ml)

0

10

5

Endometrium

0

Proliferative

2

Menses

Secretory

6

10

14

Day

18

22

26

512 Principles of Endocrine Pharmacology

bleeding, and the formation of adhesions in the peritoneal cavity. In turn, adhesion formation can lead to infertility. Because endometriosis is usually estrogen-dependent, treatment with long half-life GnRH agonists often achieves regression of the disease (see below).

Decreased Estrogen or Androgen Secretion The effects of decreased sex hormone production vary depending on the age of the patient at the onset of symptoms. Hypogonadism results if sex hormone production is impaired before adolescence. Patients with hypogonadism do not undergo sexual maturation, but proper hormone replacement can, in many cases, allow the development of secondary sexual characteristics. Menopause is a normal physiologic response to exhaustion of the ovarian follicles. Throughout a woman’s lifetime, follicles are arrested in meiosis. Only a small percentage of follicles mature during the menstrual cycle; the rest eventually become atretic. Menstrual cycles cease when all of the follicles are depleted from the ovaries. Follicle depletion leads to a decrease in estrogen and inhibins (because developing follicles are the main estrogen and inhibin source in premenopausal women) and an increase in LH and FSH (because estrogen and inhibins suppress gonadotropin release). After menopause, androstenedione continues to be converted to estrone by aromatase in peripheral (mainly adipose) tissues. However, estrone is a less potent estrogen than estradiol. Because of the relative lack of estrogen after menopause, many

women experience hot flashes, vaginal dryness, and decreased libido. Postmenopausal women are also at risk for osteoporosis. The role of estrogen in the maintenance of bone mass is discussed in further detail in Chapter 31. Men do not experience a sudden decrease in sex hormones in a manner analogous to the female menopause, but androgen secretion does decline gradually with age. Although controversy currently exists over the role of androgen therapy in normal elderly men, androgen replacement is indicated in cases of adult hypogonadism where both low testosterone levels and symptoms of hypogonadism are present.

 PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic agents have been developed to target most of the steps in gonadal physiology and pathophysiology. The relevant drug classes include modulators of anterior pituitary gland gonadotroph activity and specific antagonists of peripheral hormone action. In addition, sex hormones are often used as replacement therapy or to modify gonadotropin release (Fig. 29-6).

Inhibitors of Gonadal Hormones Synthesis Inhibitors GnRH Agonists and Antagonists

Under physiologic conditions, the hypothalamus releases GnRH in a pulsatile fashion. The frequency of GnRH pulses controls the relative release of LH and FSH by the anterior pituitary

GnRH (pulsatile; endogenous)

Anterior pituitary gland GnRH agonists (continuous; leuprolide, goserelin, nafarelin) GnRH receptor antagonists (cetrorelix, ganirelix)

LH/FSH

Ovaries / testes

Aromatase inhibitors (exemestane, formestane, anastrozole, letrozole)

Testosterone

Progesterone

5α-reductase inhibitors (finasteride, dutasteride)

Aromatase Estrogen

Testosterone

Dihydrotestosterone Mifepristone

SERMs (+/-) Gene transcription

FIGURE 29-6.

Gene transcription

Androgen receptor antagonists (flutamide, spironolactone)

Gene transcription

Pharmacologic modulation of gonadal hormone action. Pharmacologic modulation of gonadal hormone action can be divided into inhibitors of hormone synthesis and hormone receptor antagonists. Continuous administration of GnRH suppresses LH and FSH release from the anterior pituitary gland, thus preventing gonadal hormone synthesis. GnRH receptor antagonists (cetrorelix, ganirelix) are also used for this purpose. Finasteride and dutasteride inhibit the enzyme 5␣-reductase, thus preventing conversion of testosterone to the more active dihydrotestosterone. Aromatase inhibitors (exemestane, formestane, anastrozole, letrozole) inhibit production of estrogens from androgens. A number of hormone receptor antagonists and modulators prevent the action of endogenous estrogens (some SERMs), androgens (flutamide, spironolactone), and progesterone (mifepristone).

514 Principles of Endocrine Pharmacology A Bone: both X and Y cofactors expressed SERM

Estrogen

Cofactor Y Cofactor Y Estrogen receptor

Estrogen receptor

Cofactor X

Cofactor X

Nucleus

Nucleus

DNA

DNA

X Gene 1 Y Gene 2 X Y Gene 3

X Gene 1 Y Gene 2 X Y Gene 3

Only gene 1 expressed: Partial agonist

Genes 1, 2, and 3 expressed: Full agonist

B Breast: only Y cofactor expressed SERM

Estrogen

Cofactor Y Cofactor Y Estrogen receptor

Estrogen receptor Nucleus

Nucleus

DNA

DNA

X Gene 1 Y Gene 2 X Y Gene 3

X Gene 1 Y Gene 2 X Y Gene 3

Gene 2 expressed: Full agonist

No genes expressed: Antagonist

FIGURE 29-7.

A model for the tissue specificity of action of SERMs. Selective estrogen receptor modulators (SERMs) exhibit tissue-specific estrogen receptor antagonist or partial agonist activity. This tissue specificity of action seems to be explained by the following observations: (1) transcriptional coactivators and/or corepressors are expressed in a tissue-specific manner; (2) a SERM–estrogen receptor (ER) complex can associate with some coactivators or corepressors, but not others; and (3) genes can be activated or inhibited by different combinations of SERM–ER and coactivators or corepressors. In the example shown, assume that bone cells express coactivators (cofactors) X and Y, whereas breast cells express only coactivator Y. The estrogen–ER complex can associate with X and Y, whereas the SERM–ER complex can associate with only X. A. In bone cells, estrogen binding to ER and recruitment of coactivators X and Y induce expression of genes 1, 2, and 3. The SERM–ER complex cannot bind coactivator Y, and the SERM–ER–cofactor X complex induces expression of only gene 1. In bone, then, estrogen is a full agonist, whereas the SERM is a partial agonist. B. In breast cells, estrogen binding to ER and recruitment of coactivator Y induce expression of gene 2, but the SERM is unable to promote expression of any gene. In breast, then, the SERM acts as an antagonist. For simplicity, this model shows only coactivators, although corepressors are also involved in SERM action.

even cardiovascular disease. The three SERMs in current clinical use are tamoxifen, raloxifene, and clomiphene. Tamoxifen is the only SERM currently approved for use in the treatment and prevention of breast cancer. Tamoxifen has been employed in the palliative treatment of metastatic breast cancer and as adjuvant therapy after lumpectomy. Tamoxifen is an estrogen receptor antagonist in breast tissue, but a partial agonist in the endometrium and bone. These pharmacodynamic effects result in inhibition of the estrogen-dependent growth of breast cancer, but also stimulation of endometrial growth. Because of the latter effect, tamoxifen administration is associated with a four-fold to six-fold increase in the incidence of endometrial cancer. Therefore, in order to minimize the risk of iatrogenic endometrial cancer, tamoxifen is typically administered for no more than 5 years. Raloxifene is a newer SERM that possesses estrogen receptor agonist activity in bone, but antagonist activity in both breast and endometrial tissue. Its mechanism of action is

illustrated in Figure 29-7, and its molecular structure is shown in Figure 29-8. Consistent with this profile of tissue specificities, raloxifene does not appear to increase the incidence of endometrial cancer. The agonist activity of raloxifene in bone decreases bone resorption, and thus delays or prevents the progression of osteoporosis in postmenopausal women (discussed in more detail in Chapter 31). Raloxifene is approved for use in the prevention of breast cancer and the prevention and treatment of osteoporosis. In a large clinical trial comparing raloxifene and tamoxifen for the prevention of breast cancer in women at high risk, both agents resulted in a 50% reduction in the development of invasive breast cancer. Tamoxifen treatment was associated with more cases of endometrial hyperplasia, endometrial cancer, cataracts, and deep vein thrombosis than raloxifene. However, tamoxifen prevented more cases of noninvasive breast cancer than raloxifene. Clomiphene is a SERM used to induce ovulation. The drug acts as an estrogen receptor antagonist in the hypothalamus

OH

O OCCH3

C

CH

O

O

O CH3

Medroxyprogesterone acetate

Norethindrone O OCCH3

O OCCH3

C

CH

O

O

O CH3

Megestrol acetate

Norethindrone acetate

OH

OH H2C

C CH

C CH

O

Desogestrel

Levonorgestrel

OH

OH C

CH

CH

O

HO

CH3O Ethinyl estradiol

HON

Gestodene Mestranol

C CH

C CH

C

O OCCH3

OH

Norgestimate

CHAPTER 29 / Pharmacology of Reproduction 519

Androgens Androgen replacement is an effective therapy for hypogonadism. Oral testosterone is ineffective because of its high first-pass metabolism by the liver. Two esters of testosterone, testosterone enanthate and testosterone cypionate, can be administered intramuscularly. A preparation of either of these agents, injected every 2–4 weeks, increases plasma testosterone to physiologic concentrations in hypogonadal men. Transdermal testosterone patches have also been developed; this drug delivery system has the advantages that plasma testosterone levels remain relatively constant and first-pass hepatic metabolism is bypassed. Testosterone is also available in a topical gel formulation; using this preparation on a once-a-day application schedule, plasma testosterone levels gradually increase until they reach physiologic replacement levels after 1 month of application. Testosterone can also be administered as a tablet that adheres to the buccal mucosa, resulting in rapid systemic absorption of the drug. Aging men sometimes develop symptoms and signs of hypogonadism, such as decreased energy, decreased libido, gynecomastia, decreased muscle mass, and facial hair growth. Recent guidelines recommend that androgen replacement therapy be offered to men only with consistent symptoms and signs of hypogonadism and low plasma testosterone levels (⬍3.0 ng/mL). Testosterone should not be administered to men with prostate cancer, because it may stimulate growth of the tumor. Some athletes abuse androgens by self-administration at supratherapeutic levels. Androgens have been demonstrated to increase muscle mass and fat-free mass. In one survey, approximately 5% of high school athletes reported that they had used androgen supplements. Almost every type of androgen has been abused in an attempt to enhance athletic performance, including the adrenal hormone precursors androstenedione and dehydroepiandrosterone. Covert laboratories are continuously inventing new synthetic androgens that have not yet been recognized by standard drug testing programs. These “designer” androgens are meant to enhance athletic performance and to be undetectable by sports regulatory authorities. Pharmacologic doses of androgens suppress the hypothalamic-pituitary–reproduction axis, resulting in suppression of testicular function, decreased sperm production, and impaired fertility. Because many androgens can be converted to estrogens by aromatase, pharmacologic doses of androgens can also cause an increase in plasma estrogen, resulting in gynecomastia. In addition, high plasma levels of androgens are associated with erythrocytosis, severe acne, and derangements in lipid metabolism (increased low-density lipoprotein [LDL] and decreased high-density lipoprotein [HDL]). Some athletes have recently started to use injections of hCG to stimulate endogenous Leydig cell testosterone production, hoping to avoid detection by sports authorities. SERMs and aromatase inhibitors have also been used by athletes in an attempt to increase endogenous LH secretion and Leydig cell testosterone production.

 CONCLUSION AND FUTURE DIRECTIONS The male and female hormones of reproduction share significant mechanistic overlap with one another. Androgens, estrogens, and progestins are all steroid hormones that

exert their physiologic action by binding to intracellular receptors, translocating to the nucleus, and altering gene transcription. Recent evidence suggests that estrogens may also act on membrane receptors to mediate nongenomic effects. Derangements in the physiologic effects of reproductive hormones can involve disruption of the hypothalamic-pituitary–reproduction axis, inappropriate growth of hormone-dependent tissue, or decreased activity of gonadal hormones at target tissues. Currently available pharmacologic agents can modify the endocrine axis (e.g., GnRH agonists), inhibit synthesis of active hormones (e.g., 5␣-reductase inhibitors, aromatase inhibitors), or inhibit end-organ effects at the receptor level (e.g., SERMs, anti-androgens, mifepristone). Oral contraceptives, such as estrogen–progestin combinations and progestin-only contraception, disrupt the exquisite cyclicity of the menstrual cycle and thus suppress ovulation. The development of an effective male contraceptive has met a number of obstacles but should represent a major pharmacologic advance in the future. Exciting progress is also being made in the design of new SERMs that possess a variety of tissue-specific activities; such research may result in new agents effective for both prevention of breast cancer and treatment of postmenopausal osteoporosis.

Suggested Reading Anderson GL, Limacher M, Assaf AR, et al. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA 2004;291:1701–1712. (Reports the “Estrogen Alone vs. Placebo” data shown in Table 29-3.) Archer DF, Pinkerton JV, Utian WH, et al. Bazedoxifene, a selective estrogen receptor modulator: effects on the endometrium, ovaries and breast from a randomized controlled trial in osteoporotic postmenopausal women. Menopause 2009;16:1109–1115. (Bazedoxifene is one of many new SERMs being developed to treat problems associated with menopause. Bazedoxifene is an estrogen agonist in bone and an antagonist in endometrium and breast.) Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in adult men with androgen deficiency syndromes: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2006;91:1995– 2010. (Aging men should have both consistent symptoms of hypogonadism and low serum testosterone in order to be treated with androgen replacement.) Chlebowski RY, Kuller LH, Prentice RL, et al. Breast cancer after use of estrogen plus progestin in postmenopausal women. N Engl J Med 2009;360:573–587. (After the results of the WHI trial were published in 2002, there was a marked decrease in the number of menopausal women using estrogen–progestin therapy and a parallel reduction in the number of cases of diagnosed breast cancer.) Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005;352:1223– 1236. (A clinically oriented review of the polycystic ovarian syndrome, the diagnosis for the patient presented in this chapter.) Gu Y, Liang X, Wu W, et al. Multicenter contraceptive efficacy trial of injectable testosterone undecanoate in Chinese men. J Clin Endocrinol Metab 2009;94:1910–1915. (The administration of testosterone is an effective contraception. The safety profile of this approach has not been fully explored.) Struthers RS, Nicholls AJ, Grundy J, et al. Suppression of gonadotropins and estradiol in premenopausal women by oral administration of the nonpeptide gonadotropin-releasing hormone antagonist, elagolix. J Clin Endocrinol Metab 2009;94:545–551. (An orally active nonpeptide GnRH antagonist would represent an important new drug for suppressing the pituitary–reproduction axis.) Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 2002;288:321–333. (Reports the “Continuous Estrogen–Progestin vs. Placebo” data shown in Table 29-3.)

30 Pharmacology of the Endocrine Pancreas and Glucose Homeostasis Aimee D. Shu and Steven E. Shoelson

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 524-525 BIOCHEMISTRY AND PHYSIOLOGY . . . . . . . . . . . . . . . . . . . 524 Pancreatic Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524 Energy Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 Energy Repletion and the Fed State. . . . . . . . . . . . . . . 525 Fasting and Starvation . . . . . . . . . . . . . . . . . . . . . . . . 525 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526 Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526 Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526 Action at Target Tissues . . . . . . . . . . . . . . . . . . . . . . . 529 Glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 Amylin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 Glucagon-Like Peptide-1 (GLP-1) . . . . . . . . . . . . . . . . . . . 530 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530 Type 1 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

Type 2 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531 Morbidity and Mortality . . . . . . . . . . . . . . . . . . . . . . . . 532 Hypoglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 532 Therapy for Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Inhibitors of Intestinal Glucose Absorption . . . . . . . . . . 533 Insulin Replacement: Exogenous Insulin . . . . . . . . . . . 533 Insulin Secretagogues: Sulfonylureas and Meglitinides . . 535 Reduction of Hepatic Glucose Production: Biguanides . . . 535 Amylin Analogue: Pramlintide . . . . . . . . . . . . . . . . . . . 535 GLP-1-Based “Incretin” Therapies . . . . . . . . . . . . . . . 535 Insulin Sensitizers: Thiazolidinediones . . . . . . . . . . . . . 536 Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . 536 Therapy for Hyperinsulinemia . . . . . . . . . . . . . . . . . . . . . 536 Glucagon as a Therapeutic Agent . . . . . . . . . . . . . . . . . . . 537 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 537 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537

 INTRODUCTION

 BIOCHEMISTRY AND PHYSIOLOGY

This chapter reviews the physiology and pharmacology of insulin, glucagon, and the other major hormones that regulate glucose homeostasis. Because diabetes mellitus—caused by an absolute or relative deficiency in insulin secretion—is clinically the most common disease of these endocrine axes, the majority of the chapter is devoted to the physiology and pharmacology of insulin. Medical students may be interested to note that Charles Best, a fourth-year medical student in Canada, had a significant role in the identification of insulin. Along with his mentor, Frederick Banting, Best isolated a pancreatic extract from dogs that could reduce blood glucose in diabetic dogs and humans. Although the 1923 Nobel Prize in Medicine or Physiology was jointly awarded to surgeon Frederick Banting and physiologist J. J. R. MacLeod, Banting shared his award with Best.

Pancreatic Anatomy

524

The pancreas is a glandular organ that contains both exocrine and endocrine tissue. The exocrine portion—which constitutes 99% of the pancreatic mass—secretes bicarbonate and digestive enzymes into the gastrointestinal (GI) tract. Scattered within the exocrine tissue are nearly one million small islands of endocrine tissue that secrete hormones directly into the blood. These tiny endocrine glands, collectively called islets of Langerhans, include several different cell types that secrete different hormones: ␣-cells release glucagon, ␤-cells release insulin and amylin; ␦-cells release somatostatin and gastrin; and PP cells release pancreatic polypeptide.

CHAPTER 30 / Pharmacology of the Endocrine Pancreas and Glucose Homeostasis 527 Gastric emptying

GI tract

GLP-1 analogue, amylin analogue

Dietary complex carbohydrates

α-glucosidase inhibitors

Other tissues

Glucosidases

Blood

β cell Endogenous insulin (from β cell) or Exogenous insulin

Glucose (from GI tract and liver)

Glucose

Metabolism

To tissues

Glucose

Metabolism

Insulin secretion

ATP

To tissues Diazoxide

Insulin

Insulin Muscle cell

Glucose

Insulin

Adipose cell

Glycogen

Sulfonylureas, meglitinides, GLP-1 analogue, DPP-4 inhibitor

Liver cell

Glucose

Glucose

Triglyceride

Glycogen

Glucose PPARγ

Glucagon

Gluconeogenesis

Thiazolidinediones

Biguanides

GLP-1 analogue, DPP-4 inhibitor, amylin analogue

FIGURE 30-1.

Physiologic and pharmacologic regulation of glucose homeostasis. Dietary complex carbohydrates are broken down to simple sugars in the GI tract by the action of glucosidases; simple sugars are then absorbed by GI epithelial cells and transported into the blood. Glucose in the blood is taken up by metabolically active tissues throughout the body. Glucose metabolism in pancreatic ␤-cells increases cytosolic ATP, which stimulates insulin secretion. Released insulin acts on plasma membrane insulin receptors in target tissues (muscle, adipose, liver) to increase glucose uptake and storage as glycogen or triglyceride. Glucose is also taken up by other cells and tissues to fuel metabolism. In muscle and liver, insulin promotes glucose storage as glycogen. In adipose cells, insulin promotes glucose conversion to triglycerides. Peroxisome proliferator-activated receptor ␥ (PPAR␥) also promotes the conversion of glucose to triglycerides in adipocytes. Glucagon promotes both hepatic gluconeogenesis and glycogen breakdown; the newly generated glucose is transported out of the liver cell into the blood. Note that glucose from dietary complex carbohydrates and insulin secreted by pancreatic ␤-cells both enter the liver in high concentrations through the portal circulation (not shown). Pharmacologic interventions that decrease blood glucose levels include: delaying gastric emptying with a GLP-1 analogue or an amylin analogue; inhibiting intestinal ␣-glucosidases with an ␣-glucosidase inhibitor; administering exogenous insulin; augmenting ␤-cell insulin secretion with sulfonylureas, meglitinides, or incretins; suppressing glucagon and gluconeogenesis with incretins, an amylin analogue, or biguanides; and enhancing the action of insulin in adipose cells using thiazolidinediones. To treat hyperinsulinemic hypoglycemia, diazoxide inhibits pancreatic ␤-cell insulin secretion.

closed, the cell depolarizes and insulin is released. Because ATP inhibits the channel and ADP activates the channel, a high intracellular ATP/ADP ratio closes the K⫹/ATP channel. The resulting depolarization of the cell activates voltage-gated Ca2⫹ channels, leading to an influx of extracellular Ca2⫹. The increase in intracellular [Ca2⫹] signals the insulin-containing vesicles to fuse with the plasma membrane, which releases insulin into the circulation. In contrast, under conditions of relatively low extracellular glucose concentrations (e.g., in the fasting state), the ␤-cell has a low ATP/ADP ratio. In this case, the K⫹/ATP channels remain

open, and the ␤-cell is maintained in a hyperpolarized state that prevents Ca2⫹ influx and insulin secretion (Fig. 30-3). ␤-cell K⫹/ATP channels are octameric structures containing four subunits of Kir6.2 and four subunits of the sulfonylurea receptor, SUR1. The Kir6.2 tetramer forms the pore of the K⫹/ATP channel, while the associated SUR1 proteins regulate the channel’s sensitivity to ADP and pharmacologic agents. Kir6.2 binds ATP, which inhibits K⫹ conductance. SUR1 enhances the sensitivity of the Kir6.2 channel to ATP, and it also confers sensitivity to sulfonylurea and related insulin secretagogue drugs. SUR1 binds ADP–Mg2⫹ complexes,

528 Principles of Endocrine Pharmacology Dipeptide cleavage site

NH2

SUR1

Kir6.2

Inhibitors

Arg Lys

Activators

1 2

Mg2+-ADP

Sulfonylurea/ meglitinide

3

Diazoxide Cys

Cys

ATP

Cys Cys

K+/ATP channel

GLUT2 transporter Arg Arg

Proinsulin Cys

COOH

Lys

Asn

Pro

Cys

K+ conductance

Glucose

Dipeptide cleavage site

Membrane depolarization

ATP Metabolism ADP

Ca2+ influx Insulin vesicles Ca2+

COOH B chain NH2

A chain NH2 Pancreatic β cell

1

1

Insulin secretion

2

2

3

Asn

C peptide

4

Changed to lysine in insulin glulisine

FIGURE 30-3.

Cys Cys

Changed to glutamic acid in insulin glulisine

Cys Cys

Myristoylated in insulin detemir Two additional arginine residues in insulin glargine

Changed to glycine in insulin glargine

COOH 19 Cys

Insulin

Cys

COOH Asn

NH2

Lys Pro

27

Reversed in insulin lispro

26

Changed to aspartic acid in insulin aspart

FIGURE 30-2.

Processing of human insulin. Preproinsulin is synthesized and exported into the endoplasmic reticulum, where the signal peptide (not shown) is cleaved to generate proinsulin (top panel). Intramolecular disulfide bonds (cys–cys) aid in the proper folding of proinsulin. Proinsulin is transported to secretory vesicles, where prohormone convertases act on dipeptide cleavage sites in proinsulin (boxes) to generate insulin and connecting (C) peptide. Two disulfide bonds aid in holding the A-chain and B-chain of insulin together. Insulin and C-peptide are secreted together from the pancreatic ␤-cell (bottom panel). Modifications to insulin’s amino acid sequence result in the altered pharmacokinetics of the various insulin analogues; lispro, aspart, and glulisine are rapid-acting insulins, whereas glargine and detemir have slower absorption. The substitutions are: in lispro, the positions of ProB28 and LysB29 are reversed; in aspart, ProB28 is replaced by aspartic acid; in glulisine, AsnB3 and LysB29 are replaced by lysine and glutamic acid, respectively; in glargine, AsnA21 is replaced by glycine, and two extra arginines are added to the carboxyl terminus of the B-chain; and in detemir, a fatty acid (myristic acid) is esterified to the ␧amino group of LysB29.

Physiologic and pharmacologic regulation of insulin release from pancreatic ␤ cells. When the K⫹/ATP channel is open in its basal state, less insulin is released; when the K⫹/ATP channel is closed, more insulin is released. In the basal state, the plasma membrane of the ␤-cell is hyperpolarized, and the rate of insulin secretion from the cell is low. However, when glucose is available, it enters the cell via GLUT2 transporters in the plasma membrane and is metabolized to generate intracellular ATP. ATP binds to and inhibits the plasma membrane K⫹/ATP channel. Inhibition of the K⫹/ATP channel decreases plasma membrane K⫹ conductance; the resulting depolarization of the membrane activates voltage-gated Ca2⫹ channels and thereby stimulates an influx of Ca2⫹. Ca2⫹ mediates fusion of insulin-containing secretory vesicles with the plasma membrane, leading to insulin secretion. The K⫹/ATP channel, an octamer composed of Kir6.2 and SUR1 subunits, is the target of several physiologic and pharmacologic regulators. ATP binds to and inhibits Kir6.2, while sulfonylureas and meglitinides bind to and inhibit SUR1; all three of these substances promote insulin secretion. The GLP-1 mimetic exenatide, acting as an agonist at G proteincoupled GLP-1 receptors in the plasma membrane of the pancreatic ␤-cell, also stimulates glucose-dependent insulin secretion. This action of exenatide appears to be mediated by an increase in intracellular cyclic AMP and may involve an indirect effect on the K⫹/ATP channel (not shown). Mg2⫹-ADP and diazoxide bind to and activate SUR1, thereby inhibiting insulin secretion. (For clarity, only four of the eight K⫹/ATP channel subunits are shown.)

which activate the channel and further inhibit insulin secretion when the ATP/ADP ratio is low. Mutations in Kir6.2 or SUR1 can result in hyperinsulinemic hypoglycemia, because the channel remains closed and the ␤-cell remains continually depolarized even when the extracellular glucose concentration and the intracellular ATP/ADP ratio are low. In addition to blood glucose, nutrient sugars, amino acids, and fatty acids increase the intracellular ATP/ADP ratio and thereby stimulate insulin release. Acting via G protein-mediated pathways, parasympathetic activity and the GI hormones GLP-1

Insulin Glucose

Insulin receptor GLUT4 P

P

Translocation GLUT4

P

Shc

Glucose Hexokinase

P

IRS proteins

Grb-2

Grb-2

SOS

SOS

SHP-2

p85 p110 PI3-kinase

? Mitogenesis

Protein synthesis

Glucose-6phosphate

Glycogen synthesis

Glucose transport

Metabolism/ storage

CHAPTER 30 / Pharmacology of the Endocrine Pancreas and Glucose Homeostasis 535

The major danger with insulin therapy is that administration of insulin in the absence of adequate carbohydrate intake can result in hypoglycemia. While tight glycemic control that aims to maintain normoglycemia decreases the incidence of diabetic complications, it also increases the frequency of hypoglycemic attacks. Therefore, patients taking insulin, whether they are type 1 or type 2 diabetics, must be cautioned not to take too much. Indeed, it is challenging to maintain the fine balance between insufficient and excessive insulin. In type 2 diabetic patients such as Mrs. S, insulin resistance is typically more severe in muscle and liver than in fat cells. For this reason, insulin preferentially deposits calories in adipose tissue, and insulin therapy in insulin-resistant patients (especially those who are already obese, like Mrs. S) often results in weight gain. Insulin Secretagogues: Sulfonylureas and Meglitinides Sulfonylureas

Sulfonylureas have been available in the United States since the 1950s for the treatment of type 2 diabetes. Sulfonylureas stimulate insulin release from pancreatic ␤-cells, thereby increasing circulating insulin to levels sufficient to overcome the insulin resistance. At the molecular level, sulfonylureas bind to the SUR1 subunit, thereby inhibiting the ␤-cell K⫹/ ATP channel (Fig. 30-3). Sulfonylureas may act by displacing endogenous Mg2⫹-ADP, which binds to SUR1 and activates the channel. The sulfonylureas used to treat type 2 diabetes bind with a higher affinity to SUR1 than to SUR2 isoforms, accounting for their relative ␤-cell specificity. The inhibition of the K⫹/ATP channel by sulfonylureas is functionally similar to the molecular events induced physiologically in the fed state, in which increased glucose metabolism causes ␤-cell accumulation of intracellular ATP, membrane depolarization, Ca2⫹ influx, fusion of insulin-containing vesicles with the plasma membrane, and insulin secretion (see above). Sulfonylureas are orally available and are metabolized by the liver. Their major adverse effect is hypoglycemia resulting from oversecretion of insulin. Thus, these medications should be used cautiously in patients who are unable to recognize or respond appropriately to hypoglycemia, such as those with impaired sympathetic function, mental status changes, or advanced age. Studies show that sulfonylurea use is associated with a marginal decrease in circulating lipids. These agents can cause weight gain secondary to increased insulin activity on adipose tissue; this adverse effect is counterproductive in obese patients such as Mrs. S. Therefore, sulfonylureas are better suited for nonobese patients. Because first-generation sulfonylureas bind with lower affinity to SUR1 than second-generation agents, first-generation agents are administered in higher doses to achieve the same degree of glucose lowering. Sulfonylureas are generally effective, safe, and inexpensive (generically available) drugs and, along with metformin, are mainstays of treatment for type 2 diabetes.

Reduction of Hepatic Glucose Production: Biguanides Hepatic glucose production may be abnormally elevated in type 2 diabetes. Metformin acts to decrease glucose production in the liver by activating the energy-regulating enzyme AMPK. By triggering hepatic AMPK, metformin inhibits gluconeogenesis, fatty-acid synthesis, and cholesterol synthesis. Metformin also improves glucose uptake in peripheral muscle; the molecular mechanism responsible for this effect is less well understood. Metformin increases insulin signaling (i.e., it increases the activity of the insulin receptor) and is especially effective at lowering glucose in type 2 diabetics who are obese and insulin resistant. Unlike insulin and insulin secretagogues, biguanides are associated with a lowering of serum lipids and a decrease in weight. Metformin is also used for the off-label (not FDA-approved) treatment of other conditions, such as polycystic ovarian syndrome, that are associated with insulin resistance and hyperinsulinemia. The most common adverse effect of metformin is mild gastrointestinal distress, which is usually transient and can be minimized by slow titration of the dose. A potentially more serious adverse effect is lactic acidosis. Because biguanides decrease the flux of metabolic acids through gluconeogenic pathways, lactic acid can accumulate to dangerous levels in biguanide-treated patients. This complication is rarely seen with metformin (as opposed to phenformin, which is not approved for use in the United States). Lactic acidosis may occur more frequently when metformin is taken by patients who have other conditions predisposing to metabolic acidosis, such as hepatic disease, heart failure, respiratory disease, hypoxemia, severe infection, alcohol abuse, a tendency to ketoacidosis, or renal disease (the latter because biguanides are excreted by the kidneys). Biguanides do not directly affect insulin secretion, and their use is not associated with hypoglycemia. Amylin Analogue: Pramlintide Pramlintide was designed as a stable analogue of human amylin, the ␤-cell hormone that is co-secreted with insulin and helps regulate postprandial glucose levels. Type 1 diabetics lack endogenous amylin, and type 2 diabetics are relatively deficient in amylin. Thus, pramlintide is approved for use in both type 1 diabetics and insulin-requiring type 2 diabetics. Pramlintide’s structure is similar to amylin’s with the exception of three amino acid substitutions that confer improved solubility and stability (three prolines replace an alanine and two serines). Pramlintide slows gastric emptying, reduces postprandial glucagon and glucose release, and promotes satiety. It is administered as a subcutaneous injection before meals. Use of pramlintide often results in modest weight loss. The most common adverse effect is nausea, which is often limiting but may improve in some patients with prolonged use. Pramlintide is not associated with hypoglycemia unless it is used in conjunction with other agents that can cause hypoglycemia.

Meglitinides

GLP-1-Based “Incretin” Therapies

Like sulfonylureas, meglitinides stimulate insulin release by binding SUR1 and inhibiting the ␤-cell K⫹/ATP channel. Although sulfonylureas and meglitinides both act on the SUR1 subunit, these two classes of drugs bind to distinct regions of the SUR1 molecule. The absorption, metabolism, and adverse-effect profiles of meglitinides are similar to those of sulfonylureas.

GLP-1 Analogues

Exenatide is a long-acting analogue of GLP-1 that was originally isolated from the salivary gland of the Gila monster. It acts as an agonist at human GLP-1 receptors. Exenatide was approved for use in type 2 diabetics in the United States in 2005. The drug must be injected, typically twice a day, and is used in combination with metformin, a sulfonylurea, or a

CHAPTER 30 / Pharmacology of the Endocrine Pancreas and Glucose Homeostasis 537

openers of this type are known, but most are specific for SUR2 isoforms and thus are not useful to target the SUR1/ Kir6.2 channel expressed by pancreatic ␤-cells. Diazoxide binds channels containing either SUR1 or SUR2 isoforms and is therefore used not only to decrease insulin secretion by pancreatic ␤-cells but also to hyperpolarize SUR2expressing cardiac and smooth muscle cells. By maintaining these cells in a more relaxed state, off-label use of diazoxide may decrease blood pressure in hypertensive emergencies. In a rare form of genetic hyperinsulinemic hypoglycemia, a mutant SUR1 isoform is relatively insensitive to Mg2⫹-ADP but does respond to diazoxide; in most forms of this disease, however, the mutant channel is not transported to the cell surface, and diazoxide is ineffective. Octreotide is a somatostatin analogue that is longer acting than endogenous somatostatin. Like somatostatin, this agent blocks hormone release from endocrine-secreting tumors, such as insulinomas, glucagonomas, and thyrotropinsecreting pituitary adenomas. Octreotide has several other clinical indications (see Chapter 26).

reduces insulin levels. Thiazolidinediones and biguanides increase insulin sensitivity at target tissues. Amylin analogues reduce postprandial glucose levels. Octreotide, a synthetic form of somatostatin, has wide-ranging inhibitory effects on hormone secretion. Exogenous glucagon can be used to increase plasma glucose levels. Research on new pharmacologic treatments for early type 1 diabetes include efforts to develop immune modulatory therapies aimed at reversing ␤-cell dysfunction. For type 2 diabetes, agents may be developed: to inhibit the enzymes of glycogen synthesis and glycogenolysis in order to restrain glucose production (e.g., inhibitors of glycogen synthase kinase 3 to promote glycogen synthesis and inhibitors of hepatic glycogen phosphorylase to suppress glycogenolysis); to facilitate excretion of glucose in the renal proximal tubule (e.g., inhibitors of sodium–glucose cotransporter-2); or to target inflammation using small-molecule anti-inflammatory drugs or selected biologicals that block the actions of certain cytokines.

Acknowledgment Glucagon as a Therapeutic Agent Glucagon is used to treat severe hypoglycemia when oral or intravenous glucose administration is not possible. As with insulin, glucagon is administered by subcutaneous injection. The hyperglycemic action of glucagon is transient, and it requires a sufficient hepatic store of glycogen. Glucagon is also used as an intestinal relaxant before radiographic or magnetic resonance imaging (MRI) of the gastrointestinal tract. The mechanism by which glucagon mediates intestinal relaxation remains uncertain.

 CONCLUSION AND FUTURE DIRECTIONS Fuel homeostasis involves the pancreatic hormones insulin, glucagon, amylin, and somatostatin and the GI hormones GLP-1 and GIP. When the levels of these hormones are pathologically altered, an individual can become hyperglycemic (as in diabetes mellitus) or hypoglycemic. Various pharmacologic agents act at several different cellular and molecular sites to normalize blood glucose levels. ␣-Glucosidase inhibitors slow the intestinal absorption of carbohydrates. Exogenous insulin, sulfonylureas, meglitinides, and GLP1-based therapies increase insulin levels, while diazoxide

We thank Martin G. Myers, Jr. for his valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading DeWitt DE, Hirsch IB. Outpatient insulin therapy in type 1 and type 2 diabetes mellitus: scientific review. JAMA 2003;299:2254–2264. (Reviews currently available insulin preparations and their pharmacodynamic and pharmacokinetic profiles.) Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3:153– 165. (Reviews basic physiology of GLP-1 and related hormones.) Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 2003;144:5179–5183. (Reviews function and mechanism of action of probable biguanide target.) Krentz AJ, Bailey CJ. Oral antidiabetic agents: current role in type 2 diabetes mellitus. Drugs 2005;65:385–411. (Thorough review of the pharmacology of oral agents for the treatment of diabetes, with an emphasis on therapeutics.) Krentz AJ, Patel MB, Bailey CJ. New drugs for type 2 diabetes mellitus. Drugs 2008;68:2131–2162. (Reviews currently available oral and injectable pharmacologic agents, their indications, and adverse effects.) Nathan DM. Initial management of glycemia in type 2 diabetes mellitus. N Engl J Med 2002;347:1342–1349. (Clinically oriented approach to treatment of type 2 diabetes, including diet, exercise, insulin, oral agents, and combination therapy.)

31 Pharmacology of Bone Mineral Homeostasis Robert M. Neer, Ehrin J. Armstrong, and Armen H. Tashjian, Jr.

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 541-542 PHYSIOLOGY OF BONE MINERAL HOMEOSTASIS . . . . . . . . 541 Structure of Bone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541 Mineral Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542 Regulation of Bone Remodeling . . . . . . . . . . . . . . . . . . . . 542 Hormonal Control of Calcium and Phosphate . . . . . . . . . . 543 Parathyroid Hormone . . . . . . . . . . . . . . . . . . . . . . . . . 544 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546 Fibroblast Growth Factor 23 and Phosphatonins . . . . . 547 Calcitonin, Glucocorticoids, Thyroid Hormone, and Gonadal Steroids . . . . . . . . . . . . . . . . . . . . . . . . . 547 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 550 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 551 Antiresorptive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 Hormone Replacement Therapy (HRT) . . . . . . . . . . . . . 552

Selective Estrogen Receptor Modulators . . . . . . . . . . . 552 Bisphosphonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552 RANKL Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 Bone Anabolic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555 Parathyroid Hormone . . . . . . . . . . . . . . . . . . . . . . . . . 555 Treatment of Secondary Hyperparathyroidism in Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 556 Oral Phosphate Binders . . . . . . . . . . . . . . . . . . . . . . . 556 Calcitriol and Its Analogues . . . . . . . . . . . . . . . . . . . . . 556 Calcimimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556 Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Inorganic Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 557 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

 INTRODUCTION

factors—parathyroid hormone and vitamin D—control bone metabolism for the purpose of maintaining extracellular calcium homeostasis. Other hormones, such as glucocorticoids, thyroid hormone, gonadal steroids, and fibroblast growth factor 23, also have important effects on bone integrity. This section reviews the cellular and molecular mechanisms that mediate bone formation and bone resorption and the mechanisms by which hormones (especially parathyroid hormone and vitamin D) maintain plasma calcium levels within a narrow concentration range.

The 206 bones of the human skeleton are far from the lifeless structures they are commonly imagined to be. Bones are remodeled continuously and are involved in many functions besides structural support and protection of internal organs, including hematopoiesis and mineral storage. The focus of this chapter is on the critical role of bone in mineral homeostasis, the process and regulation of bone remodeling, the diseases that can result when the delicate balances of mineral homeostasis and bone remodeling are perturbed, and the pharmacologic therapies employed to treat these conditions. A key concept regarding the pharmacologic agents discussed in this chapter is the distinction between bone antiresorptive agents, which slow bone loss, and bone anabolic agents, which have the potential to increase overall bone mass.

 PHYSIOLOGY OF BONE MINERAL HOMEOSTASIS Specialized cells called osteoblasts and osteoclasts continually remodel the human skeleton in response to mechanical forces and endocrine and paracrine factors. Two of the endocrine

Structure of Bone Bone consists of 25% organic and 75% inorganic components. The organic component includes the cells (osteoblasts, osteoclasts, osteocytes, bone lining cells, bone stromal cells) and osteoid (a matrix consisting primarily of type I collagen fibers and several low-abundance proteins). The inorganic component consists of crystalline calcium phosphate salts, primarily hydroxyapatite. The chemical formula of hydroxyapatite is (Ca)5(PO4)3OH. Ninety-nine percent of the calcium in the body is stored in the skeleton, mostly as hydroxyapatite. Figure 31-1 illustrates the structure of a long bone. 541

CHAPTER 31 / Pharmacology of Bone Mineral Homeostasis 543

A

Articular cartilage

Proximal epiphysis

Epiphyseal plate Spongy trabecular bone Compact cortical bone Medullary cavity

Diaphysis

Osteon (Haversian system)

Lamellae

Distal epiphysis

Trabecular bone Central (Haversian) canal

Cortical bone

Periosteum

B Osteoblasts

Osteoclast

Resorption lacuna

Osteocyte

FIGURE 31-1. Structure of bone. A. The upper panel depicts the structure of a long bone (exemplified by the humerus). Note that the diaphysis consists of a thick outer layer or cortex of compact cortical bone surrounding the bone marrow. In the epiphysis, the cortex is thinner and surrounds trabecular bone as well as bone marrow; trabecular bone is also found in the vertebral bodies and much of the pelvis. B. The lower panel shows the detailed structure of bone. Bone remodeling is a dynamic balance between the catabolic activity of osteoclasts and the anabolic activity of osteoblasts. Osteoblasts and osteoclasts are found on all inner bone surfaces, including the endosteum that lines cortical bone and the many surfaces in trabecular bone. Bone remodeling is most intense in trabecular bone. Consequently, conditions that disrupt bone remodeling and/or bone mineralization affect trabecular bone preferentially. For example, osteoporotic fractures occur most commonly in vertebral bodies, which are predominantly trabecular bone. According to Le Chatelier’s principle, OH⫺ consumption drives this reaction to the right. This is an important mechanism exploited by osteoclasts to resorb the mineral component of bone. Demineralization of the bone matrix exposes it to proteolysis by cathepsin K, collagenases and other proteases that

are concomitantly secreted by the villi. Although this proteolysis totally degrades much of the exposed bone matrix, some of the type I collagen peptide chains escape into the circulation after partial proteolysis. The blood level of such type I collagen metabolites (e.g., NTX, CTX) is an index of type I collagenolysis and total body bone resorption. Because of its large hydroxyapatite-covered surface area, bone normally adsorbs various nonskeletal proteins and peptides from its environment, including IGF-I and TGF-␤. Demineralization exposes these adsorbed growth factors to the proteolytic enzymes secreted by osteoclast villi, but some escape proteolysis and affect the cellular activity of neighboring osteoclasts, osteoblasts, and osteocytes. After about 3 weeks of such bone resorption, cytokines and growth factors liberated from the matrix, together with hormonal and other factors (see below), begin to stimulate local accumulation of osteoblasts via proliferation, differentiation, and reduced apoptosis. These osteoblasts replace the osteoclasts in the resorption cavity (lacuna) and begin to refill the cavity with concentric layers, or lamellae, of unmineralized organic matrix (osteoid) (Fig. 31-3). As osteoblasts fill the cavity with new osteoid, they also secrete alkaline phosphatase, which hydrolyzes phosphate esters including pyrophosphate (a potent inhibitor of bone mineralization). The hydrolysis of pyrophosphate also increases the local concentration of inorganic phosphate. Together, the alkaline phosphatase-catalyzed hydrolysis of pyrophosphate and the liberation of inorganic phosphate promote the crystallization of calcium phosphate salts and mineralization of the bone matrix. As osteoblasts continue to lay down matrix, some eventually become completely surrounded by it and are then called osteocytes (Fig. 31-1). Osteocytes help control the balance between bone formation and resorption via their secretion of sclerostin (a protein that inhibits bone formation) and other factors. Genetic mutations that delete or inactivate sclerostin increase bone formation, without a corresponding increase in bone resorption. Such uncoupling leads to a marked increase in bone mass and bone strength in humans and experimental animals. A monoclonal antibody that inactivates sclerostin, and thereby increases bone mass and bone strength throughout the skeleton, is being evaluated as a potential treatment for osteoporosis. Mature osteocytes normally alter their secretion of sclerostin in bone regions subjected to mechanical loads, and thereby play a critical role in skeletal adaptations to gravity and other mechanical loads by localizing bone remodeling responses to such loads.

Hormonal Control of Calcium and Phosphate Calcium is essential for many important physiologic processes, such as neurotransmitter release, muscle contraction, and blood coagulation, and deviations in extracellular calcium levels can have serious consequences. Therefore, the blood calcium level is tightly regulated. Inorganic phosphate concentrations must also be regulated, in part because changes in plasma inorganic phosphate concentrations affect plasma calcium levels (see below). Three main hormones—parathyroid hormone (PTH), vitamin D, and FGF-23—mediate calcium and phosphate homeostasis. In addition, calcitonin, glucocorticoids, thyroid hormone, and gonadal steroids have lesser effects on calcium and phosphate homeostasis. Table 31-1 summarizes the mechanisms and effects of these hormones on calcium and phosphate homeostasis.

544 Principles of Endocrine Pharmacology

FIGURE 31-2.

Calcium intake 1000 mg

PTH

1,25(OH)2D

(exogenous; once-daily)

300 mg

300 mg

(Absorption)

100 mg

(Accretion)

Plasma calcium

300 mg

(Secretion)

Small intestine

(Resorption)

CT (exogenous)

800 mg

200 mg Renal excretion

Fecal excretion PTH

(stimulates Ca2+ reabsorption and enhances PO4 excretion)

Parathyroid Hormone The most important endocrine regulator of calcium homeostasis is parathyroid hormone, an 84-amino acid peptide hormone secreted by the parathyroid glands. The secretion of PTH is finely regulated in response to plasma calcium levels. Calcium-sensing receptors reside on the plasma membrane Osteoblast precursor

PTH, shear stress, TGF-β

Osteoblast precursor

1 5 RANKL

Mature osteoblasts 2

Osteoclast precursor Mature osteoclast

4

RANK

Osteoblast precursor

FIGURE 31-3.

TGF-β, IGF-1, growth factors, cytokines

3 M-CSF

Interaction of osteoblasts and osteoclasts in bone remodeling. Bone resorption and bone formation are coupled by the interactions between osteoblasts and osteoclasts: (1) Factors such as parathyroid hormone (PTH), shear stress, and transforming growth factor ␤ (TGF-␤) cause osteoblast precursors to express the osteoclast differentiation factor RANK-ligand (RANKL). (2) RANKL binds to RANK, a receptor expressed on osteoclast precursors. (3) The RANKL–RANK binding interaction, together with macrophage colony-stimulating factor (M-CSF), causes osteoclast precursors to differentiate into mature osteoclasts. (4) As mature osteoclasts resorb bone, matrix-bound factors such as TGF-␤, insulin-like growth factor 1 (IGF-1), other growth factors, and cytokines are released. (5) These liberated factors stimulate osteoblast precursors to develop into mature osteoblasts, which begin to refill the resorption cavities excavated by the osteoclasts.

Bone PTH (endogenous; continuous)

Daily whole-body calcium balance. In a state of whole-body calcium balance, the fluxes of calcium include net uptake of 200 mg per day from the GI tract and excretion of 200 mg per day by the kidneys. Calcitriol [1,25(OH)2D3] enhances absorption of Ca2⫹ from the GI tract. Continuous secretion of parathyroid hormone (PTH) increases bone formation and (even more) bone resorption, and stimulates renal tubular reabsorption of calcium; both effects raise plasma Ca2⫹. Continuous secretion of PTH also enhances renal clearance of inorganic phosphate (PO4). In contrast, once-daily injection of PTH (in blue) stimulates new bone formation (accretion) more than it stimulates bone resorption and has only transient (and consequently minor) effects on renal clearance of Ca2⫹ and PO4. Exogenous calcitonin (CT; also in blue) inhibits bone resorption.

of chief cells in the parathyroid gland; when bound by extracellular calcium ions, these G protein-coupled receptors mediate increases in the level of intracellular free calcium, which, in turn, decreases secretion of preformed PTH. By this mechanism, high plasma calcium concentration suppresses PTH secretion, while low plasma calcium stimulates PTH secretion. (Note: In many other secretory tissues, an increase in intracellular calcium enhances secretion. Thus, the parathyroid chief cell is unusual in its response to changes in intracellular calcium.) PTH acts on three organs to raise the plasma calcium concentration: it acts directly on kidney and bone, and indirectly on the gastrointestinal (GI) tract (Fig. 31-4). The most rapid physiologic effects of PTH are to increase reabsorption of calcium and to decrease reabsorption of inorganic phosphate by the kidney tubules. These actions decrease renal clearance of calcium while increasing renal clearance of inorganic phosphate. In this manner, PTH raises plasma calcium levels and decreases plasma inorganic phosphate concentrations. Another important, although slower, effect of PTH results from its direct actions on bone cells. Physiologic levels of PTH stimulate cell surface PTH receptors on osteoblasts, causing these cells to increase their expression of the osteoclast differentiation factor RANKL (Fig. 31-3) and decrease their expression of its antagonist OPG. The resulting increase in osteoclastic activity increases bone resorption and thereby increases the release of calcium and inorganic phosphate into the circulation. PTH also induces bone marrow stromal cells to secrete cytokines such as IL-6, and these cytokines ultimately stimulate osteoclast proliferation and bone resorption. Finally, PTH raises plasma calcium by an indirect effect on the intestine. PTH stimulates the kidney not only to increase calcium reabsorption and decrease phosphate reabsorption, as described above, but also to increase the enzymatic conversion of 25-hydroxy vitamin D to 1,25-dihydroxy vitamin D (calcitriol). This hydroxylation takes place in cells of the proximal renal tubules. Calcitriol, in turn, increases small intestinal absorption of calcium and (to a lesser extent) inorganic phosphate (discussed below). Although the release of skeletal calcium and inorganic phosphate could be considered catabolic, PTH simultaneously stimulates new bone formation by promoting differentiation of osteoblast precursors to mature osteoblasts and

Thyroid gland

Parathyroid glands

Plasma [Ca2+]

Plasma [Ca2+] PTH

Osteoclastic activity liberates PO4 and Ca2+ Bone

Kidney

PO4/ Ca2+ reabsorption Hydroxylation of 25(OH) vitamin D to 1,25(OH)2 vitamin D

Mucosal Ca2+ uptake and transport proteins Ca2+ absorption Intestine

546 Principles of Endocrine Pharmacology

extracellular PTH increase bone formation more than bone resorption and cause a net increase in bone mass. Consequently, intermittent PTH administration by once-daily injection or by some other drug delivery technology increases bone matrix production, bone mass, bone mineral density, and bone strength (see below). In contrast, continuous elevation of extracellular PTH increases bone formation equally, but increases bone resorption more, and causes net bone loss in patients with primary or secondary hyperparathyroidism. Vitamin D Despite its name, vitamin D3 is produced in the skin and is not required in the diet if sun exposure is generous. Because it is produced endogenously and travels in the blood to effect responses in distant target tissues, vitamin D3 is more correctly considered a hormone. The term vitamin D applies to two related compounds, cholecalciferol and ergocalciferol. Cholecalciferol, or vitamin D3, is generated nonenzymatically in the skin when 7-dehydrocholesterol absorbs a photon of short ultraviolet light (UV-B; Fig. 31-5). Ergocalciferol, or vitamin D2, is produced when ergosterol in plants absorbs such a photon. Vitamins D2 and D3 are each added to dairy products and some other foods; each is available as a dietary supplement; and each is available (in much higher doses) as a prescription drug. Vitamins D2 and D3 have equal biological activities, and “vitamin D” in subsequent paragraphs refers to both the D2 and D3 forms of the hormone. Whether from an endogenous (skin) or an exogenous (dietary) source, vitamin D travels to the liver, where it is either stored or converted to calcifediol [25-hydroxy vitamin D, or 25(OH)D] by the first of two enzymatic hydroxylation steps. The second enzymatic hydroxylation converts calcifediol to the final, active form of vitamin D called calcitriol [1␣,25-dihydroxy vitamin D, or 1,25(OH)2D]. This second hydroxylation takes place in many tissues, particularly in the proximal tubule of the kidney (where it is PTH-dependent), but does not take place in the intestines because they lack the enzyme required for the second hydroxylation. Calcitriol’s primary effect on calcium balance is in the small intestine, where it increases the absorption of dietary calcium. Calcitriol enhances Ca2⫹ absorption by acting on nuclear receptors in the enterocyte to up-regulate the expression of genes coding for a number of brush border proteins. Calcitriol also promotes the transcellular transport of Ca2⫹ through the enterocyte by inducing the expression of: (1) a calcium uptake pump on the luminal surface of the enterocyte; (2) calbindin, an intracellular Ca2⫹-binding protein; and (3) an ATP-dependent Ca2⫹ pump that extrudes Ca2⫹ from the enterocyte into the surrounding capillaries. Because enterocytes do not express the enzyme needed to form 1,25(OH)2D from 25(OH)D, their absorption of calcium is regulated by blood levels of 1,25(OH)2D, which in turn depend on renal tubular function and blood levels of PTH. Calcitriol has important effects on other target organs, including the parathyroid gland, bone, kidneys, and the immune system. Calcitriol binds to nuclear receptors in parathyroid cells, and thereby inhibits PTH synthesis and release. In bone, calcitriol increases osteoclast number and activity, resulting in increased bone resorption. High blood levels of calcitriol, and lower levels of certain calcitriol analogues, increase bone formation. In the distal tubule of the kidney, calcitriol increases the reabsorption of both calcium and phosphate. In the immune system, calcitriol production by macrophages may act

H

H

HO

7-dehydrocholesterol Skin

UV-B

H

HO

Vitamin D3 (cholecalciferol) Circulation Dietary Vitamin D3 (animal sources) Vitamin D2 (plant sources)

Vitamin D storage

Side-chain of Vitamin D2 (ergocalciferol)

25-hydroxylase

Liver OH

H

HO

25-hydroxy vitamin D (calcifediol)

1α-hydroxylase

PTH, hypophosphatemia

Kidney

OH

H

HO

OH

1,25-dihydroxy vitamin D (calcitriol)

FIGURE 31-5. Photobiosynthesis and activation of vitamin D. Both endogenous and exogenous vitamin D are converted to 25-hydroxy vitamin D in the liver and then to calcitriol in the kidney. Calcitriol is the active metabolite of vitamin D. Endogenous vitamin D3 is synthesized in the skin from 7-dehydrocholesterol, in a reaction that is catalyzed by ultraviolet light (UV-B). Exogenous vitamin D can be provided as D3 (from animal sources) or as D2 (from plant sources); D3 and D2 have the same biological activity. Parathyroid hormone (PTH) increases the activity of 1␣-hydroxylase in the kidney and thereby stimulates the conversion of 25hydroxy vitamin D to calcitriol, as does hypophosphatemia.

Growth spurt

Increased production of cytokines Longer lifespan of osteoclasts ( apoptosis)

Perimenopause in women

Shorter lifespan of osteoblasts ( apoptosis)

Bone mass

Activation of osteoclasts

Shorter lifespan of osteocytes ( apoptosis)

Men Deeper, larger resorption cavities in bone

Mechanosensing

Women More fragile bone

Bone fracture

0

25

50

Age (years)

75

100

Microdamage in bone

CHAPTER 31 / Pharmacology of Bone Mineral Homeostasis 551 Chronic kidney disease Oral phosphate binders FGF-23

1,25(OH)2 D production Active vitamin D analogues

Phosphate retention

Plasma phosphate and binding to calcium

Active vitamin D analogues

GI Ca2+ absorption

Plasma [Ca2+] Synthesis of PTH Secretion of PTH Cinacalcet Parathyroid gland hyperplasia

Synthesis of PTH Secretion of PTH Degradation of PTH Ca2+ receptors on parathyroid cells and Set-point for Ca2+ regulation Synthesis of PTH Parathyroid gland hyperplasia Cinacalcet Hyperparathyroidism

Bone resorption Osteomalacia Osteitis fibrosa cystica

activity in the kidney—and the calcimimetic cinacalcet— which adjusts the sensitivity of the calcium-sensing receptor on parathyroid chief cells (see below). Hyperphosphatemia, resulting from decreased renal excretion of phosphate, further exacerbates the hypocalcemia of chronic kidney disease. Hyperphosphatemia induces hypocalcemia by altering the equilibrium for hydroxyapatite formation and dissolution, as described in Equation 31-1. Hyperphosphatemia leads to the formation of toxic calcium phosphate precipitates in extraskeletal tissues, as in tumoral calcinosis, but in chronic kidney disease, hyperphosphatemia also stimulates increased secretion of FGF-23. Elevated blood FGF-23 suppresses renal secretion of 1,25-(OH)2D and has the toxic cardiovascular effects described above. Paradoxically, osteomalacia may coexist with ectopic calcium phosphate deposits, because bone matrix does not mineralize normally. Although metabolic acidosis due to chronic kidney disease does inhibit bone mineralization, other mineralization inhibitors are also involved but are not yet adequately defined.

 PHARMACOLOGIC CLASSES AND AGENTS Significant advances have occurred in recent years in the treatment of osteoporosis and chronic kidney disease. For osteoporosis, the relevant pharmacologic agents can be divided into two main categories: drugs that inhibit bone resorption and drugs that stimulate bone formation. Antiresorptive agents consist of hormone replacement therapy (HRT), selective estrogen receptor modulators, bisphosphonates, RANKL antagonists, calcitonin, and cathepsin K inhibitors (in development). Bone

FIGURE 31-8. Pathophysiologic basis for osteomalacia and osteitis fibrosa cystica in chronic kidney disease. In chronic kidney disease, compromised renal function leads to decreased 1,25(OH)2 vitamin D synthesis and decreased phosphate excretion. The decrease in 1,25(OH)2 vitamin D causes decreased gastrointestinal (GI) absorption of Ca2⫹, while the increased phosphate retention causes an increase in the levels of plasma phosphate, which complexes with Ca2⫹. By these two mechanisms, chronic kidney disease leads to hypocalcemia. Hypocalcemia stimulates secretion of parathyroid hormone (PTH). Decreased levels of 1,25(OH)2 vitamin D stimulate PTH synthesis and parathyroid gland hyperplasia, and lead to a decreased number of Ca2⫹ receptors on parathyroid gland chief cells and an elevated setpoint for Ca2⫹ regulation. Hyperphosphatemia may stimulate increased synthesis and secretion of PTH directly and also increase levels of FGF-23, leading to decreased levels of 1,25(OH)2 vitamin D. This combination of complex regulatory events leads to hyperparathyroidism, a syndrome characterized by increased bone resorption, increased amounts of unmineralized osteoid, and osteitis fibrosa cystica. Oral phosphate binders lower plasma phosphate levels by preventing dietary phosphate absorption. Active vitamin D analogues bypass the defect in renal 1␣-hydroxylase activity that accompanies chronic kidney disease. Calcimimetics (cinacalcet) modulate the activity of the Ca2⫹-sensing receptor on chief cells, such that the receptor is activated at lower plasma Ca2⫹ concentrations.

anabolic agents consist of fluoride and parathyroid hormone. For chronic kidney disease, the relevant pharmacologic agents include drugs that lower plasma phosphate levels (oral phosphate binders) and drugs that decrease parathyroid hormone synthesis and secretion (vitamin D, vitamin D analogues, and calcimimetics). Oral calcium and vitamin D also have an important role in the prevention and treatment of osteoporosis, rickets, and hypoparathyroidism.

Antiresorptive Agents Antiresorptive agents prevent or arrest bone loss by suppressing osteoclastic bone resorption. However, because bone resorption and bone formation are closely coupled processes, a decrease in one typically leads to a decrease in the other, via molecular mechanisms that are unclear. As a result, hormone replacement therapy (HRT), selective estrogen receptor modulators, bisphosphonates, RANKL antagonists, and calcitonin induce little increase in bone tissue. The increase in bone mineral density seen during the first 12–18 months of therapy with these drugs represents filling of resorption cavities produced during the previous period of excessive bone resorption, mineralization of this new bone, and completion of mineralization (secondary mineralization) in old bone formed and partially mineralized during the 12–18 months preceding antiresorptive therapy. After the first 12–18 months of therapy with these agents, bone mineral density increases slowly, reflecting the slow formation and mineralization of new bone when resorption is suppressed. Cathepsin K inhibitors are an unusual exception, because they suppress osteoclastic bone resorption without suppressing bone formation.

OH

H H

H

HO 17β-estradiol

OH

S

HO

O N O Raloxifene

-O

O

O

P

P

HO

O

O-

-O

O

O

P

P

HO

OH

OOH

R1 R2

Pyrophosphate Bisphosphonate

Bisphosphonate R1

R2

Etidronate

OH

CH3

Pamidronate

OH

CH2

CH2

NH2

Alendronate

OH

CH2

CH2

CH2

Ibandronate

OH

CH2

CH2 N

NH2

CH3 (CH2)4 CH3 N Risedronate

OH

CH2

Zoledronate

OH

CH2

Tiludronate

H

S

N

N

Cl

556 Principles of Endocrine Pharmacology

to fracture and osteitis fibrosa cystica. In contrast, although intermittent exposure of bone cells to PTH also increases bone remodeling, more new bone is formed than old bone resorbed. Thus, once-daily subcutaneous administration of PTH favors bone anabolism, while continuous exposure to PTH favors bone catabolism. Native PTH is an 84-amino acid peptide, but N-terminal fragments containing the first 31–34 amino acids of PTH retain essentially all the important functional properties of the native protein. The 1–34 fragment has been shown in clinical trials to act as a powerful anabolic agent that builds new bone. Because PTH(1–34) is a peptide, the bioavailability of this agent is close to zero when administered orally. The currently available formulation is a subcutaneous injection that is designed to be self-administered. Alternative dosage forms (e.g., transcutaneous) are in advanced stages of clinical development. Human PTH(1–34) is approved under the generic name teriparatide for the treatment of osteoporosis in postmenopausal women, idiopathic and hypogonadal osteoporosis in men, and glucocorticoid-induced osteoporosis in patients of either sex, and approved for the reduction of spine and nonspine fractures in postmenopausal osteoporotic women at high risk of fracture. Full-length human PTH(1–84) is approved for such uses in some countries, but not in the United States (because of hypercalcemia and other adverse effects from the marketed dose). Because prolonged treatment of rodents with either of these peptides causes dramatic bone overgrowth followed by osteosarcomas, teriparatide is used only in patients at high risk of fractures. However, there is no evidence that any parathyroid hormone increases osteosarcomas in humans. In humans, the anabolic skeletal effects of teriparatide are attenuated by concomitant alendronate therapy. It is unclear whether concomitant therapy with other bisphosphonates, or prior therapy with any bisphosphonate, does the same.

Treatment of Secondary Hyperparathyroidism in Chronic Kidney Disease There are currently three pharmacologic approaches to preventing and modifying the metabolic sequelae of chronic kidney disease—oral phosphate binders, calcitriol and its analogues, and calcimimetics. Oral Phosphate Binders In patients with chronic kidney disease, or chronic hyperphosphatemia of any cause, the increased plasma phosphate can complex with circulating calcium. The resulting decrease in plasma calcium concentration can lead to hyperparathyroidism, and the precipitation of calcium phosphate in extraskeletal tissues can impair their function. Dietary phosphate restriction and oral phosphate binders can limit both processes. Aluminum hydroxide was one of the first agents used to treat hyperphosphatemia. Aluminum precipitates phosphate in the gastrointestinal tract, forming nonabsorbable complexes. Although effective at lowering plasma phosphate levels, this approach has been abandoned (except in cases of refractory hyperphosphatemia) because of aluminum toxicity: over the course of years, chronic use of aluminum-based phosphate binders can lead to chronic anemia, osteomalacia, and neurotoxicity.

Oral preparations of calcium carbonate and calcium acetate can control plasma phosphate. These agents, when administered with meals, bind to dietary phosphate and thereby inhibit its absorption. At the doses required for phosphate binding, however, these agents can also cause iatrogenic hypercalcemia and may increase the risk of vascular calcification. Sevelamer is a nonabsorbable cationic ion-exchange resin that binds intestinal phosphate, thereby decreasing the absorption of dietary phosphate. Sevelamer also binds bile acids, leading to interruption of the enterohepatic circulation and to decreased cholesterol absorption. Its principal disadvantage is its expense. Sevelamer is used to treat hyperphosphatemia in patients with chronic kidney disease. Sevelamer is also used to correct hyperphosphatemia in patients with the hyperphosphatemia-hyperostosis syndrome (also known as tumoral calcinosis with hyperphosphatemia), who are deficient in FGF-23 secretion or action (Table 31-2). Calcitriol and Its Analogues Because impaired synthesis of 1␣-vitamin D derivatives is one of the main homeostatic disturbances leading to secondary hyperparathyroidism in chronic kidney disease, vitamin D is a logical replacement therapy in this disease. Three active (i.e., 1␣-hydroxylated) vitamin D congeners are approved for treatment of secondary hyperparathyroidism. All of these agents bypass the need for 1␣-hydroxylation in the kidney and are therefore useful in the treatment of bone diseases that complicate renal failure. Active vitamin D increases dietary absorption of calcium, and the resulting increase in plasma calcium suppresses the secretion of PTH by chief cells of the parathyroid gland. In addition, these agents bind to and activate vitamin D receptors on the chief cells, and thereby suppress PTH gene transcription and parathyroid hyperplasia. Care should be taken to avoid hypercalcemia when administering any of the active vitamin D congeners. Calcitriol [1,25(OH)2D3] is the dihydroxylated form of vitamin D3. Calcitriol is available in oral and intravenous forms; some data suggest that the intravenous formulation may be more effective in patients on hemodialysis. Calcitriol should not be administered to patients with chronic kidney disease until hyperphosphatemia has been controlled with diet and/or drugs, because the addition of calcitriol can cause increased plasma levels of both calcium and phosphate. Paricalcitol [19-nor-1,25(OH)2D2] is a synthetic analogue of vitamin D. Doxercalciferol [1␣-(OH)D2] is the 1␣hydroxylated form of vitamin D2; it is 25-hydroxylated to the fully active 1,25-dihydroxy form in the liver. Both paricalcitol and doxercalciferol may lower plasma PTH levels without significantly raising plasma calcium levels. Calcimimetics Although vitamin D and its analogues can be effective in the treatment of secondary hyperparathyroidism, these agents can also lead to unwanted hypercalcemia and hyperphosphatemia. The so-called calcimimetics—agents that modulate the activity of the calcium-sensing receptor on chief cells— are effective treatments for hyperparathyroidism that do not cause these unwanted effects. Cinacalcet, the first Food and Drug Administration (FDA)-approved calcimimetic, binds to the transmembrane region of the calcium-sensing receptor, and thereby modulates receptor activity by increasing its sensitivity to calcium. Because the cinacalcet-bound receptor is activated

558 Principles of Endocrine Pharmacology

mass because of increased bone resorption or decreased bone formation or (2) the formation of architecturally unsound bone because of excessively rapid bone formation (woven bone) or deficient bone mineralization (rickets and osteomalacia). In turn, structural weakening of the bone predisposes to bone fracture or deformity. Bone disorders can be treated by correcting the underlying hormonal or mineral imbalances (e.g., vitamin D, calcium) or by modulating bone remodeling (e.g., SERMs, bisphosphonates, RANKL antagonists). Pharmacologic interventions directed at the physiology of bone remodeling can be divided into two main categories: antiresorptive agents and bone anabolic agents. The majority of drugs currently approved by the FDA for the treatment of osteoporosis are antiresorptive agents. These drugs act by inhibiting osteoclastic bone resorption, and thus slowing the loss of bone mass. However, these drugs do not stimulate new bone formation and do not increase true bone mass (matrix plus mineral). Hence, antiresorptive agents do not represent optimal therapy for individuals who have already sustained severe loss of bone mass. The only FDA-approved bone anabolic agent is once-daily PTH, which acts by increasing bone formation and is therefore the most beneficial agent for patients with very low bone mass. The structurally related natural protein, PTH-related protein, has similar effects in animals, and a synthetic analogue of PTHrP increases bone mass in humans and is undergoing additional trials in humans. Most drugs that reduce bone resorption subsequently reduce bone formation. Two important exceptions are currently undergoing clinical trials in humans: a fully-humanized monoclonal antibody that neutralizes sclerostin and an oral inhibitor of cathepsin K. The former increases bone formation without increasing bone resorption, and the latter decreases bone resorption without decreasing bone formation. The action of these agents suggests that it is possible to uncouple bone resorption from bone formation and thereby effectively treat osteoporosis.

Acknowledgment We thank Allen S. Liu and Ariel Weissmann for their valuable contributions to this chapter in the First and Second

Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Andress DL. Vitamin D treatment in chronic kidney disease. Semin Dial 2005;18:315–321. (Reviews progression of chronic kidney disease and indications for vitamin D therapy.) Bergwitz C, Juppner H. Disorders of phosphate homeostasis and tissue mineralisation. Endocr Dev 2009;16:133–156. (Current understanding of the pathophysiology, diagnosis, and treatment of abnormal phosphate homeostasis and tissue mineralization.) Bilezikian JP. Efficacy of bisphosphonates in reducing fracture risk in postmenopausal osteoporosis. Am J Med 2009;122(Suppl 2):14S–21S. (Summarizes effects on fracture incidence, effects of less-frequent dosing regimens, and efficacy during long-term treatment.) Cranney A, Weiler HA, O’Donnell S, Puil L. Summary of evidence-based review on vitamin D efficacy and safety in relation to bone health. Am J Clin Nutr 2008;88:513S–519S. (Current clinical evidence for use of vitamin D in prevention and treatment of osteoporosis.) Drake MT, Clarke BL, Khosla S. Bisphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc 2008;83:1032–1045. (Selective review of medical literature from the preceding decade.) Ebeling PR. Osteoporosis in men. N Engl J Med 2008;358:1474–1482. (Review of an underappreciated public health problem.) Maclean C, Newberry S, Maglione M, et al. Systematic review: comparative effectiveness of treatments to prevent fractures in men and women with low bone density or osteoporosis. Ann Intern Med 2008;148:197–213, 423–425, 884–887. (Excellent overview of the comparative effectiveness of various agents for the treatment of osteoporosis.) Querfeld U. The therapeutic potential of novel phosphate binders. Pediatr Nephrol 2005;20:389–392. (Review of agents used to lower serum phosphate levels.) Rahmani P, Morin S. Prevention of osteoporosis-related fractures among postmenopausal women and older men. Can Med Assn J 2009;181: 815–820. (Focus on prevention of fractures rather than prevention of bone loss.) Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest 2005;115:3318–3325. (Current understanding of osteoporosis pathophysiology.) Rosen CJ. Postmenopausal osteoporosis. N Engl J Med 2005;353:595–603. (Succinct overview of the clinical management of osteoporosis.) Steddon SJ, Cunningham J. Calcimimetics and calcilytics—fooling the calcium receptor. Lancet 2005;365:2237–2239. (New approaches to pharmacologic modulation of the calcium-sensing receptor.)

V Principles of Chemotherapy

32 Principles of Antimicrobial and Antineoplastic Pharmacology Quentin J. Baca, Donald M. Coen, and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 563-564 MECHANISMS OF SELECTIVE TARGETING . . . . . . . . . . . . . 564 Unique Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565 Selective Inhibition of Similar Targets . . . . . . . . . . . . . . . . 565 Common Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 PATHOGENS, CANCER CELL BIOLOGY, AND DRUG CLASSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Fungi and Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Carcinogenesis and Cell Proliferation . . . . . . . . . . . . . 568 Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 Log Cell Kill Model . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 MECHANISMS OF DRUG RESISTANCE . . . . . . . . . . . . . . . . . 572 Genetic Causes of Drug Resistance . . . . . . . . . . . . . . . . . 572 Reduced Intracellular Drug Concentration . . . . . . . . . . 572 Target-Based Mechanisms . . . . . . . . . . . . . . . . . . . . . 573 Insensitivity to Apoptosis . . . . . . . . . . . . . . . . . . . . . . . 573 Practices That Promote Drug Resistance . . . . . . . . . . . . . 574

METHODS OF TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . 574 Combination Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . 574 Prophylactic Chemotherapeutics . . . . . . . . . . . . . . . . . . . 574 INHIBITORS OF FOLATE METABOLISM: EXAMPLES OF SELECTIVE TARGETING AND SYNERGISTIC DRUG INTERACTIONS . . . . . . . . . . . . . . . . . . 574 Folate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Inhibitors of Folate Metabolism . . . . . . . . . . . . . . . . . . . . 575 Unique Drug Targets: Antimicrobial Dihydropteroate Synthase Inhibitors . . . . . . . . . . . . . . 575 Selective Inhibition of Similar Targets: Antimicrobial Dihydrofolate Reductase Inhibitors . . . . . . . . . . . . . . . 576 Common Targets: Antineoplastic Dihydrofolate Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Synergy of DHFR Inhibitors and Sulfonamides . . . . . . . 577 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 578 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578

 INTRODUCTION

tuberculosis (1.46 million), and malaria (1.0 million). In the developed world, although infectious disease mortality is increasing, cancer (along with heart disease and stroke) is a more significant cause of death. The most deadly cancers in the United States presently include lung cancer (159,390 estimated deaths in 2009), colon cancer (49,920), breast cancer (40,610), pancreatic cancer (35,240), and prostate cancer (27,360). Both infectious and neoplastic patterns of disease will likely change as increasingly effective treatments and preventive measures are developed and distributed. This chapter focuses on the principles of antimicrobial and antineoplastic pharmacology, but there are also many important and effective nonpharmacologic strategies to combat microbes and cancer. These strategies include public health measures, vaccinations, and screening procedures. Most public health and vaccination programs aim to prevent infections rather than treat existing infections. Smallpox, for example, was eradicated worldwide in 1977 through aggressive vaccination programs, although concerns have been raised about the potential

Although infectious diseases and cancers have different underlying etiologies, from a pharmacologic perspective, the broad principles of treatment are similar. The common thread in these pharmacologic strategies is the targeting of selective differences between the microbe or cancer cell and the normal host cell. Because both microbes and cancer cells can evolve resistance to drug therapies, the development of new treatments is also a continually evolving process. Infectious diseases and cancers are among the most deadly afflictions plaguing human societies. The World Health Organization (WHO) has estimated that, in the year 2004, five major infectious diseases caused almost 11 million of the 59 million total deaths worldwide, and malignant neoplasms were responsible for 7.4 million deaths. Among the infectious diseases, the most common causes of mortality worldwide included lower respiratory infections (4.18 million), diarrheal diseases (2.16 million), HIV/AIDS (2.04 million),

563

566 Principles of Chemotherapy

Infections: DNA Replication, Transcription, and Translation). Macrolide antibiotics such as erythromycin bind to the 50S bacterial ribosomal subunit and block translocation by preventing emergence of the protein from the ribosome. Aminoglycoside antibiotics such as streptomycin and gentamicin bind to the 30S bacterial ribosomal subunit and disrupt the decoding of mRNA. More generally, the bacterial protein synthesis inhibitors include a wide variety of individual drugs with diverse mechanisms, and the selectivity and dose-limiting toxicities of these drugs are often class- and/or drug-specific. For example, the macrolides rarely cause serious adverse effects, whereas some of the aminoglycosides have dose-limiting ototoxicity and nephrotoxicity. Some adverse effects appear to result from drug binding to human mitochondrial ribosomes in addition to bacterial ribosomes. Thus, selective inhibition of similar targets, as exemplified by DHFR inhibitors and protein synthesis inhibitors, can result in effects characterized by therapeutic indices that range from low to high, depending on the individual drug or drug class under consideration.

Common Targets When the host and pathogen or cancer share common biochemical and physiologic pathways, a basis for selectivity may be found if the pathogen or cancer requires a metabolic activity or is affected by its inhibition to a greater degree than the host. These relatively minor differences are often exploited in cancer pharmacology, explaining the narrow therapeutic indices of many of these drugs. Tumor cells arise from normal cells that have been transformed by genetic mutations into cells with dysregulated growth. These cells utilize the same machinery for growth and replication as do normal cells. Therefore, selective inhibition of cancer cell growth is a major challenge. Recent discoveries have identified a number of mutated or overexpressed proteins in cancer cells, and selective inhibitors of these proteins are entering clinical use with increasing frequency (see Chapter 39, Pharmacology of Cancer: Signal Transduction). Nonetheless, it is still the case that the basis for the selectivity of most currently used antineoplastic drugs arises not from biochemical differences, but rather from variations in cancer cell growth behavior and from increased susceptibility of cancer cells to induction of apoptosis or senescence. Cancer, as a disease of persistent proliferation, requires continued cell division. Therefore, drugs targeting processes involved in DNA synthesis, mitosis, and cell cycle progression may kill rapidly cycling cancer cells preferentially over their normal relatives. (An important correlate to this statement is that many chemotherapeutic strategies are more successful against rapidly growing than slowly growing cancers.) Antimetabolites such as 5-fluorouracil (5-FU) inhibit DNA synthesis in dividing cells (see Chapter 38, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance). 5-FU inhibits thymidylate synthase, the enzyme responsible for converting dUMP to dTMP, a pyrimidine building block of DNA. As a pyrimidine analogue, 5-FU is also incorporated into growing RNA and DNA strands, thereby interrupting the synthesis of these strands. By causing DNA damage, 5-FU induces the cell to activate its apoptotic pathway, resulting in programmed cell death. 5-FU is toxic to all human cells undergoing DNA synthesis and, thus, is selectively toxic

both for rapidly cycling tumor cells (therapeutic effect) and for high-turnover host tissues such as the bone marrow and gastrointestinal mucosa (adverse effect). These examples illustrate the importance of studying the cell biology, molecular biology, and biochemistry of microbes and cancer cells to identify specific targets for selective inhibition. Clinically, an awareness of drug mechanisms and the basis of drug selectivity can help to explain the narrow or broad therapeutic indices that have an impact on drug dosing and treatment strategies. Understanding the selectivity of drugs for their targets is also important in combating drug resistance. Thus, the fundamental pharmacologic principles of drug–receptor interactions, therapeutic and adverse effects, and drug resistance form the basis for selective targeting in antimicrobial and antineoplastic drug therapy.

 PATHOGENS, CANCER CELL BIOLOGY, AND DRUG CLASSES Pharmacologic interventions target specific differences between the host and the microbial pathogen or cancer cell. This section examines some of the unique characteristics evolution has bestowed on organisms, and the major drug classes that target these molecular differences among host cells, pathogens, and cancer cells.

Bacteria Bacteria are organisms that often contain unique targets for pharmacologic intervention. Some of these drug targets have been discussed previously and are illustrated in Figure 32-1. Currently available drugs act to interrupt bacterial DNA replication and repair (this chapter and Chapter 33), transcription and translation (Chapter 33), and cell wall synthesis (Chapter 34). Depending on the role of the drug target in bacterial physiology, antibacterial drugs can produce bacteriostatic or bactericidal effects. Drugs that inhibit the growth of the pathogen without causing cell death are called bacteriostatic. These drugs target metabolic pathways that are necessary for bacterial growth but not for bacterial survival. Most protein synthesis inhibitors (aminoglycosides are an exception) have a bacteriostatic effect. The clinical effectiveness of these drugs relies on an intact host immune system to clear the nongrowing (but viable) bacteria. In contrast, bactericidal drugs kill bacteria. For example, cell wall synthesis inhibitors (e.g., penicillins and cephalosporins) cause bacterial lysis when the bacteria grow in or are exposed to hypertonic or hypotonic environments. Thus, bacterial infections in immunocompetent hosts can often be treated with bacteriostatic drugs, whereas the treatment of bacterial infections in immunocompromised hosts often requires bactericidal drugs. Bacteriostatic and bactericidal effects are also important to consider when antibiotics are used clinically in combination (see Chapter 40, Principles of Combination Chemotherapy). The combination of a bacteriostatic drug with a bactericidal drug can result in antagonistic effects. For example, the bacteriostatic drug tetracycline inhibits protein synthesis and thereby retards cell growth and division. The action of this drug antagonizes the effects of a cell wall synthesis inhibitor, such as penicillin, which requires bacterial growth in order to be effective. In contrast, the combination of two bactericidal drugs can be synergistic; that is, the

CHAPTER 32 / Principles of Antimicrobial and Antineoplastic Pharmacology 567

Peptidoglycan cell wall PABA Inhibitors of cell wall synthesis Fosfomycin Cycloserine Vancomycin Penicillins Cephalosporins

Monobactams Carbapenems Ethambutol Pyrazinamide Isoniazid

Pteridine Plasmid THF

DHF

Protein Ribosome 50S Inhibitors of transcription and translation Rifampin Aminoglycosides Spectinomycin Tetracyclines Macrolides

Chloramphenicol Lincosamides Streptogramins Oxazolidinones Pleuromutilins

Purines

Pyrimidines

Inhibitors of DNA synthesis and integrity Sulfonamides Trimethoprim Quinolones

30S

mRNA DNA

FIGURE 32-1. Sites of action of antibacterial drug classes. Antibacterial drug classes are often divided into three general groups. Drugs in one group inhibit specific enzymes involved in DNA synthesis and integrity: sulfonamides and trimethoprim inhibit the formation or use of folate compounds that are necessary for nucleotide synthesis; quinolones inhibit bacterial type II topoisomerases. Drugs targeting transcription and translation inhibit bacterial processes that mediate RNA and protein synthesis: rifampin inhibits bacterial DNA-dependent RNA polymerase; aminoglycosides, spectinomycin, and tetracyclines inhibit the bacterial 30S ribosomal subunit; macrolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins inhibit the bacterial 50S ribosomal subunit. A third group of drugs inhibits specific steps in bacterial cell wall synthesis: fosfomycin and cycloserine inhibit early steps in peptidoglycan monomer synthesis; vancomycin binds to peptidoglycan intermediates, inhibiting their polymerization; penicillins, cephalosporins, monobactams, and carbapenems inhibit peptidoglycan cross-linking; and ethambutol, pyrazinamide, and isoniazid inhibit processes necessary for synthesis of the cell wall and outer membrane of Mycobacterium tuberculosis. There are several clinically useful antibacterial drugs that do not fit into one of these three groups; one recent example is daptomycin. The development of resistance is a problem for all antibacterial agents. Many bacteria carry plasmids (small, circular segments of DNA) with genes that confer resistance to an antibacterial agent or class of agents. PABA, para-aminobenzoic acid; DHF, dihydrofolate; THF, tetrahydrofolate. effect of the combination is greater than the sum of the effects of each drug alone (at the same doses of the two drugs). For example, a penicillin–aminoglycoside combination can have a synergistic effect because inhibition of bacterial cell wall synthesis by the penicillin allows increased entry of the aminoglycoside. The combination of two bacteriostatic drugs can also be synergistic (see Synergy of DHFR Inhibitors and Sulfonamides, below).

Fungi and Parasites Eukaryotes, which include pathogenic fungi (yeasts and molds) and parasites (protozoa and helminths) as well as all multicellular organisms, are more complex than bacteria. Cells in these organisms contain a nucleus and membranebound organelles, as well as a plasma membrane. Eukaryotic cells reproduce by mitotic division rather than binary fission. Because of the similarities among human, fungal, and parasitic cells, infections caused by fungi and parasites can be more difficult to target than bacterial infections. However, the burden of disease from these organisms is vast. Parasitic infections caused by protozoa and helminths (worms) affect some 3 billion people worldwide, especially in less developed countries where morbidity and mortality can be devastating. In both developed and less developed parts of the world, increasing numbers of patients are immunocompromised from AIDS, cancer chemotherapy, organ transplants, and old age. Such patients are especially susceptible

to fungal and parasitic infections, which are becoming more prominent and will require greater attention in the future. The currently available antifungal drugs can be divided into four main classes. As mentioned above, polyenes (e.g., amphotericin, nystatin) and azoles (e.g., miconazole, fluconazole) selectively target ergosterol in the fungal cell membrane, and echinocandins (e.g., caspofungin, micafungin) inhibit the synthesis of ␤-(1,3)-D-glucans in the fungal cell wall. Pyrimidines such as 5-fluorocytosine inhibit DNA synthesis. Another class of miscellaneous antifungals, mostly acids, are used only topically because of their unacceptable systemic toxicity. As with antibacterials, antifungals can be fungistatic or fungicidal; this distinction is usually determined empirically. For example, the azoles interfere with fungal cytochrome P450-mediated ergosterol metabolism. Many azoles (e.g., itraconazole and fluconazole) are fungistatic. Newer azole agents (e.g., voriconazole and ravuconazole) may have fungicidal activity against some fungal species. As compared to fungistatic drugs, fungicidal drugs are more efficacious and faster acting and allow more favorable dosing regimens. Antifungal drugs are discussed in further detail in Chapter 35. Parasites exhibit complex and diverse life cycles and metabolic pathways, and the treatment of parasitic infections utilizes a wide array of antiparasitic drugs (see Chapter 36, Pharmacology of Parasitic Infections). Malaria is an example of a complex parasite that, while theoretically susceptible to numerous classes of drugs, is becoming resistant

568 Principles of Chemotherapy

to many currently available therapies. Malaria is transmitted when the female Anopheles mosquito deposits Plasmodia sporozoites in the human bloodstream. The parasites leave the circulation and develop into tissue schizonts in the liver. The tissue schizonts rupture, releasing merozoites that again enter the circulation to infect red blood cells (erythrocytes). The parasites then mature to trophozoites and, finally, to mature schizonts. The mature schizonts are released into the bloodstream when the erythrocytes rupture, causing the typical cyclic fever associated with malaria. Antimalarial drugs target different stages of the protozoal life cycle; several classes of drugs can be used, depending on the local pattern of resistance. Aminoquinolines (such as the previous firstline drug, chloroquine) inhibit the polymerization of heme within the erythrocyte; it is thought that nonpolymerized heme is toxic to intraerythrocytic Plasmodia. Dihydrofolate reductase inhibitors, protein synthesis inhibitors, artemisinins, and other classes of drugs are also used in malaria treatment. As chloroquine resistance is now common and resistance to most antimalarial agents has increased, the WHO recommends against all single-agent treatment regimens in the first line of therapy for malaria. Instead, combination therapies are now recommended as first-line treatments. Resistance to artemisinin and its derivatives has been observed, and the WHO recommends artemisinin-based combination treatments both to increase the efficacy of therapy and to reduce the spread of drug-resistant malaria. Combinations of an artemisinin-based drug with amodiaquine, mefloquine, or sulfadoxine-pyrimethamine are recommended. Malaria is but one example that illustrates the complexities of both the parasitic life cycle and the use of drugs to treat parasitic infections.

Viruses Viruses are noncellular organisms that typically consist of a nucleic acid core of RNA or DNA enclosed in a proteinaceous capsid. Some viruses also possess a host cellderived lipid envelope containing viral proteins. Viruses lack the capability to synthesize proteins themselves, relying instead on the host cell machinery. Most viruses also encode distinct or even unique proteins not normally produced by human cells, however. Many of these proteins are involved in the viral life cycle, mediating virus attachment and entry into the host cell, viral capsid uncoating, viral genome replication, viral particle assembly and maturation, and release of viral progeny from the host cell. These virus-specific processes are often targeted by antiviral drugs. A schematic diagram of the general viral life cycle is presented to illustrate the stages of viral replication that can be targeted by antiviral drugs (Fig. 32-2). Because these targets are present only during active viral replication, viruses that exhibit latency are not well controlled by antiviral drugs. One distinct viral protein is the HIV protease. This enzyme cleaves viral precursor proteins to generate the structural proteins and enzymes necessary for virus maturation. Without HIV protease, only immature and noninfective virions (individual virus particles) are produced. HIV protease inhibitors structurally mimic natural substrates of the protease, but contain a noncleavable bond. These drugs are competitive inhibitors at the active site of the enzyme (see Chapter 37, Pharmacology of Viral Infections). In combination with other

classes of anti-HIV drugs, protease inhibitors helped to revolutionize the treatment of patients with HIV/AIDS. Several classes of drugs target distinct proteins that are encoded by the influenza virus. Zanamivir and oseltamivir target a viral neuraminidase that is vital for virion release from host cells. Amantadine and rimantadine act on the influenza virus membrane protein M2 (a proton channel) to inhibit viral uncoating. Although these anti-influenza drugs are highly effective inhibitors of the viral neuraminidase and proton channel, respectively, they have not revolutionized influenza therapy to the extent that anti-HIV drugs have for HIV. Because most flu infections are identified clinically when the immune system has already begun to eradicate the virus, these drugs have only a limited effect on flu symptoms. This example illustrates the point that even selective inhibitors with high therapeutic indices do not necessarily become highly effective drugs in the clinic. Currently, the most important antiviral drugs are the polymerase inhibitors. Most viruses use a viral polymerase, either an RNA or DNA polymerase, to replicate their genetic material. Polymerase inhibitors are especially effective against human herpesviruses, HIV, and hepatitis B virus. Two types of polymerase inhibitors are the nucleoside analogues and the nonnucleoside reverse transcriptase (NNRT) inhibitors. Nucleoside analogues (such as zidovudine [AZT or ZDV] and acyclovir) become phosphorylated and thereby activated by viral or cellular kinases (phosphorylating enzymes), at which point they competitively inhibit the viral polymerase and, in some cases, are incorporated into the growing DNA strand. Selectivity is dependent on the relative affinities of the nucleoside analogue for the viral and cellular kinases and polymerases. Nonnucleoside reverse transcriptase inhibitors (such as efavirenz) inhibit viral reverse transcriptase, preventing DNA replication. Mutations in viral polymerase genes are a major mechanism of resistance to polymerase inhibitors. Chapter 37 provides a detailed discussion of the pharmacology of antiviral drugs.

Cancer Cells Cancer is a disease of cell proliferation in which normal cells are transformed by genetic mutation into cells with dysregulated growth. Neoplastic cells compete with normal cells for energy and nutrition, resulting in deterioration of normal organ function. Cancers also impinge on vital organs by mass effects. Carcinogenesis, chemotherapy, and the log cell kill model of tumor regression are discussed below to provide an overview of cancer pharmacology. Chapters 38 and 39 should be read with these principles in mind, and Chapter 40 provides integrated examples of the clinical applications of combination antineoplastic chemotherapy. Carcinogenesis and Cell Proliferation Carcinogenesis occurs in three main steps—transformation, proliferation, and metastasis. Transformation denotes a change in phenotype from a cell with normal growth controls to a cell with dysregulated growth. Nonlethal genetic damage (mutations) can be inherited in the germ line, can occur spontaneously, or can be caused by environmental agents such as chemicals, radiation, or viruses. If the DNA damage is not repaired, the mutated genes (e.g., genes involved in growth regulation and DNA repair) can express altered gene

CHAPTER 32 / Principles of Antimicrobial and Antineoplastic Pharmacology 569

Virus Receptor Attachment and entry inhibitors Maraviroc Enfuvirtide (T-20)

Attachment and entry Host cell

Ion channel blockers Amantadine Rimantadine

Uncoating

Polymerase inhibitors Acyclovir Zidovudine Efavirenz Integrase inhibitors Raltegravir

Genome replication

RNA synthesis Host ribosome

Protein synthesis Assembly and maturation

Protease inhibitors Saquinavir Ritonavir

Egress and release

Neuraminidase inhibitors Zanamivir Oseltamivir

FIGURE 32-2. Stages of the viral life cycle targeted by antiviral drug classes. The viral life cycle begins with attachment of the virus to a host cell receptor and entry of the virus into the cell. The virus then uncoats, sometimes in an endosomal compartment. The uncoated viral nucleic acid undergoes genome replication; viral genes are transcribed (RNA synthesis); and virally coded RNA is translated into proteins on host cell ribosomes. The replicated viral genome and viral proteins are assembled into a virion (viral particle), which is then released from the host cell. The process of virion assembly and/or release is often accompanied by maturation of the virus into an infective agent that is able to repeat this life cycle with a new host cell. The anti-HIV drugs maraviroc and enfuvirtide (T-20) block the attachment and entry of HIV into host cells. The ion channel blockers amantadine and rimantadine inhibit influenza virus uncoating. Polymerase inhibitors are a large class of antiviral agents that include acyclovir, zidovudine, and efavirenz; these drugs inhibit viral genome replication by interfering with viral DNA polymerase (acyclovir) and reverse transcriptase (zidovudine and efavirenz). The anti-HIV drug raltegravir inhibits viral genome replication by interfering with the viral integrase. Protease inhibitors, such as the anti-HIV drugs saquinavir and ritonavir, inhibit viral maturation. Neuraminidase inhibitors block the release of influenza virus particles from the host cell.

products that allow abnormal cell growth and proliferation. Mutations can activate growth-promoting genes, inactivate growth-inhibiting genes, alter apoptosis-regulating genes, confer immortalization, and inactivate DNA repair genes. Expression of altered gene products and/or loss of regulatory proteins can cause genetic instability and dysregulated growth. Most cancers are initially clonal (i.e., genetically identical to a single precursor cell), but evolve to heterogeneity as new mutations increase the genetic variation among daughter cells. When progeny cells with higher survival capacity are selected, increased cell proliferation ensues, and the tumor progresses to greater and greater heterogeneity. Thus, carcinogenesis, the progression from a normal cell to a malignant tumor, is a multistep process that requires an accumulation of multiple genetic alterations. As more is learned about the molecular basis for carcinogenesis, these genetic differences can be targeted for selective drug therapy. The growth of transformed cells into a tumor requires proliferation, or an increase in the number of cells.

Dividing human cells progress through a cell cycle (or mitotic cycle) consisting of distinct phases. The two key events in the cell cycle are the synthesis of DNA during S phase and the division of the parent cell into two daughter cells during mitosis or M phase. The phase between cell division and DNA synthesis is called gap 1 (G1), and the phase between DNA synthesis and mitosis is called G2. Proteins called cyclins and cyclin-dependent kinases (CDKs) govern progression through the phases of the cell cycle; mutations in cyclin and/or CDK genes can result in neoplastic transformation. A proliferating cancer cell has three potential fates: the daughter cell can become quiescent by entering a resting phase called G0; the cell can enter the cell cycle and proliferate; or the cell can die. The ratio of the number of cells that are proliferating to the total number of cells in the tumor is called the growth fraction. An average tumor growth fraction is about 20%, because only one in five cells participates in the cell cycle at any given time. Most antineoplastic drugs

570 Principles of Chemotherapy

target dividing cells. Hence, tumor cells in a quiescent (G0) state, such as nutrient-starved cells in the center of a large tumor, are not easily killed by chemotherapy. Small or rapidly growing cancers (i.e., cancers with high growth fractions, such as leukemias) often respond more favorably to chemotherapy than do large bulky tumors. Unfortunately, cells in normal tissues characterized by high growth fractions, such as the bone marrow and gastrointestinal mucosa, are also killed by antineoplastic drugs, resulting in doselimiting toxicities. Tumor cells do not proliferate in isolation. Transformed cancer cells secrete and induce a variety of chemical mediators to induce a specialized local environment. These chemical mediators include growth factors such as epidermal growth factor (EGF), and inhibitors of growth factor signaling have been developed for clinical use as cancer chemotherapeutic agents. Some tumors create a protective fibrous connective tissue stroma; for example, this property makes breast cancer nodules palpable. Most solid tumors also require the induction of blood vessel growth (angiogenesis) to deliver nutrients into the center of the tumor; for this reason, angiogenesis inhibitors represent a valuable class of antineoplastic drugs. Cancer cells may acquire the capability to invade tissues and metastasize throughout the body. In order to metastasize, tumor cells must acquire mutations that allow invasion into tissues and vessels, seeding of cavities, spread through lymph or blood vessels, and growth in a new environment.

Aggressive, rapidly growing primary tumors are generally more likely to metastasize than indolent, slowly growing tumors. In the process of gaining mutations, tumor cells can also evolve differential receptor expression patterns and drug sensitivities. Often, although the primary tumor may respond well to chemotherapy, the more dedifferentiated metastatic cells respond poorly. Thus, metastatic spread often represents a poor prognostic sign. Chemotherapy By the time a typical solid tumor is clinically evident, it contains at least 109 cells, has progressed to heterogeneity, and has developed surrounding stroma. The tumor may or may not have metastasized from its site of origin (“primary site”) to one or more secondary sites. These factors can render the cancer difficult to treat pharmacologically. Many of the traditional chemotherapeutic agents interfere with cell proliferation and rely on rapid cell cycling and/or promotion of apoptosis for their relative selectivity against cancer cells (Fig. 32-3). As noted above, tumors are most sensitive to chemotherapy when they are growing rapidly, primarily because they are progressing through the cell cycle. These metabolically active cells are thus susceptible to drugs that interfere with cell growth and division (the mitotoxicity hypothesis). Many antineoplastic drugs interfere with the cell cycle at a particular phase; such drugs are called cell-cycle specific. Other antineoplastic drugs act independently of the cell cycle and are called cell-cycle nonspecific (Fig. 32-4).

Purine precursors

Pyrimidine precursors Folate

N

N N

N

N

N R

R

Purine nucleotides Inhibitors of DNA synthesis and integrity

Pyrimidine nucleotides Microtubules

Antimetabolites and folate pathway inhibitors Topoisomerase inhibitors

mRNA

Inhibitors of microtubule function Vinca alkaloids Taxanes

DNA DNA damaging agents Alkylating agents Antitumor antibiotics Platinum complexes

FIGURE 32-3. Antineoplastic drug classes. Many cancer cells divide more frequently than normal cells, and cancer cells can often be killed preferentially by targeting three critical processes in cell growth and division. DNA-damaging agents alter the structure of DNA and thereby promote apoptosis of the cell. These drugs include alkylating agents (which covalently couple alkyl groups to nucleophilic sites on DNA), antitumor antibiotics (which cause free radical damage to DNA), and platinum complexes (which cross-link DNA). Inhibitors of DNA synthesis and integrity block intermediate steps in DNA synthesis; these agents include antimetabolites and folate pathway inhibitors (which inhibit purine and pyrimidine metabolism) and topoisomerase inhibitors (which induce damage to DNA during winding and unwinding). Inhibitors of microtubule function interfere with the mitotic spindle that is required for cell division. This group of drugs includes vinca alkaloids, which inhibit microtubule polymerization, and taxanes, which stabilize polymerized microtubules. Additional classes of antineoplastic agents—such as hormones, tumor-specific monoclonal antibodies, growth factor receptor antagonists, signal transduction inhibitors, proteasome inhibitors, and angiogenesis inhibitors—are not shown (see Chapter 39).

CHAPTER 32 / Principles of Antimicrobial and Antineoplastic Pharmacology 571

Inhibitors of microtubule function M

Antitumor antibiotics

Glucocorticoids G1

G2

S Topoisomerase inhibitors

Antimetabolites and folate pathway inhibitors

Alkylating agents Platinum complexes (cell-cycle nonspecific)

FIGURE 32-4. Cell-cycle specificity of some antineoplastic drug classes. The cell cycle is divided into four phases. Cell division into two identical daughter cells occurs during mitosis (M phase). Cells then enter the gap 1 (G1) phase, which is characterized by active metabolism in the absence of DNA synthesis. Cells replicate their DNA during the synthesis (S) phase. After completion of S phase, the cell prepares for mitosis during the gap 2 (G2) phase. Some antineoplastic drugs exhibit specificity for different phases of the cell cycle, depending on their mechanism of action. Inhibitors of microtubule function affect cells in M phase; glucocorticoids affect cells in G1; antimetabolites and folate pathway inhibitors affect cells in S phase; antitumor antibiotics affect cells in G2; topoisomerase inhibitors affect cells in S phase and G2. Alkylating agents and platinum complexes affect cell function in all phases and are therefore cell-cycle nonspecific. The differential cell-cycle specificity of the various drug classes allows them to be used in combination to target different populations of cells. For example, cell-cycle specific drugs can be administered to target actively replicating neoplastic cells, whereas cell-cycle nonspecific agents can be used to target quiescent (nonreplicating) neoplastic cells. Additional classes of antineoplastic agents—such as hormones, tumor-specific monoclonal antibodies, growth factor receptor antagonists, signal transduction inhibitors, proteasome inhibitors, and angiogenesis inhibitors—are not shown (see Chapter 39).

Inhibitors of DNA synthesis, such as antimetabolites and folate pathway inhibitors, are S-phase specific. Microtubule poisons, such as taxanes and vinca alkaloids, interfere with spindle formation during M phase. The alkylating agents that damage DNA and other cellular macromolecules act during all phases of the cell cycle. These various classes of drugs can be administered in combination, using cell-cycle specific drugs to target mitotically active cells and cell-cycle nonspecific agents to kill both cycling and noncycling tumor cells (see Chapter 40). The mitotoxicity hypothesis of anticancer therapy leaves some puzzles unresolved, however. Although cancer chemotherapy is often toxic to the bone marrow, gastrointestinal mucosa, and hair follicles, these tissues usually recover, while (in successful treatment) cancers with similar growth kinetics are eradicated. It has now been established that almost all chemotherapeutic drugs also cause apoptosis of cancer cells. DNA damage is normally sensed by molecules, such as p53, that arrest the cell cycle in order to allow time for the damage to be repaired. If the damage is not repaired,

a cascade of biochemical events is triggered, which can result in apoptosis (programmed cell death). Therefore, a cancer cell that is defective in its capability for DNA repair may undergo apoptosis, whereas a normal cell can repair its DNA and recover. Cancers that express wild-type p53, such as most leukemias, lymphomas, and testicular cancers, are often highly responsive to chemotherapy. In contrast, cancers that acquire a mutation in p53, including many pancreatic, lung, and colon cancers, are often minimally responsive or even resistant to DNA-damaging drugs, because apoptosis is not triggered in response to DNA damage. Advances in cancer cell biology over the past several decades have led to the development of new classes of therapeutic agents that are targeted more specifically to the molecular pathways responsible for the dysregulated growth of cancer cells. The concept is emerging that specific cancers may become dependent on one particular growth factor or signal transduction pathway for their growth and survival and that selective targeting of these pathways may provide the basis for selective killing of cancer cells while sparing normal cells (which are less dependent on any one particular pathway). This concept and the many new classes of antineoplastic agents that have recently been developed—including tumorspecific monoclonal antibodies, growth factor receptor antagonists, signal transduction inhibitors, proteasome inhibitors, and angiogenesis inhibitors—are discussed in Chapter 39. Log Cell Kill Model The log cell kill model is based on experimentally observed rates of tumor growth and tumor regression in response to chemotherapy. Tumor growth is typically exponential, with a doubling time (i.e., time required for the total number of cancer cells to double) that depends on the type of cancer. For example, testicular cancer often has a doubling time of less than 1 month, whereas colon cancer tends to double every 3 months. In solid tumors, the cancer may grow exponentially until a clinically observable tumor size is achieved. The log cell kill model states that the cell destruction caused by cancer chemotherapy is first-order; that is, that each dose of chemotherapy kills a constant fraction of cells. If the tumor starts with 1012 cells and 99.99% are killed, then 108 malignant cells will remain. The next dose of chemotherapy will then kill 99.99% of the remaining cells, and so on. Unlike antibacterial drugs, which can often be used in a constant high dose until the bacteria are eradicated, most antineoplastic drugs must be used intermittently to reduce toxic side effects. Intermittent dosing allows partial recovery of normal cells, but also provides time for cancer cell regrowth and for evolution of drug resistance in the cancer cells. As shown in Figure 32-5, intermittent “cycles” of chemotherapy are administered until all the cancer cells are killed or the tumor develops resistance. Drug-resistant cells continue to grow exponentially despite treatment, eventually resulting in death of the host. Improvements in the rates of eradication of malignant cell populations are likely to require either higher doses of chemotherapeutic agents (which are limited by toxicity and drug resistance) or initiation of therapy at a time when the tumor contains fewer cells (which implies earlier detection). Adjuvant therapies, such as surgery and radiation, are other important modalities used to reduce the number of tumor cells before chemotherapy is initiated. Surgery and radiation may also recruit more tumor cells into the cell cycle and thus increase the susceptibility of these cells to cell-cycle specific agents.

Number of cancer cells

572 Principles of Chemotherapy

1012 10

A Death

Detectable cancer Local treatment

10

108

Palliation

Nondetectable cancer

D

106

Resistance or toxicity

104 102

B Cure

C Cure

Surgery Radiation therapy

Time

FIGURE 32-5.

Log cell kill model of tumor growth and regression. The log cell kill model predicts that the effects of antineoplastic chemotherapy can be modeled as a first-order process. That is, a given dose of drug kills a constant fraction of tumor cells, and the number of cells killed depends on the total number of cells remaining. The four curves (A–D ) represent four possible outcomes of antineoplastic therapy. Curve A is the growth curve of untreated cancer. The cancer continues to grow over time, eventually resulting in the death of the patient. Curve B represents curative local treatment (surgery and/or radiation therapy) before metastatic spread of the malignancy. Curve C represents local treatment of the primary tumor, followed immediately by systemic chemotherapy administered in cycles (down arrows) to eradicate the remaining metastatic cancer cells. Note that each cycle of chemotherapy reduces the number of cancer cells by a constant fraction (here, by about two “logs,” or by about 99%) and that some cancer growth occurs as the normal tissues are given time to recover between cycles of chemotherapy. Curve D represents local treatment followed by systemic chemotherapy that fails when the tumor becomes resistant to the drugs or when toxic drug effects occur that are intolerable to the patient. Note that 109 to 1010 cancer cells must typically be present for a tumor to be detectable; for this reason, multiple cycles of chemotherapy are required to eradicate the cancer, even when there is no detectable tumor remaining.

 MECHANISMS OF DRUG RESISTANCE Having provided a general introduction to the pharmacology of drug targets in bacteria, fungi, parasites, viruses, and cancer, the discussion now turns to the mechanisms of drug resistance, which is a major problem in all of antimicrobial and antineoplastic pharmacology. Although resistance to current drug therapies is emerging relatively rapidly, the rate of introduction of new drugs (especially antimicrobial drugs) is relatively slow. Formerly curable diseases such as gonorrhea and typhoid fever are becoming more difficult to treat, and old killers such as tuberculosis and malaria are growing increasingly resistant worldwide. In some parts of China, up to 99% of gonorrhea isolates are multidrug resistant. In the United States, 60% of hospital-acquired (nosocomial) infections due to Gram-positive bacteria are caused by drug-resistant microbes. Tuberculosis, the fourth leading cause of infectious disease deaths worldwide, currently has an estimated 5% overall multidrug resistance (MDR) rate, although the rate of MDR in new cases of tuberculosis ranges from 14.6% (Uzbekistan) to 22.3% (Azerbaijan) in some Asian countries. The appearance of MDR tuberculosis in the United States is of special concern because of the airborne spread of this organism. Despite these ominous trends, only five new classes of antibiotics—oxazolidinones (linezolid), lipopeptides (daptomycin), pleuromutilins (retapamulin), streptogramins (quinupristin/dalfopristin),

and glycylcyclines (tigecycline)—have entered clinical use in the past four decades. The numerous examples of rapidly emerging drug-resistant organisms suggest that this problem must be addressed promptly. Because pathogens and cancer cells are primed to evolve rapidly in response to adaptive pressure, resistance can eventually appear with the use of any antimicrobial or antineoplastic drug. In a population of microbes or transformed cells, the cells that contain random mutations promoting fitness will survive. Thus, high cell number, rapid growth rate, and high mutation rate all promote the development of a heterogeneous population of cells that can acquire resistance through mutational escape. Because the use of a drug inherently selects for organisms that can survive in the presence of high concentrations of that drug, resistance is an omnipresent consequence of drug therapy. In many cases, the emergence of drug resistance confounds effective treatment.

Genetic Causes of Drug Resistance The recent explosion in drug resistance has both genetic and nongenetic causes. Genetic mechanisms of resistance arise from chromosomal mutations and from exchange of genetic material. Table 32-2 lists the major mechanisms of genetic drug resistance that can be caused by either chromosomal mutation or genetic exchange. Chromosomal mutations typically occur in the gene(s) that code(s) for the drug target or in genes that code for drug transport or metabolism systems. These mutations can then be transferred to daughter cells (vertical transmission) to create drug-resistant pathogens or cancer cells. Alternatively, bacteria can acquire resistance by gaining genetic material from other bacteria (horizontal transmission). For example, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are able to cause highly feared nosocomial infections because these bacteria have acquired resistance genes. Bacteria acquire genetic material by three main mechanisms: conjugation, transduction, and transformation. In conjugation, chromosomal or plasmid DNA is transferred directly between bacteria. DNA can also be transferred from one cell to another by a bacterial virus, or bacteriophage, in a process called transduction. In transformation, naked DNA in the environment is taken up by the bacteria. Drug resistance in bacteria is most often caused by the transfer of plasmids, which are extrachromosomal strands of DNA that contain drug resistance genes. Transfer of a DNA plasmid is especially important for drug resistance because this mechanism occurs at high rates both within and between bacterial species and because multidrug resistance genes can be transferred. Reduced Intracellular Drug Concentration Drugs must reach their targets in order to be effective. When an insufficient amount of drug reaches the target, the growth of pathogens or cancer cells and the emergence of resistant strains are allowed. Microbes and cancer cells have evolved a number of mechanisms to inactivate drugs before the drugs can bind to their targets. Many bacteria are resistant to penicillins and cephalosporins because they produce a hydrolytic enzyme, ␤-lactamase, which cleaves the ␤-lactam ring and thereby disables the drug’s active site. A single ␤lactamase enzyme can hydrolyze 103 penicillin molecules

574 Principles of Chemotherapy

design of new drugs and the evolution of changes leading to drug resistance.

Practices That Promote Drug Resistance One of the most important causes of drug resistance is the overprescription of antibiotics that are not indicated for the clinical situation. Overprescription is a problem not only in humans, but also in the treatment and prophylaxis of animal infections. Such widespread use promotes drug resistance, which is then transferred from one bacterium to another by the mechanisms described above. Other mechanisms of resistance involve pharmacologic and anatomic drug barriers, such as the wall of an abscess or the blood–brain barrier. Poor patient adherence can also promote resistance, as can the erratic drug availability found in parts of the developing world (and even in some communities in the developed world). International travel promotes a global disease community, ensuring that the multidrug-resistant tuberculosis found in Russia or Peru will eventually emerge in hospitals in the United States. Finally, demographic shifts and other trends have created large populations that are susceptible to infections, such as immunocompromised cancer patients, AIDS patients, and the elderly population.

 METHODS OF TREATMENT Combination Chemotherapy The development of drug resistance depends on such factors as the number of microorganisms or cancer cells in the pretreatment population, the rate of replication or “generation time” of the organism or cell, the intrinsic rate of mutation in the population, and the fitness of the resistant organism or cell. Compared to treatment with a single agent, treatment with a combination of drugs can significantly decrease the probability that resistance will develop. Combination chemotherapy is the standard-of-care in tuberculosis and HIV therapy and most antineoplastic drug regimens. There are several major reasons to administer multiple drugs simultaneously in a combination chemotherapy regimen; the rationales are discussed in further detail in Chapter 40. First, the use of multiple drugs with different mechanisms of action targets multiple steps in microbial or cancer cell growth, leading to the maximum possible rate of cell killing. Second, the use of combinations of drugs that target different pathways or molecules in the pathogen or cancer cell makes it more difficult for resistance to develop. Even if the likelihood of development of a resistance mutation to one drug is relatively high, the concurrent emergence of separate mutations against several different drugs is less likely. Third, the use of lower doses of synergistically acting drugs in the combination can reduce drug-associated adverse effects. This is especially important in antimicrobial chemotherapy, where synergistic activity of drug combinations has been clearly demonstrated. Fourth, because many antineoplastic drugs have different dose-limiting adverse effects (toxicities), it is often possible to give each drug to its maximally tolerated dose and thereby achieve increased overall cell killing. Finally, the concept of combination chemotherapy is being redefined as new treatments become available. In the future, immunotherapies, hormone therapies, and biotherapies will become increasingly integrated into combination chemotherapy regimens (see Chapter 53, Protein Therapeutics).

Prophylactic Chemotherapeutics In most instances, antimicrobial and antineoplastic drugs are used to treat overt disease. These classes of drugs can also be used to prevent diseases from occurring (chemoprophylaxis), both before a potential exposure and after a known exposure. The potential benefit of chemoprophylaxis must always be weighed against the risk of creating drug-resistant pathogens or cancer cells and the potential for toxicity attributable to the chemoprophylactic agent. Antimicrobial chemoprophylaxis is frequently used in high-risk patients to prevent infection. Travelers to malaria-infested areas, for example, often take prophylactic antimalarial drugs such as mefloquine (see Chapter 36). Chemoprophylaxis is also used in some types of surgery to prevent wound infections. Antibiotics are commonly administered prophylactically during surgical procedures that could release bacteria into the wound site, such as colon resection. Antibiotics have also been used prophylactically before dental procedures in patients at high risk for endocarditis, because such procedures can produce a transient bacteremia. In certain situations, immunocompromised patients are given antibacterial, antifungal, antiviral, and/or antiparasitic drugs prophylactically to prevent opportunistic infections. For example, acyclovir can protect previously infected immunocompromised patients against disease caused by reactivation of latent herpes simplex virus. Chemoprophylaxis or preemptive therapy can also be used in healthy persons after exposures to certain pathogens. Prophylactic therapy after known or suspected exposure to gonorrhea, syphilis, bacterial meningitis, HIV, and other infections can often prevent disease. The risk of seroconversion after a single needle stick exposure to HIV-infected blood is approximately 0.3% (95% confidence interval [CI] ⫽ 0.2–0.5%). Although limited data are available regarding the reduction of risk achievable with prophylaxis, the Centers for Disease Control and Prevention (CDC) currently recommends postexposure treatment with a two- or three-drug antiretroviral therapy regimen (e.g., zidovudine [AZT] and lamivudine [3TC] for basic postexposure treatment or AZT, 3TC, and lopinavir/ritonavir or another HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor for expanded postexposure treatment) for 4 weeks. Two-drug regimens are recommended for lower risk exposures (to minimize adverse effects), and the addition of a third drug is appropriate after a high-risk exposure (e.g., large-bore hollow needle stick, deep puncture wound, visible blood on device, or needle stick from an individual with a high viral load or a symptomatic HIV infection). Zidovudine has also been shown to reduce maternal transmission of HIV, representing chemoprophylaxis for the fetus (see Chapter 37).

 INHIBITORS OF FOLATE METABOLISM: EXAMPLES OF SELECTIVE TARGETING AND SYNERGISTIC DRUG INTERACTIONS Folic acid is a vitamin that participates in a number of enzymatic reactions involving the transfer of one-carbon units. These reactions are essential for the biosynthesis of DNA and RNA precursors; the amino acids glycine, methionine, and glutamic acid; the formyl-methionine initiator tRNA; and other essential metabolites. Given the importance of folate metabolism

A Folic acid

H2 N

N

N H N

N N

H N

OH Pteridine moiety

COOH

O

COOH

PABA

Glutamate

B PABA analogues

NH2

NH2

NH2

O

O

S

S O

NH N

O

O N

N

S O

NH

O

NH2

Sulfanilamide

Sulfadiazine

Sulfamethoxazole

C Folate analogues N

H2 N

N

N

N N H N

NH2 O

COOH COOH

Methotrexate

O H2N

N

O

H2 N

N N

N O NH2

Trimethoprim

NH2

Pyrimethamine

Cl

Bacteria Pteridine + PABA Dihydropteroate synthase

Sulfonamides

Dihydropteroic acid Glutamate

Bacteria and humans

Dihydrofolate Dihydrofolate reductase (DHFR)

Tetrahydrofolate

Trimethoprim Methotrexate Pyrimethamine

5-Fluorouracil Flucytosine

Purines

Methionine Glycine fMet-tRNA

Thymidine

DNA RNA

Proteins

DNA

33 Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation Marvin Ryou and Donald M. Coen

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 581-582 BIOCHEMISTRY OF BACTERIAL DNA REPLICATION, TRANSCRIPTION, AND TRANSLATION . . . . . . . . . . . . . . . . . 581 DNA Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 DNA Replication, Segregation, and Topoisomerases . . . . . 582 Bacterial Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 Bacterial Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . 583 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 586 Inhibitors of Topoisomerases: Quinolones . . . . . . . . . . . . . 587

Inhibitors of Transcription: Rifamycin Derivatives . . . . . . . 587 Inhibitors of Translation . . . . . . . . . . . . . . . . . . . . . . . . . . 588 Antimicrobial Drugs Targeting the 30S Ribosomal Subunit . . . . . . . . . . . . . . . . . . . . . 589 Antimicrobial Drugs Targeting the 50S Ribosomal Subunit . . . . . . . . . . . . . . . . . . . . . 592 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 595 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

 INTRODUCTION

genetic information. To transmit all of the genetic information in a cell to two progeny cells, the parental DNA must be copied in its entirety (replicated), and the two resulting copies must be segregated—one copy going to each progeny cell. In order to express the genes that are embedded in the DNA, these specific portions of the DNA are copied (transcribed) into RNA. Some RNAs (mRNAs) are then read (translated) by the protein synthesis machinery in order to produce proteins. Other RNAs, such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), perform complex functions essential to protein synthesis. It is important to note that the following discussion of these bacterial processes is greatly simplified to emphasize the steps that are inhibited by antibiotics.

The central dogma processes—DNA replication, transcription, and translation—are generally similar in bacteria and humans. DNA is replicated and transcribed into RNA, and messenger RNA is translated into protein. However, there are important differences in the biochemistry of bacterial and human central dogma processes, and these differences can be exploited for the development and clinical use of antibiotics. Three such differences are targeted by the currently available antibacterial chemotherapeutic drugs: (1) topoisomerases, which regulate supercoiling of DNA and mediate segregation of replicated strands of DNA; (2) RNA polymerases, which transcribe DNA into RNA; and (3) ribosomes, which translate messenger RNA (mRNA) into protein. This chapter briefly reviews the biochemistry of central dogma processes in bacteria and discusses certain relevant differences between these processes in bacteria and humans. With this background, the chapter discusses the mechanisms by which pharmacologic inhibitors interrupt bacterial DNA replication, transcription, and translation.

 BIOCHEMISTRY OF BACTERIAL DNA REPLICATION, TRANSCRIPTION, AND TRANSLATION The central dogma of molecular biology begins with the structure of DNA, which is the macromolecule that carries

DNA Structure DNA is composed of two strands of polymerized deoxyribonucleotides that wind around one another in a “double helix” conformation. The 5⬘-hydroxyl group of each nucleotide’s deoxyribose ring is joined by a phosphate group to the 3⬘hydroxyl group of the next nucleotide, thereby forming the phosphodiester backbone of each side of the double helical “ladder” (Figs. 33-1 and 33-2). The purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C), which are covalently linked to the deoxyribose ring, associate with one another (A with T, G with C) via hydrogen bonds to form the “rungs” of the ladder (Fig. 33-2). It is the linear sequence of bases that encodes the genetic information 581

5'

O

O H

4'

H

3'

2'

O

H

H

β-D-2-deoxyribose

5' End

base

O O

1'

H

P

O

O

-

O

H O

H

H

O

H

P

H

O

base O

-

O

H O

H

H

O

H

P

H

O

O

O3'-5' Phosphodiester bond

base

H

H

H

O

H

H

3' End

CHAPTER 33 / Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation 583

A

O H

H N

N

N

H

N

deoxyribose

N

N

O Thymine

N

deoxyribose Adenine B

H N H O

N

N deoxyribose

H

N

N

O H

N

N

Cytosine

N

deoxyribose

H

Guanine

C

base O

O H O

H

H

O

H

P

se

H

base

O

O

OH O

H

H

O

H

P

H

O -

O

FIGURE 33-2. Hydrogen bonding between DNA strands. A and B. The dashed lines indicate hydrogen bonds between complementary bases on opposite DNA strands. Adenine (A) and thymine (T) form two hydrogen bonds, while guanine (G) and cytosine (C) form three hydrogen bonds. C. These A-T and G-C base pairs form the “rungs” of the DNA double helical “ladder.” Note that the deoxyribose moieties and phosphodiester bonds are located on the outside of the DNA double helix, while the purine and pyrimidine bases stack in the center of the DNA molecule. are both more complex and more versatile than type I topoisomerases, and the type II enzyme serves as a more frequent molecular target for chemotherapeutic agents. The mechanism of action of a type II topoisomerase proceeds in two steps. First, the enzyme binds a segment of DNA and forms covalent bonds with phosphates from each strand, thereby nicking both strands. Second, the enzyme causes a second stretch of DNA from the same molecule to pass through the break, relieving supercoiling (Fig. 33-4).

This passage of double-stranded DNA through a doublestranded break is what permits separation of intertwined copies of DNA following replication and, thereby, segregation of DNA into progeny cells. There are two main bacterial type II topoisomerases. The first to be identified, DNA gyrase, is a bacterial type II topoisomerase that is unusual in that it can introduce negative supercoils before the DNA strands separate, and thereby neutralize positive supercoils that form as the strands unwind. The second major type II topoisomerase is topoisomerase IV. DNA gyrase is particularly crucial for segregation in some bacteria, while topoisomerase IV is the critical enzyme in other bacteria. Because supercoiling is important for transcription as well as segregation, topoisomerases influence this central dogma process as well. Given their multiple functions, topoisomerases are usually engaged with DNA, and this is important for their roles as drug targets. These enzymes are not only important as antibacterial drug targets, but also as targets for cancer chemotherapy (see Chapter 38).

Bacterial Transcription Gene expression begins with transcription, which involves the synthesis of single-stranded RNA transcripts from a DNA template. Transcription is catalyzed by the enzyme RNA polymerase. In bacteria, five subunits (2 ␣, 1 ␤, 1 ␤⬘, and 1 ␴) associate to form the holoenzyme. As discussed below, the ␴ subunit is instrumental for initiating transcription, while the rest of the RNA polymerase enzyme—also known as the core enzyme—contains the catalytic machinery for RNA synthesis. The process of transcription occurs in three stages: initiation, elongation, and termination (Fig. 33-5). During initiation, the RNA polymerase holoenzyme separates the strands of a short segment of double-helical DNA after its ␴ subunit recognizes an upstream site. Once the double helix is unwound to form a single-stranded template, RNA polymerase initiates RNA synthesis at a start site on the DNA. During elongation, RNA polymerase synthesizes a complementary RNA strand by joining together ribonucleoside triphosphates via phosphodiester bonds. In the process, the ␴ subunit dissociates from the holoenzyme. RNA synthesis proceeds in the 5⬘→3⬘ direction, with the nascent RNA strand emerging from the enzyme, until a termination sequence is reached. The RNA polymerase enzyme differs between bacteria and humans and thus can serve as a selective target for antibacterial drug action. In bacteria, one RNA polymerase synthesizes all of the RNA in the cell (except for the short RNA primers needed for DNA replication, which are made by primase). Furthermore, bacterial RNA polymerase is composed of only five subunits. In contrast, eukaryotes express three different nuclear RNA polymerases, and each enzyme is considerably more complex in its subunit structure than the bacterial counterpart. For example, eukaryotic RNA polymerase II, which synthesizes the precursors of mRNA, consists of 12 subunits.

Bacterial Protein Synthesis Once the mRNA transcripts are synthesized, these transcripts are translated by the bacterial translational machinery. Although the overall process of translation is similar between bacteria and higher organisms, there are a number

584 Principles of Chemotherapy Strand-rotation mechanism

FIGURE 33-3.

Regulation of DNA supercoiling by type I topoisomerases. Two mechanisms have been proposed for the action of type I topoisomerases. In the strand-rotation model, type I topoisomerase binds to opposite strands of the DNA double helix. The topoisomerase then nicks one strand and remains bound to one of the nicked ends (filled green circle). The unbound end of the nicked strand is able to unwind by one or more turns and is then joined (religated) to its parent strand. In the strand-passage model, type I topoisomerase binding to the DNA double helix results in melting (separation) of the two DNA strands. The DNA-bound topoisomerase then introduces a nick into one strand, while remaining bound to each end of the broken DNA strand (filled green circles). The broken strand is then passed through the helix and joined (religated), resulting in a net unwinding of the DNA. Camptothecins, which are used in cancer chemotherapy (see Chapter 38), inhibit the joining of the broken strand of DNA after strand passage.

Strand-passage mechanism

Bind

Bind Melt

Type I topoisomerase

Break Rotate

Break

Rotate Join

Pass Join

A

Camptothecins

B

C

G-segment ATPase domain

T-segment

B' domain ATP

Type II topoisomerase

ATP

ATP

A' domain ADP + Pi F

E

ATP

ATP

D

ATP

ATP

ATP

ATP

Quinolones (inhibit bacterial enzyme) Anthracyclines Epipodophyllotoxins (inhibit human enzyme) Amsacrine

Regulation of DNA supercoiling by type II topoisomerases. A. The type II topoisomerase enzymes contain A⬘, B⬘, and ATPase domains. The A⬘ and B⬘ domains engage a segment of the DNA double helix (G-segment). B. Interaction with the G-segment induces a conformational change in the type II isomerase, causing it to “lock” around the DNA G-segment. C. ATP binds to the ATPase domains of the topoisomerase, and a second segment of the DNA double helix (T-segment) enters and is “locked” into the B⬘ domains. D. Once the enzyme is engaged with both DNA segments, the topoisomerase cuts both strands of the G-segment DNA. E. This double-stranded break in the G-segment allows the T-segment to pass through the G-segment to the opposite side of the topoisomerase. F. The T-segment is released from the topoisomerase, and the G-segment nick is religated. ATP is hydrolyzed to ADP, ADP dissociates from the topoisomerase, and the cycle begins anew. The result of each cycle is to change the coiling of DNA or, when two separate circular DNA molecules are involved, to resolve catenanes. Quinolone antibiotics inhibit passage of the T-segment and religation of the G-segment by bacterial type II topoisomerases. At therapeutic concentrations, quinolones also promote topoisomerase subunit dissociation, resulting in double-stranded breaks in the DNA and killing of the bacteria. Several classes of cancer chemotherapeutic agents, including the anthracyclines, epipodophyllotoxins, and amsacrine, inhibit passage of the T-segment and religation of the G-segment by human type II topoisomerases, thereby causing double-stranded DNA breaks and inducing apoptosis of the cancer cells (see Chapter 38).

FIGURE 33-4.

CHAPTER 33 / Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation 585

RNA polymerase (α2ββ'σ holoenzyme) A Initiation

Start

B Elongation

α2ββ'

3'

Nascent RNA 5'

Elongation site Template strand σ

Movement of polymerase C Termination

RNA

the ribosome that are responsible for the ribosome’s key activities, namely, decoding the mRNA, linking together amino acids, and translocating the translation machine. The 70S ribosome contains two sites that bind tRNAs during translation: the P or “peptidyl” site, which contains the growing peptide chain, and the A or “aminoacyl” site (also known as the “acceptor” site), which binds incoming tRNA molecules carrying the various amino acids (Fig. 33-6). (There is also an E or “exit” site, which binds the tRNAs that have been used during translation before they are ejected from the ribosome.) Translation, like transcription, can be divided into three steps (Fig. 33-7). During initiation, the components of the translation system assemble together. First, the mRNA joins with the 30S subunit of the bacterial ribosome and with a specific tRNA molecule linked to formylated methionine (fMet), the first amino acid encoded by every bacterial mRNA. The tRNA-formylated methionine molecule (fMettRNAf) binds to its initiation codon (AUG) on the mRNA. Next, the 50S subunit joins with the 30S subunit to form the complete 70S ribosome. The fMet-tRNAf now occupies the P site of the 70S ribosome. Elongation involves the addition of amino acids to the carboxyl end of the growing polypeptide chain, as the ribosome moves from the 5⬘-end to the 3⬘-end of the mRNA that is being translated. tRNA molecules carrying specific amino acids (aminoacyl tRNAs) enter the ribosomal A site and base-pair to their complementary codons on the mRNA. Utilization of the correct tRNA requires not only anticodoncodon recognition between tRNA and mRNA, respectively, but also decoding functions provided by the 16S rRNA in the 30S ribosomal subunit. Peptidyl transferase, an enzyme whose activity derives from the 23S rRNA of the 50S

α2ββ' 70S ribosome

P

FIGURE 33-5.

Bacterial transcription. A. During initiation, the RNA polymerase holoenzyme (␣2␤␤⬘␴) searches for and recognizes promoter sequences on DNA. The holoenzyme then separates the strands of the double helical DNA, exposing the start site for transcription, and begins synthesis of the new RNA strand. B. During elongation, the core enzyme (without the ␴ subunit) extends the new RNA strand in the 5⬘→ 3⬘ direction, using the unwound DNA strand as a template. RNA polymerase separates the strands of the DNA double helix as it moves along the template strand, extruding the 5⬘ end of the transcript behind it. Rifampin blocks elongation by complexing with the ␤ subunit of RNA polymerase (not shown). C. Upon reaching a termination sequence, the DNA, core enzyme, and newly synthesized RNA separate from one another.

of pharmacologically exploitable differences in the details of the mechanisms. In particular, the number and composition of the rRNA molecules differ between bacterial and human ribosomes. Thus, bacterial ribosomes can also serve as selective targets for antibiotics. The ribosome of a representative bacterium, Escherichia coli, has a sedimentation coefficient of 70S and is composed of a 30S subunit and a 50S subunit. The 30S subunit contains a single 16S rRNA molecule and 21 different proteins, while the 50S subunit contains two rRNA molecules—23S rRNA and 5S rRNA—and more than 30 different proteins. Importantly, it is the rRNA rather than the protein components of

A

50S subunit (23S rRNA, 5S rRNA, more than 30 proteins) Macrolides Chloramphenicol Lincosamides Streptogramins Oxazolidinones Pleuromutilins 30S subunit (16S rRNA, 21 proteins) Aminoglycosides Spectinomycin Tetracyclines

FIGURE 33-6.

The bacterial 70S ribosome. The bacterial 70S ribosome consists of a 30S subunit and a 50S subunit. Each subunit is composed of ribosomal RNA (rRNA) and numerous proteins. The rRNAs are responsible for most of the important activities of the ribosome and are the targets of antibiotic drugs that inhibit translation. Aminoglycosides, spectinomycin, and tetracyclines bind to and inhibit the activity of 16S rRNA in the 30S subunit. Macrolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins bind to and inhibit the activity of 23S rRNA in the 50S subunit. A, aminoacyl site (site of binding of aminoacyl tRNA); P, peptidyl site (site of binding of tRNA that is covalently joined to the elongating peptide chain).

586 Principles of Chemotherapy fMet fMet-tRNA mRNA

+

50S

30S

P

A

Initiation complex 70S ribosome

Amino acid Charged tRNA

tRNA binding Tetracyclines (30S)

tRNA P

A

Aminoglycosides (30S) Chloramphenicol (50S, A site) Lincosamides (50S, A and P sites) Oxazolidinones (50S, A site) Streptogramins (50S, A and P sites) Pleuromutilins (50S, A and P sites)

P

Decoding

Peptide bond formation

A

tRNA binding

Charged tRNA P

A

Translocation and peptide movement (egress)

Spectinomycin (30S)

FIGURE 33-7.

Macrolides (50S, polypeptide exit tunnel)

Bacterial translation. Bacterial translation begins with the assembly of a complex containing a 30S ribosomal subunit, mRNA, formylmethionine-linked tRNA, and a 50S ribosomal subunit. This assembly step is dependent on the binding of fMet-tRNA f to an initiator codon in the mRNA. The assembled 70S ribosome contains the aminoacyl (A) and peptidyl (P) sites. The A site accepts incoming triplet codons of mRNA and allows the corresponding amino acid-linked tRNA (i.e., charged tRNA) to bind to its corresponding triplet. The decoding function of 16S rRNA helps ensure that the mRNA codon binds to the correct tRNA. Once a charged tRNA has entered the A site, the peptidyl transferase activity of the 23S rRNA catalyzes the formation of a peptide bond between the amino acid occupying the A site and the carboxy-terminus of the nascent peptide residing in the P site. Once the peptide bond has formed, the tRNA–mRNA complex translocates from the A site to the P site, the tRNA molecule that had occupied the P site dissociates from the P site, and the elongating polypeptide chain moves out through the exit tunnel. The A site is now empty, and introduction of the next charged tRNA molecule into the A site completes the cycle. Translation continues until a stop codon is encountered in the mRNA, at which point the newly synthesized protein is released from the ribosome. Pharmacologic agents that inhibit translation interfere with the activities of the bacterial ribosome. Aminoglycosides bind to rRNA in the 30S subunit and enable the binding of incorrect tRNAs to mRNA; tetracyclines block aminoacyl tRNA binding to the A site; chloramphenicol, lincosamides, oxazolidinones, streptogramins, and pleuromutilins inhibit the peptide bond formation activity of the 50S subunit. Spectinomycin and macrolides inhibit peptide translocation. The binding site of a second component of the streptogramins overlaps with that of the macrolides (not shown).

subunit (i.e., peptidyl transferase is a ribozyme), catalyzes the formation of a peptide bond between fMet and the next amino acid. The peptide bond links fMet to the next amino acid, which, in turn, is linked to the tRNA in the A site (i.e., the tRNA in the A site has “accepted” the fMet). After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3⬘-end of the mRNA. In the process, the tRNAf that was originally linked to the fMet is ejected from the P site (and binds to the E site), the tRNA that is now linked to two amino acids shifts from the A site to the unoccupied P site, the A site becomes available, and the growing peptide emerges from the exit tunnel of the ribosome. This process is known as translocation. In this manner, polypeptide chain elongation results from multiple cycles of aminoacyl tRNA binding to the A site, peptide bond formation, and translocation. During termination, proteins called release factors recognize the termination codon in the A site and activate discharge of the newly synthesized protein and dissociation of the ribosome–mRNA complex. In at least some cases, this process seems to involve structural mimicry of tRNAs by the release factors. Three general points are worth noting about bacterial translation. First, the two ribosomal subunits demonstrate segregated functions: the 30S subunit is responsible for faithful decoding of the mRNA message, while the 50S subunit catalyzes peptide bond formation. Translocation, however, seems to involve both subunits. Second, the catalytic machinery resides in the RNA component of the ribosome, not in the ribosomal proteins. In other words, it is the rRNA that “does the work.” Third, inhibitors of protein synthesis block the process of translation at different steps.

 PHARMACOLOGIC CLASSES AND AGENTS There are three general categories of drugs that target bacterial DNA replication, transcription, and translation: (1) quinolones, (2) rifamycin derivatives, and (3) drugs that target bacterial ribosomes. Quinolone antibiotics are broad-spectrum agents; they not only inhibit certain topoisomerases but also convert these enzymes into DNA-damaging agents. Rifamycin derivatives bind to and inhibit bacterial RNA polymerase. One such derivative, rifampin, is a mainstay in the therapy of tuberculosis. A number of classes of drugs bind bacterial ribosomes to inhibit protein synthesis. Specifically, aminoglycosides, spectinomycin, and tetracyclines bind the 30S ribosomal subunit, while macrolides, chloramphenicol, lincosamides, streptogramins, oxazolidinones, and pleuromutilins target the 50S ribosomal subunit. These inhibitors of protein synthesis generally act on both Gram-positive and Gram-negative organisms and are therefore in wide clinical use (see Chapter 34 for a discussion of Gram-positive and Gram-negative bacteria). Elucidation of the mechanisms of action of the agents described below has depended crucially on the field of bacterial genetics. In particular, the molecular targets of antibiotics have been identified by the isolation of bacteria that are resistant to the particular antibiotic (e.g., rifampin), followed by showing that the target molecule (e.g., RNA polymerase) exhibits biochemical resistance to the antibiotic, and, finally, showing that the drug-resistance mutation lies within the gene encoding the target. More recent work, using nuclear magnetic resonance spectroscopy and x-ray crystallography,

O COOH

CH3COO CH3O

OH

OH

OH

O

OH NH

N

N N N

O

O

OH O

Nalidixic acid

Rifampin

O F

COOH

CH3COO CH3O

OH

OH

OH

N

O

O NH

N O

HN

NH

O N O

N

Ciprofloxacin

Rifabutin

N

NH HO NH OH

HO O

O CHO HO HO HO

HN

OH O

HO HO

H 2N

NH H2N

O

O

NH2

O HO

H2 N NH2 O O

NHCH3

OH

HO

HO

NHCH3

Streptomycin

H N

HO

HO

H

H NH

O

H

O HO

Gentamicin A

OH H

NH2 O

OH

OH N H

O

Doxycycline

O

N

NH2 OH OH O

OH OH O

O

Tetracycline

OH

OH

N OH

Spectinomycin

H

H

O

O

H N

H

H

N OH

O N H

NH2 OH

O

OH OH O

Tigecycline

O

590 Principles of Chemotherapy A

B C1054

G530

A1493

PAR

A1492 S12

C

D ASL

ASL

mRNA

mRNA PAR

Cell wall E

Inner membrane

Outer membrane

F Incorrect amino acid incorporated

Polysome

Aminoglycoside G

H Membrane pore (abnormal protein)

Aminoglycoside "monosome" (nonfunctional)

CHAPTER 33 / Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation 593 O

HO

OH OH

OH

N

O

O

HO

O

OH O

CHCl2

HN O2N

O

O OH

O

O

Chloramphenicol

Erythromycin A O

CH3 N

H

H

C

NH

O

HO

C

N

Cl HN

CH3CH2CH2 H

H

O

CH O H

O

OH H H

O H SCH3

HO

N N

N

O

CH3

O

O H N

S

N O O

NH

N

OH

Clindamycin

Quinupristin

O OH N O O

O O

O O

N

N

N

N

H N

N O

S O

F

O

O

O

Linezolid

Dalfopristin

OH N

O H

S O

O

Retapamulin

FIGURE 33-11. Structures of antimicrobial drugs targeting the 50S ribosomal subunit. Chloramphenicol, erythromycin (a macrolide), clindamycin (a lincosamide), quinupristin (a streptogramin), dalfopristin (a streptogramin), linezolid (an oxazolidinone), and retapamulin (a pleuromutilin) each inhibit bacterial translation by targeting the 50S ribosomal subunit.

alternatives are not available, as in the case of resistance or serious drug allergy. Chloramphenicol binds to 23S rRNA and inhibits peptide bond formation, apparently by occupying a site that interferes with proper positioning of the aminoacyl moiety of tRNA in the A site (Fig. 33-12B). Microbes have developed resistance to chloramphenicol by two major mechanisms. Low-level resistance has emerged in large chloramphenicol-susceptible populations by the selection of mutants with decreased permeability to

the drug. The more clinically significant type of chloramphenicol resistance has arisen from the spread of specific plasmid-encoded acetyltransferases (at least three types of which have been characterized) that inactivate the drug. The fundamental mechanism underlying the toxicity of chloramphenicol appears to involve inhibition of mitochondrial protein synthesis. One manifestation of this toxicity is the gray baby syndrome, which can occur when chloramphenicol is administered at high doses to newborn infants. Because newborns lack an effective glucuronic acid

594 Principles of Chemotherapy

Lincosamides

A

The major lincosamide in clinical use is clindamycin (Fig. 33-11). Clindamycin blocks peptide bond formation, apparently through interactions with both the A site (like chloramphenicol) and the P site (Fig. 33-12B). The most important indications for clindamycin are the treatment of serious anaerobic infections caused by Bacteroides and the treatment of mixed infections involving other anaerobes. Clindamycin has been implicated as a potential cause of pseudomembranous colitis caused by Clostridium difficile superinfection. An infrequent member of the normal fecal flora, C. difficile is selected for during the administration of clindamycin or other broad-spectrum oral antibiotics. C. difficile elaborates a cytotoxin that can cause colitis characterized by mucosal ulcerations, severe diarrhea, and fever. This serious adverse effect is a major concern in the use of oral clindamycin. Streptogramins B

Macrolides Clindamycin

Chloramphenicol CC-puromycin CHCl2 P-site tRNA

A-site tRNA

FIGURE 33-12. Mechanism of action of erythromycin, clindamycin, and chloramphenicol revealed by crystallographic analysis of drug binding to the 50S ribosomal subunit. A. Erythromycin binds to a specific segment of 23S rRNA and blocks the exit tunnel from which nascent peptides emerge. B. Clindamycin and chloramphenicol have partially overlapping binding sites on the 50S ribosomal subunit. These sites are near the macrolide binding site. The positions of the A-site tRNA and P-site tRNA are also shown. conjugation mechanism for the degradation and detoxification of chloramphenicol, the drug can accumulate to toxic levels and cause vomiting, flaccidity, hypothermia, gray color, respiratory distress, and metabolic acidosis. More frequently, chloramphenicol causes dose-related, reversible depression of erythropoiesis and gastrointestinal distress (nausea, vomiting, and diarrhea). Aplastic anemia, a rare but potentially fatal toxicity, occurs via an idiopathic mechanism that is unrelated to dose. Of special interest are the adverse effects that chloramphenicol can cause in tandem with other drugs. Like the macrolides, chloramphenicol increases the half-life of certain drugs, such as phenytoin and warfarin, by inhibiting the cytochrome P450 enzymes that metabolize these drugs. Chloramphenicol also antagonizes the bactericidal effects of penicillins and aminoglycosides, as do other bacteriostatic inhibitors of microbial protein synthesis.

In 1999, the FDA approved the first drug in the streptogramin class of protein synthesis inhibitors. The drug is a mixture of two distinct chemicals: dalfopristin, a group A streptogramin, and quinupristin, a group B streptogramin (Fig. 33-11). Dalfopristin/quinupristin was approved for the treatment of serious or life-threatening infections caused by vancomycin-resistant Enterococcus faecium or Streptococcus pyogenes. The streptogramins inhibit protein synthesis by binding to the peptidyl transferase center of bacterial 23S rRNA. Mutations and modifications affecting this region can confer resistance. The binding site for the B component overlaps with that of the macrolides, and it is thought that, like the macrolides, the streptogramins block emergence of nascent peptides from the ribosome. The A component binds to a location overlapping both the A and P sites in the peptidyl transferase center, and it can inhibit peptidyl transferase in vitro. Streptogramins are unusual among the 50S antibiotics in that they are bactericidal against many, but not all, susceptible bacterial species. A clear explanation for this phenomenon remains elusive; the current hypothesis is that, unlike the other 50S antibiotics, the streptogramins induce a conformational change in the ribosome that is reversible only after subunit dissociation. Oxazolidinones

In 2000, the FDA approved linezolid (Fig. 33-11), the first drug in the oxazolidinone class of antibacterial agents. Linezolid demonstrates excellent activity against drug-resistant Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA), penicillin-resistant streptococcus, and vancomycin-resistant enterococcus (VRE). Although there was initially controversy regarding the precise mechanism of action of linezolid, crystallographic analyses have located the binding site of the drug in a pocket in the A site where the amino acid moiety in aminoacyl tRNA normally binds. Moreover, mutations in 23S rRNA can confer drug resistance. These results and those of biochemical studies suggest that linezolid blocks productive interactions of aminoacyl tRNAs with the A site in the peptidyl transferase active center. Pleuromutilins

In 2007, the FDA approved retapamulin (Fig. 33-11), the first drug in the pleuromutilin class of antibiotics. This drug is used as a topical treatment for bacterial skin infections,

CHAPTER 33 / Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation 595

and its mechanism of action is relatively well understood. Like linezolid, pleuromutilins bind to a pocket in the A site of the peptidyl transferase active center where aminoacyl tRNA normally binds. Distinct from linezolid, pleuromutilin binding also extends into the P site. Thus, the binding site of pleuromutilins is similar to that of group A streptogramins. The locations of mutations conferring resistance to pleuromutilins are consistent with this binding site. These compounds inhibit peptide bond formation, but once elongation is under way and the A and P sites are occupied, pleuromutilins are no longer active. The fact that three of the most recently developed antibiotics inhibit the ribosome emphasizes the continuing value of this complex structure as a target for new drug development. There is much continuing effort to discover new protein synthesis inhibitors, and this work is aided by the availability of structures of ribosomes bound to drugs.

 CONCLUSION AND FUTURE DIRECTIONS A number of classes of antibiotics target the bacterial machinery responsible for the central dogma processes, disrupting bacterial gene expression at multiple steps. Most of these drugs demonstrate selective binding to bacterial enzymes or RNAs and have relatively few adverse effects. All are associated with some degree of toxicity, however, and some (e.g., chloramphenicol) have limited clinical use because of their potential to cause life-threatening adverse effects. Several of these antibiotic classes—the quinolones, rifamycin derivatives, and several of the protein synthesis inhibitors— are bactericidal, but most protein synthesis inhibitors are

bacteriostatic. Drug resistance is a persistent and serious problem for all of these agents. Although the emergence of resistance is an expected consequence of antibiotic use, judicious drug administration, multidrug therapies, and the continued development of new antibacterial agents can combat the emergence of resistance. The development of the new glycylcycline, streptogramin, oxazolidinone, and pleuromutilin classes of bacterial ribosome inhibitors represents an important advance in the search for drugs that are effective against resistant bacteria. Further elucidation of the mechanism of action of these drugs will both inform the basic biology of translation and define new biochemical targets for pharmacologic intervention.

Suggested Reading Campbell EA, Korzheva N, Mustaev A, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 2001;104:901–912. (Mechanism of rifampin action.) Kohanski MA, Dwyer DJ, Wierzbowski J, et al. Mistranslation of membrane proteins and two-component system activation trigger antibioticmediated cell death. Cell 2008;135:679–690. (Presents a new model for bactericidal action of aminoglycosides.) Ogle JM, Murphy FV, Tarry MJ, et al. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 2002;111:721– 732. (Structural basis for the mechanism of aminoglycoside-induced codon misreading.) Steitz TA, Moore PB. RNA, the first macromolecular catalyst: the ribosome is a ribozyme. Trends Biochem Sci 2003;28:411–418. (Reviews function of RNA as a target of antibiotic action in the 50S subunit.) Walsh CT. Antibiotics: actions, origins, resistance. Washington, DC: ASM Press; 2003. (Reviews antibiotic synthesis, action, and mechanisms of resistance.)

34 Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis Tania Lupoli, David C. Hooper, Ramy A. Arnaout, Daniel Kahne, and Suzanne Walker

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 599-600 BIOCHEMISTRY OF BACTERIAL CELL WALL SYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599 Cell Wall Structure and Function . . . . . . . . . . . . . . . . . . . 599 Peptidoglycan Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . 601 Synthesis of Murein Monomers . . . . . . . . . . . . . . . . . . 601 Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Cross-Linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 Mycobacterial Cell Wall Synthesis . . . . . . . . . . . . . . . . . . 604 Autolysins and Cell Wall Degradation . . . . . . . . . . . . . . . . 606 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 606 Inhibitors of Murein Monomer Synthesis . . . . . . . . . . . . . 606 Fosfomycin and Fosmidomycin . . . . . . . . . . . . . . . . . . 606

Cycloserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607 Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607 Inhibitors of Murein Polymerization . . . . . . . . . . . . . . . . . 607 Vancomycin, Teicoplanin, and Telavancin . . . . . . . . . . 607 Inhibitors of Polymer Cross-Linking . . . . . . . . . . . . . . . . . 608 ␤-Lactam Antibiotics: General Considerations . . . . . . . 608 ␤-Lactam Antibiotics: Specific Agents . . . . . . . . . . . . . 610 Inhibitors of Cell Membrane Stability . . . . . . . . . . . . . . . . 612 Antimycobacterial Agents. . . . . . . . . . . . . . . . . . . . . . . . . 612 Ethambutol, Pyrazinamide, and Isoniazid. . . . . . . . . . . 612 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 613 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613

 INTRODUCTION

 BIOCHEMISTRY OF BACTERIAL CELL WALL SYNTHESIS

In 1928, Alexander Fleming made a chance discovery that would revolutionize the treatment of bacterial infections. He observed that certain molds produce a compound that inhibits the growth of bacteria. The compound he isolated was penicillin, the first in a long line of antibiotics that act by inhibiting the biosynthesis of peptidoglycan, the major component of the bacterial cell wall. The unique chemical and structural properties of peptidoglycan make it an attractive and prominent target for antibacterial chemotherapy. The emergence and spread of antibiotic resistance increasingly complicate the clinical use of cell wall synthesis inhibitors, however. This chapter reviews the biochemistry of peptidoglycan synthesis and describes the mechanisms of action, uses, and limitations of the antibiotics that interfere with this pathway. These limitations include resistance, toxicity, and drug–drug interactions. Antibiotics that target other essential components of the bacterial cell wall are also discussed.

Cell Wall Structure and Function Peptidoglycan, named for its peptide and sugar composition, is a three-dimensional meshwork of peptide–cross-linked sugar polymers that surrounds the bacterial cell just outside its cytoplasmic membrane (Fig. 34-1). Peptidoglycan is also known as murein, after the Latin murus (wall). Nearly all clinically important bacteria produce peptidoglycan. The major exceptions are Mycoplasma pneumoniae, which can cause atypical pneumonia, and the intracellular form (or “reticulate body”) of Chlamydia trachomatis, which can cause a sexually transmitted disease. Peptidoglycan is critically important for the survival of bacteria, which experience large fluctuations in osmotic pressure depending on their environment. The peptidoglycan layers wrapped around the cell provide the tensile strength required to withstand high turgor pressures that would otherwise cause the plasma

599

Gram-positive bacteria

Gram-negative bacteria

Mycobacteria

Lipopolysaccharide Murein

Pore

Pore Outer membrane Lipoprotein Periplasm

Extractable phospholipids Mycolic acids Arabinogalactan Murein

Murein Cytoplasmic membrane

Cytoplasmic membrane

Cytoplasmic membrane

CHAPTER 34 / Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis 601

binding capacity and accessibility of the thick murein layer are much greater in Gram-positive organisms, these bacteria stain purple. The outer membrane of Gram-negative bacteria not only limits the penetration of Gram stain into the periplasm, but it also prevents the penetration of many other molecules, including antibiotics that target peptidoglycan synthesis, such as vancomycin and bacitracin. Hence, although Gramnegative organisms contain the molecular targets for these antibiotics, they are not susceptible. To enable uptake of hydrophilic nutrients and excretion of hydrophilic waste products, Gram-negative bacteria contain outer membrane porins—beta barrel proteins that traverse the outer membrane and allow certain molecules to pass in and out (see Fig. 34-1). Porins are important pharmacologically because it is through these pores that most hydrophilic antibiotics that have activity against Gram-negative organisms gain access to the murein layer and to the structures beneath this layer. Also important pharmacologically are the lipopolysaccharides (LPS) that compose the outer leaflet of the outer membrane of Gram-negative bacteria. Lipopolysaccharides are amphipathic molecules that protect bacteria from toxic hydrophilic host molecules such as bile salts. Lipopolysaccharides are also important for bacterial adherence to host cells and evasion of the host immune response. Polymyxin is a topically used antibiotic that facilitates its own entry into the periplasm by binding to LPS and disrupting the integrity of the outer membrane. Once in the periplasm, polymyxin permeabilizes the inner membrane, discharging the membrane potential so that cells no longer produce the energy required for survival. Although polymyxin is too toxic for systemic use in people, its mechanism of action suggests that it may be possible to develop less toxic molecules that breach the outer membrane and allow the passage of antibiotics to their molecular targets in Gram-negative bacteria. Gram-positive bacteria do not contain an outer membrane; the extracellular enzymes involved in cell wall synthesis are therefore accessible to a wider range of antibiotics than can penetrate Gram-negative organisms. However, the cell wall of Gram-positive organisms is not simply composed of peptidoglycan; there is a set of other cell wall polymers that play important roles in adherence to host tissue and other aspects of pathogenicity. These include lipoteichoic acids and wall teichoic acids, anionic polymers that are typically composed of acyclic sugar-phosphate repeats functionalized with D-alanine and cyclic sugars such as glucose. Lipoteichoic acids are anchored in the bacterial membrane and extend up into the peptidoglycan layers. Wall teichoic acids are covalently attached to peptidoglycan and extend through and beyond its outermost layer. These polymers are important for host infection, and the biosynthetic pathways are possible targets for antibiotics. In some Gram-positive organisms, including Staphylococcus aureus, the peptidoglycan layers are also functionalized with proteins that are required for pathogenesis. These proteins are covalently attached to uncross-linked peptides in peptidoglycan by enzymes called sortases. Sortases have also been suggested as targets for antibiotics that could prevent the spread of infection. These important structural differences between the cell envelopes of Gram-negative and Gram-positive bacteria lead to differential access of antibiotics to cellular targets and also present different opportunities for the development of new antibiotics. However, peptidoglycan biosynthesis, which is

conserved among Gram-negative and Gram-positive organisms, remains the most important antibacterial cell envelope target. In fact, the peptidoglycan biosynthetic pathway is one of a very small number of broad-spectrum antibacterial targets that exist in bacterial pathogens. The other broad-spectrum targets include DNA synthesis, RNA synthesis, and protein synthesis (see Chapter 33, Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation). Of these processes, only peptidoglycan synthesis is unique to bacteria.

Peptidoglycan Biosynthesis Peptidoglycan biosynthesis occurs in three major stages. The first stage is intracellular and involves the synthesis of murein monomers from amino acids and sugar building blocks; the second and third stages involve the export of these murein monomers to the surface of the inner membrane, followed by their polymerization into linear peptidoglycan polymers and their cross-linking into two-dimensional lattices and threedimensional mats (Fig. 34-2). Because the details of bacterial cell wall synthesis can be daunting, it is helpful to keep in mind the three major stages—monomer synthesis, glycan polymerization, and polymer cross-linking—during the discussion that follows. In principle, any of the biochemical steps in peptidoglycan synthesis could be targets for antibiotics; in practice, clinically used antibiotics target only a few of the steps in these stages. A vast number of secondary metabolites produced by soil and marine microorganisms also block peptidoglycan synthesis, providing a reservoir of structurally and functionally novel compounds for possible clinical development as our existing antibiotics fail due to the spread of resistance. Synthesis of Murein Monomers The “murein monomer” is a disaccharide comprising Nacetylglucosamine connected via a beta linkage to the C4 hydroxyl of N-acetyl muramic acid, which is functionalized on the C3 lactate moiety with a peptide (Fig. 34-2). The first phase of peptidoglycan synthesis takes place in the cytoplasm and involves the conversion of UDP-N-acetylglucosamine (UDP-NAG), a nucleotide-sugar used as a building block in many cell wall polymers, to UDP-N-acetyl muramic acid pentapeptide (UDP-NAM-peptide; also known as the Park nucleotide). The first two enzymes in this process, MurA and MurB, convert the C3 hydroxyl of NAG to lactate. MurA, also known as enolpyruvate transferase, transfers enolpyruvate from phosphoenolpyruvate (PEP) to UDPNAG to form UDP-NAG pyruvate enol ether (Box 34-1). Second, the flavoenzyme MurB (also known as UDP-NAGenolpyruvate reductase) reduces the double bond to produce UDP-NAM, which has a free carboxylate to serve as the handle for the peptide chain. UDP-NAM is a sugar unique to bacteria, and its biosynthesis thus provides opportunities for selective antibiotics. One clinically used antibiotic that blocks the biosynthesis of UDP-NAM is fosfomycin, a PEP analog that inhibits MurA. The peptide component of UDP-NAM-peptide is assembled on the C3 lactate from amino acids and dipeptides by a series of ATP-dependent ligases. MurC, MurD, and MurE sequentially add the amino acids L-alanine, D-glutamate, and a diamino acid—either L-lysine or diaminopimelic acid (DAP)—to UDP-NAM. DAP differs from lysine in having a carboxyl group as well as an amine on the side chain. Most Gram-positive bacteria use L-lysine, whereas a minority of Gram-positive and all known Gram-negative bacteria use DAP. This is noteworthy

602 Principles of Chemotherapy A Murein monomer synthesis (cytoplasmic phase)

HO HO HO

O O

-OOC NH

O O AcHN O P O P O O- O-

N O

OPO3-2

PEP

HO HO O

NADPH

O

rA

Mu

OH OH

rB

HO

Mu

O

L-Ala, D-Glu, L-Lys, ATP ATP ATP

O O N O

O

rC

OH OH

UDP-NAG

HO HO O

NH

O O AcHN O P O P O O- O-

Mu

rE

rD

Mu

Mu

O

HN

O N

O

H N

Fosfomycin Fosmidomycin

O

HO

D-Ala-D-Ala

HO

DdlB, ATP D-Ala + D-Ala Alanine racemase L-Ala + L-Ala

NH2 O

N O

O

OH OH NH

O

Cycloserine

NH

NH

O O AcHN O P O P O O- O-

O

OH OH

HO

O

O

HN

O

O

O

UDP-NAM

MurF, ATP

NH

O O AcHN O P O P O O- O-

O

HO HO O

O

H N O

NH2 O H N

HN

O OH

O

Park nucleotide

B Completion of murein monomer synthesis, and murein monomer export, polymerization, and crosslinking β-lactams: Penicillins Cephalosporins Monobactams Carbapenems

NAG NAM L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala (L-Gly)5

NAG

NH

O D-Ala

D-Ala

γ-D-Glu

L-Ala NH HO HO HO

O

TP

O HO O

AcHN

O AcHN

P2O7-3

HO O HO

O

O HO O

AcHN

NH HO HO HO

O O O AcHN O P O P O O- O-

γ-D-Glu

L-Ala

L-Ala

PGT

NH

O

O

(L-Gly)5 L-Lys

γ-D-Glu

L-Ala

NH

O

D-Ala

(L-Gly)5 L-Lys

γ-D-Glu

NH2

L-Ala

D-Ala

D-Ala

(L-Gly)5 L-Lys

(L-Gly)5

NAG

D-Ala

D-Ala

NAM L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala

(L-Gly)5 L-Lys

γ-D-Glu

D-Ala

NAG

D-Ala

(L-Gly)5 L-Lys

Periplasm

Vancomycin Teicoplanin

NH

O

O HO O O O AcHN AcHN P2O7-3

HO O HO

O

O O HO O

AcHN

HO HO HO

O O O AcHN O P O P O O- O-

O HO O

O AcHN

O O O AcHN O P O P O O- O-

Cytoplasmic membrane P2O7-3

HO PO4-2 Bactoprenyl HO O phosphate

Dephosphorylase

HN L-Ala

O

O O O O AcHN O P O P O O- O-

N O OH OH

γ-D-Glu L-Lys

Bacitracin

HO HO O

NH O

MraY

HN

HO HO HO

MurG

O AcHN

HO O O

HN

L-Ala γ-D-Glu

Park nucleotide

O

O O O AcHN O P O P O O- O-

O

O O O AcHN O P O P O O- O-

HO HO HO

Staph FemABX

L-Ala

UDP-NAG

UDP

L-Lys

D-Ala

D-Ala

D-Ala

D-Ala

D-Ala

D-Ala

HO O O

HN

O

O O O AcHN O P O P O O- O-

L-Ala

γ-D-Glu

L-Lys

O AcHN

Gly-tRNA

tRNA

γ-D-Glu L-Lys (L-Gly)5

Lipid II

D-Ala

Murein monomer

D-Ala

Cytoplasm

FIGURE 34-2.

Bacterial cell wall biosynthesis and its inhibition by pharmacologic agents. Bacterial cell wall biosynthesis can be divided into three major stages. A. In the cytoplasmic phase of murein monomer synthesis, glucose is amidated and phosphorylated to glucosamine-1-phosphate (not shown), which is acetylated and conjugated to a uridine diphosphate (UDP) nucleotide by the enzyme GlmU (not shown) to form UDP-N-acetylglucosamine (UDP-NAG). Addition of phosphoenolpyruvate (PEP) by enolpyruvate transferase (MurA) and reduction of the resulting product by MurB form UDP-N-acetyl muramic acid (UDP-NAM). NAG and NAM are the two sugar building blocks for subsequent cell wall synthesis. MurC, MurD, and MurE sequentially add the amino acids L-alanine, D-glutamate, and L-lysine to UDP-NAM. In some bacteria, diaminopimelic acid (DAP) is added instead of L-lysine. Alanine racemase converts L-alanine to D-alanine, and D-Ala-D-Ala ligase B (DdlB) forms the dipeptide D-Ala-D-Ala. This dipeptide is then added to the L-Ala-D-Glu-L-Lys (or L-Ala-D-Glu-DAP) tripeptide by MurF, resulting in a UDP-NAM molecule linked to five amino acids (Park nucleotide). Fosfomycin and fosmidomycin are selective inhibitors of MurA. Cycloserine inhibits both alanine racemase and D-Ala-D-Ala ligase B, thereby preventing the addition of alanine residues to the growing peptide chain. B. The NAM–pentapeptide complex is transferred from UDP to the lipid carrier bactoprenol by the enzyme MraY, and NAG is added from UDP-NAG by MurG. In some bacteria, one to five amino acids can then be added to L-lysine or DAP to form a branched peptidoglycan; the amino acids are added from amino acyl tRNA. (Here, as an example, five glycine residues are added from glycyl-tRNA.) This completes the synthesis of the murein monomer. In the murein monomer export and polymerization stage, the bactoprenol–peptidoglycan complex is transported from the bacterial inner membrane to the periplasmic space, where peptidoglycan glycosyltransferases (PGTs) join the murein monomer to the growing peptidoglycan chain. Simultaneously, the bactoprenol is liberated to facilitate another round of murein monomer translocation. Bactoprenyl diphosphate is dephosphorylated to bactoprenyl phosphate by dephosphorylase, regenerating the form of the lipid carrier that can react with the Park nucleotide. In the final stage of cell wall biosynthesis, adjacent glycopeptide polymers are cross-linked in a reaction catalyzed by bacterial transpeptidases (TPs). In the example shown, a transpeptidase cross-links a glycine pentapeptide on one peptidoglycan chain to a D-Ala residue on an adjacent peptidoglycan chain; as shown in detail in Figure 34-3, the terminal D-Ala residue is displaced in this reaction. Bacitracin inhibits bactoprenol dephosphorylation and thereby interrupts murein monomer synthesis and export. Vancomycin, telavancin (not shown), and teicoplanin bind the D-Ala-D-Ala terminus of the bactoprenol-conjugated murein monomer unit and thereby prevent the PGT-mediated addition of murein monomer to the growing peptidoglycan chain. The ␤-lactam antibiotics (penicillins, cephalosporins, monobactams, and carbapenems) inhibit the transpeptidase enzymes that cross-link adjacent peptidoglycan polymers.

604 Principles of Chemotherapy

Polymerization is catalyzed by enzymes called peptidoglycan glycosyltransferases (PGTs, or formerly, transglycosylases). These enzymes are processive and catalyze several rounds of elongation by addition of disaccharide subunits to the reducing end of the growing polymer without releasing it. With each glycosylation reaction, bactoprenyl diphosphate is released and returns to the inner surface of the cytoplasmic membrane, where it loses a phosphate group to form bactoprenyl phosphate; this step is catalyzed by a dephosphorylase. Bactoprenyl phosphate is now ready to accept another Park nucleotide (Fig. 34-2B). The PGTs are often found as N-terminal catalytic domains in bifunctional proteins that also include a C-terminal transpeptidation domain; however, they can also be found as monofunctional PGTs (known as MGTs). Most bacteria contain a number of structurally related PGTs, some bifunctional and some monofunctional. Their enzymatic activities are similar in vitro, but they are presumed to play different roles in cells. For example, in rod-shaped organisms, some PGTs are dedicated to the synthesis of side-wall peptidoglycan, whereas others are dedicated to the synthesis of septal peptidoglycan. Nevertheless, these enzymes can partially substitute for one another, complicating a detailed understanding of their specific roles. One possible way of understanding this biological complexity is that bacteria have evolved to have multiple overlapping systems to ensure survival should specific problems arise in an individual machine. This partial redundancy can be both an advantage and a disadvantage from the standpoint of antibiotic treatment, depending on the particular case. Cross-Linking In the final stage of cell wall synthesis, murein chains are cross-linked to one another by enzymes called transpeptidases (TPs). Because transpeptidases were first identified as the molecular targets of penicillin, they are also called penicillin-binding proteins (PBPs). The PGT domain couples murein monomers to produce glycan strands. These oligosaccharide chains must then be cross-linked through their stem peptides to produce the murein found in bacterial cell walls. The transpeptidation reaction takes place in two steps: activation and coupling. In the activation step, a serine hydroxyl in the active site of a TP enzyme attacks the D-Ala-D-Ala amide bond of one of the stem peptides on the glycan polymer, forming a covalent enzyme-peptidoglycan intermediate and releasing alanine. In the coupling step, a free amino group on the terminal amino acid of the interbridge peptide (glycine for many Gram-positive bacteria) or on DAP (Gram-negative bacteria) then attacks this intermediate, producing a new amide bond cross-link between the two stem peptides and regenerating the active enzyme (Figs. 34-2B and 34-3). Penicillin, a ␤-lactam, apparently mimics the terminal D-Ala-D-Ala substrate: it binds in the TP active site, where it then reacts with the serine nucleophile to form a covalent enzyme–penicillin complex (Fig. 34-3). This modification inactivates the enzyme, thereby resulting in lower degrees of cell wall cross-linking; in turn, this compromises the integrity of the cell wall and eventually causes cell lysis (see discussion below). Bacteria typically contain several TPs with different but overlapping specificities. As described for PGTs, these different enzyme isoforms are used to build different parts of the wall. Escherichia coli, for example, has six

transpeptidases, some of which build the cylindrical middle of this rod-shaped bacteria, and others of which build its hemispherical ends. It is believed that differences in the number and type of cross-links and glycan chain length give each bacterial species its characteristic shape and size and the cell wall of each species its characteristic thickness. Consistent with this hypothesis, it has been found that the suite of transpeptidases differs from species to species and especially between rods such as E. coli and C. perfringens and spherical cocci such as streptococci and staphylococci. It is thought that, in some cases, bacteria exploit the presence of multiple TPs in order to develop antibiotic resistance in the clinic. A major form of resistance develops in S. aureus when strains acquire a resistant TP that is capable of cross-linking peptidoglycan even when exposed to the ␤-lactam methicillin, which typically inactivates TPs in a manner similar to penicillin (see discussion below). The cell wall produced by methicillin-resistant S. aureus (MRSA) in the presence of drug has lower levels of cross-linking than in the absence of drug, which is presumed to be due to the inefficiency of the resistant TP. One possible strategy for overcoming MRSA is to further weaken the cross-linking ability of this resistant TP.

Mycobacterial Cell Wall Synthesis The cell wall structures described above are found in the vast majority of clinically relevant bacteria, including Grampositive cocci such as streptococci and staphylococci, Gramnegative rods such as E. coli and Pseudomonas aeruginosa, and Gram-positive rods such as C. perfringens. However, a discussion of cell wall structure would not be complete without mentioning the unusual cell envelopes of the Corynebacteriae, a group of bacteria that includes the important pathogens Mycobacterium tuberculosis and Mycobacterium leprae. These bacteria are classified as high G⫹C (i.e., a high percentage of guanine and cytosine in their DNA) Gram-positives, but their cell envelopes have characteristics of both Gram-positive and Gram-negative bacteria. Unlike other Gram-positives, the Corynebacteriae have an outer membrane. The NAM sugars in the peptidoglycan layer that surrounds the cytoplasmic (inner) membrane have covalently attached NAG-arabinogalactan polymers, to which are attached mycolic acids. The mycolic acids have long alkyl chains containing as many as 90 carbons, and these alkyl chains form a waxy layer that make the bacteria resistant to acid decolorization (acid-fast). The mycolic acids are essential for the assembly of the outer membrane, but the organizational details are unclear. In addition to mycolic acids, the outer membrane of mycobacteria contains secreted phospholipids, called extractable lipids (see Fig. 34-1). Mycobacteria have outer membrane porins, but their structures are different from the porins found in Gramnegative bacteria. The synthesis of NAG-arabinogalactan begins with the transfer of a molecule of NAG phosphate from UDP-NAG to mycobacterial bactoprenyl phosphate. Next, a molecule of the sugar rhamnose is added, followed by the addition of the several galactose and arabinose units that make up arabinogalactan. Arabinosyl transferase catalyzes the addition of the arabinose units. Mycolic acid is a long, complex, branched fatty acid. The starting materials for its synthesis include a number of long, saturated hydrocarbon chains that

CHAPTER 34 / Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis 605

Normal transpeptidation

Two peptidoglycan chains

HO O HO

HO O O

O AcHN

O

Penicillin action HO O HO

O

AcHN

AcHN

O

HN

HO O O

O

L-Ala γ-D-Glu N H

H 2N

D-Ala-D-Ala

COOH

Ser

Enzyme

(L-Gly)4 L-Lys

N H

OH

O

S N

O

HO

γ-D-Glu

O

H N

R

O

L-Ala

Ser Enzyme

O

H N

(L-Gly)5 L-Lys

AcHN

O

O

HN HO

O

D-Ala D-Ala

Gly

Activation step HO O HO

HO O O

O AcHN

Enzymepeptidoglycan intermediate

O

HO O HO

O

AcHN

AcHN

O

HN

HN

L-Ala

O

O

R

AcHN O O

O

L-Ala

γ-D-Glu

γ-D-Glu

O

(L-Gly)5 L-Lys N H

O

H 2N

Ser

N H

Enzyme

O

H 2N

(L-Gly)4 L-Lys

Ser

NH

S O HN

COOH

Enzyme

D-Ala D-Ala

Gly

O +

HO O O

O

“Dead-end” enzyme penicillin complex

OH

Displaced D-Ala

Coupling step

HO O HO

Crosslinked peptidoglycan chains

O AcHN

HO O O

O

O

AcHN

O

HN

HO O HO

O AcHN

HO O O

HN

L-Ala

H N

(L-Gly)5 L-Lys N H

O

γ-D-Glu

O N H

D-Ala-L-Gly crosslink

O

AcHN

O

L-Ala

γ-D-Glu

O

(L-Gly)4 L-Lys

+ HO Ser Enzyme

D-Ala D-Ala

FIGURE 34-3. Transpeptidase action and its inhibition by penicillin. The left side of the figure shows the mechanism by which transpeptidases catalyze transpeptidation, a reaction that occurs in bacteria but not in mammalian cells. A nucleophilic hydroxyl group in the active site of the transpeptidase (Enzyme) attacks the peptide bond between the two D-Ala residues at the terminus of a pentapeptide moiety on one peptidoglycan chain (top panel). The terminal D-alanine residue is displaced from the peptidoglycan chain, and an enzyme-D-alanine-peptidoglycan intermediate is formed. This intermediate is then attacked by the amino terminus of a polyglycine pentapeptide linked at its carboxy terminus to L-lysine or diaminopimelic acid on an adjacent peptidoglycan chain (see Fig. 34-2) (middle panel). As the enzyme is liberated from the intermediate, a new peptide bond (cross-link) is formed between the terminal glycine residue on one peptidoglycan chain and the enzymeactivated D-alanine residue on the adjacent peptidoglycan chain. The free enzyme can then catalyze another transpeptidation reaction (bottom panel). The right side of the figure shows the mechanism by which penicillin interferes with transpeptidation, leading to the formation of a penicilloyl-enzyme “dead-end complex.” In this form, the enzyme is incapable of catalyzing further transpeptidation (cross-linking) reactions. are synthesized from two-carbon units carried by acetyl CoA. The enzyme fatty acid synthetase 1 (FAS1) catalyzes the formation of these saturated hydrocarbon chains, and the enzyme fatty acid synthetase 2 (FAS2) catalyzes the linkage of these chains. The linked product then undergoes several enzymatic transformations to become mycolic acid. Mycolic acid is eventually added to NAG-arabinogalactan, which, in turn, is attached to NAM to organize and form a major component of the mycobacterial outer membrane (Figs. 34-1 and 34-4).

In principle, any step in this process is susceptible to pharmacologic intervention. As discussed below, standard antimycobacterial treatment regimens include antibiotics that target both the synthesis of NAG-arabinogalactan and the early reactions of mycolic acid synthesis. The mycobacterial cell envelope is thick, asymmetric, and highly impermeable to both hydrophilic and hydrophobic substances. M. tuberculosis is among the most challenging pathogens to eradicate because its cell envelope resists entry

606 Principles of Chemotherapy

O

O N

SCoA

NH 2

Acetyl CoA

N

Pyrazinamide

FAS1

O

O N H

NH2

R

N

OH

Fatty acids Isoniazid

FAS2

Mycolic acids

Phospholipids

FIGURE 34-4. Mycolic acid synthesis and antimycobacterial drug action. Mycolic acids are produced by the cross-linking of fatty acid chains derived from acetyl coenzyme A (Acetyl CoA). Each of the arrows in this simplified representation denotes multiple synthetic steps; the focus is on the fatty acid synthetases (FAS1 and FAS2) because of their importance as drug targets. Specifically, FAS1 is inhibited by pyrazinamide, and FAS2 is inhibited by isoniazid.

of many antibiotics, and the organism grows very slowly; note that cell-wall-active antibiotics are typically most effective against bacteria that are actively growing and making new cell wall rapidly. Special treatment regimens, involving long-term therapy with combinations of antibiotics, are required to cure tuberculosis.

Autolysins and Cell Wall Degradation Although it provides stability, the cell wall is a dynamic structure; it is continuously modified by synthetic and degradative enzymes that are finely tuned to allow the sacculus to grow and divide without lysing. For bacteria to grow, bacterial cell walls must expand; for expansion to occur, new murein units must be incorporated into the existing cell wall. This is difficult to accomplish in a “finished” cell wall, which is composed of specific lengths of glycan polymers with particular degrees of cross-linked stem peptides. In addition, for a bacterium to divide, its cell wall must at some point be broken to allow two daughter cells to separate. Bacteria address these issues by using highly regulated autolysins. These enzymes

punch small holes in the cell wall that allow for remodeling and expansion. Different autolysins exhibit preferences for different bonds in murein. Similar to the synthetic enzymes, many are functionally redundant, but play necessary roles in the cell. For example, in E. coli, three autolysins called NAML-alanine amidases cleave the stem peptide from murein during division to promote daughter cell separation. Loss of these three amidases causes a noticeable defect in cell division, while loss of one usually has little or no effect. New murein synthesis and autolysin-mediated destruction must be carefully balanced for bacteria to survive. Indeed, studies have shown that unilaterally blocking murein synthesis (for example, by drugs like penicillin) results in autolysin-mediated autolysis and cell death. The molecular events that initiate autolysis are poorly understood. The current belief is that specific proteins recruit the degradative machinery only after the cell has assembled the machinery responsible for cell wall synthesis. This ordered recruitment ensures that degradation does not occur unless new cell wall is being made. The bactericidal effect of cephalexin, a firstgeneration cephalosporin, involves targeting the synthetic machinery by specifically inhibiting the transpeptidation step of cell wall synthesis and subverting this regulatory mechanism (see discussion below). Cephalexin does not perturb the normal assembly of the synthetic machinery, but instead simply inactivates this complex by inhibiting transpeptidase enzymes. Apparently, the regulatory mechanisms of the cell can determine only that the machinery for new cell wall synthesis is present and not whether it is functional. As a result, the cell recruits the degradative machinery without ongoing synthesis, and lysis ensues. Many of the ␤-lactams discussed in this chapter interfere with the balance between cell wall synthesis and degradation.

 PHARMACOLOGIC CLASSES AND AGENTS The pharmacology of the drug classes that inhibit bacterial cell wall synthesis is discussed in the same order as the biochemistry of cell wall synthesis (Fig. 34-2). Although drugs have been identified that inhibit a number of steps in the biochemistry of cell wall synthesis, the polymer crosslinking (transpeptidation) step is, by far, the most clinically important biochemical target. For this reason, most of the discussion focuses on the panoply of agents that inhibit the cross-linking of peptidoglycan polymers.

Inhibitors of Murein Monomer Synthesis Fosfomycin and Fosmidomycin Two agents inhibit the production of murein monomers by inhibiting the synthesis of UDP-NAM from UDP-NAG (Fig. 34-2A). Fosfomycin (also written phosphomycin) is a phosphoenolpyruvate (PEP) analogue that inhibits bacterial enolpyruvate transferase (also known as MurA) by covalent modification of the enzyme’s active site. Given that PEP is a key intermediate in (mammalian) glycolysis, it may come as a surprise that this agent does not interfere with carbohydrate metabolism in human cells; this selectivity of antibacterial action is likely caused by structural differences between the mammalian and bacterial enzymes that act on PEP. Thus, fosfomycin has no appreciable effect on human enolase, pyruvate kinase, or carboxykinase, and the drug is

CHAPTER 34 / Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis 607

relatively nontoxic. Fosfomycin has been shown to have antibacterial synergy in vitro with ␤-lactams, aminoglycosides, and fluoroquinolones. Fosfomycin enters the cell via transporters for glycerophosphate or glucose-6-phosphate that are normally used by bacteria to take up these nutrients from the environment. Fosfomycin is especially effective against Gram-negative bacteria that infect the urinary tract, including E. coli and Klebsiella and Serratia species, because it is excreted unchanged in the urine. A single 3-g oral dose has been shown to be as effective as multiple doses of other agents in the treatment of urinary tract infections. As a rule, fosfomycin is less effective against Gram-positive bacteria because these bacteria generally lack selective glycerophosphate and glucose-6-phosphate transporters. Although resistance is typically caused by mutations in these transporters, a temperature-sensitive E. coli strain has been found in which a mutation in enolpyruvate transferase results in reduced affinity of the enzyme for PEP and therefore for fosfomycin. Adverse effects of fosfomycin are uncommon; between 1% and 10% of patients develop headache, diarrhea, or nausea. Significant drug interactions are also rare; the drug can precipitate when co-ingested with antacids or calcium salts, and its absorption can be decreased by co-administration with promotility agents such as metoclopramide. Fosmidomycin, another PEP analogue, acts by the same mechanism as fosfomycin, and resistance typically arises via mutations in glycerophosphate or glucose-6-phosphate transporters. Again, however, there are exceptions: at least one strain of resistant E. coli appears to contain a protein that actively pumps fosmidomycin out of the cell. Cycloserine Cycloserine, a structural analogue of D-Ala, is a secondline agent used to treat multidrug-resistant M. tuberculosis infection (Fig. 34-5). Cycloserine inhibits both the alanine racemase that converts L-Ala to D-Ala and the D-Ala-D-Ala ligase that joins together two D-Ala molecules (Fig. 34-2A). Cycloserine is an irreversible inhibitor of these enzymes and, in fact, binds these enzymes more tightly than does their natural substrate, D-Ala. Resistance to cycloserine occurs by multiple mechanisms, some of which are still unknown; known mechanisms include overexpression of alanine racemase and mutations in the alanine uptake system. As with many small molecules, including fosfomycin, cycloserine is excreted in the urine. Adverse effects include seizures, neurological syndromes including peripheral neuropathy, and psychosis. Patients with underlying neuropsychiatric disease, alcoholism, and chronic kidney disease should avoid

OH

OH

H2N N

H2N

O

O D-Cycloserine

FIGURE 34-5.

D-Alanine

Structure of cycloserine. Cycloserine is a structural analogue of D-alanine that inhibits the racemic interconversion of L-alanine to D-alanine by alanine racemase. Cycloserine also inhibits the activity of D-Ala-D-Ala ligase B, the enzyme that catalyzes the formation of the D-Ala-D-Ala dipeptide that is subsequently utilized in the synthesis of murein monomers (see Fig. 34-2A).

the drug. Alcohol, isoniazid, and ethionamide potentiate its toxicity; pyridoxine may mitigate cycloserine-induced peripheral neuropathy. Cycloserine inhibits the hepatic metabolism of phenytoin. Bacitracin So named because it was first identified in a species of Bacillus, bacitracin is a peptide antibiotic that interferes with the dephosphorylation of bactoprenyl diphosphate, rendering the bactoprenol lipid carrier useless for further rounds of murein monomer synthesis and export (Fig. 34-2B). Bacitracin is therefore notable among the anti-cell wall agents for having a lipid, rather than a protein or peptide, as its target. Bacitracin inhibits dephosphorylation by forming a complex with bactoprenyl diphosphate that involves bacitracin’s imidazole and thiazoline rings. This interaction requires a divalent metal ion, usually Zn2⫹ or Mg2⫹; hence, drugs that act as metal chelators could interfere with the activity of bacitracin. Due to its significant kidney, neurological, and bone marrow toxicity, bacitracin is not used systemically. It is most commonly used topically for superficial dermal or ophthalmologic infections. Because bacitracin is not absorbed orally, it remains within the gut lumen and is occasionally administered orally to treat Clostridium difficile colitis or to eradicate vancomycin-resistant enterococci (VRE) in the gastrointestinal tract. It should not be co-administered with other nephrotoxic medications or neuromuscular blocking agents, since the latter may result in synergistic neuromuscular blockade.

Inhibitors of Murein Polymerization Vancomycin, Teicoplanin, and Telavancin Vancomycin and teicoplanin are glycopeptides with bactericidal activity against Gram-positive rods and cocci. Telavancin is a related lipoglycopeptide with a spectrum of action similar to that of vancomycin. Gram-negative rods are resistant to the action of these drugs. These agents interrupt cell wall synthesis by binding tightly to the D-Ala-D-Ala terminus of the murein monomer unit, inhibiting peptidoglycan polymerization and thereby blocking the addition of murein units to the growing polymer chain. Telavancin has an additional lipid side chain that interacts with the bacterial cell membrane; this lipid anchor both enhances drug binding to the D-Ala-D-Ala terminus and effects depolarization of the membrane, resulting in greater antibacterial potency than vancomycin. Intravenous vancomycin is most commonly used to treat sepsis or endocarditis caused by methicillinresistant Staphylococcus aureus (MRSA) (see discussion below). Intravenous telavancin is used to treat serious skin infections involving staphylococci and streptococci. Oral vancomycin is used to treat gastrointestinal infections with C. difficile; like bacitracin (see above), the drug is poorly absorbed and therefore remains within the gastrointestinal tract. Teicoplanin is not used clinically in the United States. As a rule, the toxicity of vancomycin causes this agent to be used only when an infection is found to be resistant to other agents. Its adverse effects include skin flushing or rash—the so-called red-man syndrome—which is due to release of histamine and can be avoided by slowing the rate of intravenous infusion or preadministering antihistamines. Vancomycin has also been associated with nephrotoxicity and ototoxicity, particularly when other nephrotoxic or ototoxic

A

B H N

R

S

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H N

R

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N O

COOH

COOH

Penicillins

H N

R1

β-lactamases cleave this bond

S

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OH

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R2 COOH

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O COOH

Cephalosporins

Clavulanic acid H N

R O

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SR N O COOH Carbapenems

Sulbactam

610 Principles of Chemotherapy

Antibody N Beta-lactam antibiotic NH2

Modified human protein (antigenic)

Human protein (non-antigenic)

FIGURE 34-7. ␤-Lactam toxicity. In the absence of modification, human proteins are generally nonantigenic. ␤-Lactams can modify amino groups on human proteins, creating an immunogenic ␤-lactam hapten. This new antigenic determinant can be recognized as “nonself” by antibodies of the host immune system.

with amino groups on human proteins to create a hapten–carrier complex (Fig. 34-7). The ␤-lactam–protein conjugate can then provoke a hypersensitivity response. The most dreaded of these reactions is anaphylaxis, which typically occurs within an hour of administration and leads to bronchospasm, angioedema, and/or cardiovascular collapse. Urticaria, morbilliform drug rash, serum sickness, and drug fever may also occur. Proteins on the surface of red blood cells can also be modified by penicillin, leading to drug-induced autoimmune hemolytic anemia. Rarely, ␤-lactam antibiotics cause druginduced lupus. For most individuals, this process is strongly dose-dependent: the likelihood of a hypersensitivity reaction increases with each administration of a ␤-lactam. ␤-Lactams of a given class often cross-react with each other, but ␤-lactams of one class are less often cross-reactive with ␤-lactams of another class. Patients with a penicillin allergy should not receive ampicillin or other penicillins due to the high risk of cross-reactivity. Patients with a penicillin allergy other than anaphylaxis may receive a cephalosporin. Aztreonam (a monobactam) is unique in that it has no cross-reactivity with either penicillins or carbapenems; however, cross-reactivity between aztreonam and ceftazidime (a cephalosporin), due to a shared side chain, has been seen. Although allergic reactions to carbapenems can occur in patients with penicillin allergy, they are infrequent. -Lactam Antibiotics: Specific Agents Penicillins

As noted above, there are four structurally distinct subclasses of ␤-lactam antibiotics (see Fig. 34-6A). The first of these subclasses, the penicillins, can be further divided into five groups according to their spectra of action. The first group of penicillins includes penicillin G, which is intravenously administered, and penicillin V, its gastric acid-stable oral counterpart. Penicillin G is in more widespread use than penicillin V; the latter is used mostly to treat mixed aerobic–anaerobic infections of the head and neck, such as dental abscesses. Additionally, penicillin V is used to prevent recurrent rheumatic fever in patients with a prior episode and recurrent streptococcal cellulitis in patients with lymphedema. Penicillin G is used to treat serious

infections with Gram-positive bacteria such as pneumococcus and S. pyogenes (some strains of each), Gram-negative diplococci such as Neisseria species (except penicillinaseproducing N. gonorrhoeae), Gram-positive rods of the genus Clostridium, most anaerobes (except Bacteroides), and spirochetes such as syphilis and Leptospira. High-dose penicillin G may cause seizures, in addition to the already mentioned hypersensitivity reactions and rash. All penicillins can cause acute interstitial nephritis. Drug–drug interactions are rare, but the anticoagulant effects of warfarin may be potentiated by concomitant penicillin administration. The second group consists of the antistaphylococcal penicillins, including oxacillin, cloxacillin, dicloxacillin, nafcillin, and methicillin. These drugs are structurally resistant to staphylococcal ␤-lactamase, which is encoded by plasmid genes in most clinical isolates. Because of their relative hydrophobicity, however, antistaphylococcal penicillins lack activity against Gram-negative bacteria. (Recall also that methicillin binds to only a single transpeptidase.) Thus, these agents are used mostly for skin and soft-tissue infections or documented methicillin-sensitive S. aureus infections. Use of the oral antistaphylococcal penicillins (cloxacillin and dicloxacillin) is limited by their gastrointestinal adverse effects (nausea, vomiting, and antibioticassociated diarrhea) as well as secondary development of C. difficile colitis. Adverse effects of intravenous (IV) nafcillin include phlebitis at the injection site; agranulocytosis and acute interstitial nephritis occur at a higher rate than with the other penicillins. Oxacillin can cause hepatotoxicity, which is reversible with discontinuation of the drug. The utility of antistaphylococcal penicillins in treating S. aureus has been compromised by the emergence of MRSA strains. When a case of MRSA is found in the hospital, special precautions are taken to prevent its spread to other patients. Patients with MRSA infection are typically treated with vancomycin. Ampicillin and amoxicillin are members of the third group of penicillins, the amino penicillins, which have a positively charged amino group on the R side chain (see Fig. 34-6A). This positive charge enhances diffusion through porin channels but does not confer resistance to ␤-lactamases. These agents are effective against a variety of Gram-positive cocci, Gram-negative cocci such as Neisseria gonorrhoeae and N. meningitidis, and Gram-negative rods such as E. coli and Haemophilus influenzae, but their spectrum is limited by sensitivity to most ␤-lactamases. IV ampicillin is used most commonly to treat invasive enterococcal infections and Listeria meningitis; oral amoxicillin is used to treat uncomplicated ear, nose, and throat infections, to prevent endocarditis in high-risk patients undergoing dental work, and as a component of combination therapy for Helicobacter pylori infection. Nonurticarial rash is the most common adverse effect. The spectrum of both agents is broadened when they are co-administered with ␤-lactamase inhibitors such as clavulanic acid (with amoxicillin) or sulbactam (with ampicillin) to treat ␤-lactamase-producing organisms such as S. aureus, H. influenzae, E. coli, Klebsiella, and anaerobes. Sulbactam itself has activity against Acinetobacter. Agents in the fourth group of penicillins, the carboxy penicillins, are also broad in spectrum. The carboxyl group on the R side chain provides a negative charge that confers resistance to some ␤-lactamases but is less effective than a positively charged amino group in facilitating diffusion through

CHAPTER 34 / Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis 611

porin channels. To overcome this limitation in diffusion, high doses are used. Resistance to the chromosomally encoded ␤-lactamases of Enterobacter and Pseudomonas adds these organisms to the spectrum of the carboxy penicillins. This group has two members, carbenicillin and ticarcillin. A fifth group, the ureido penicillins, is represented by piperacillin and mezlocillin. These drugs have both positive and negative charges on their R side chains and are generally more potent than the carboxy penicillins. Their spectrum of action is similar to that of the carboxy penicillins; in addition, they have activity against Klebsiella and enterococci. Cephalosporins

Cephalosporins differ structurally from penicillins by having a six-membered rather than a five-membered accessory ring attached to the ␤-lactam ring (Fig. 34-6A). First-generation cephalosporins (cefazolin and cephalexin) are active against Gram-positive species as well as the Gramnegative rods Proteus mirabilis and E. coli, both of which cause urinary tract infections, and Klebsiella pneumoniae, which causes pneumonia in addition to urinary tract infections. These agents are sensitive to many ␤-lactamases but are not degraded by the chromosomally encoded ␤-lactamase of K. pneumoniae and the common staphylococcal ␤-lactamase. Cephalexin and cefazolin are both used to treat skin and soft-tissue infections; cefazolin is also used for surgical prophylaxis. Second-generation cephalosporins can be divided into two groups. Cefuroxime, which represents the first group, has increased activity against H. influenzae compared to the first-generation cephalosporins; cefotetan and cefoxitin, which represent the second group, demonstrate increased activity against Bacteroides. Also, second-generation cephalosporins are generally resistant to more ␤-lactamases than are first-generation cephalosporins. Thus, cefuroxime is often used to treat community-acquired pneumonia, and cefotetan is used to treat intra-abdominal and pelvic infections, including pelvic inflammatory disease. Adverse effects of these agents include diarrhea, mild liver enzyme elevation, and hypersensitivity reactions; rarely, agranulocytosis or interstitial nephritis can occur. Third-generation cephalosporins (ceftriaxone and cefotaxime) are resistant to many ␤-lactamases and are thus highly active against Enterobacteriaceae (E. coli, indolepositive Proteus, Klebsiella, Enterobacter, Serratia, and Citrobacter) as well as Neisseria and H. influenzae. The third-generation cephalosporins are less active against Grampositive organisms than are the first-generation drugs; despite that, they have good activity against penicillin-intermediate S. pneumoniae (although cephalosporin resistance can occur). Common uses include treatment of lower respiratory tract infection, community-acquired meningitis due to S. pneumoniae, uncomplicated gonococcal infection, culture-negative endocarditis, and complicated Lyme disease. In addition to the adverse effects already mentioned, ceftriaxone can cause cholestatic hepatitis, albeit uncommonly. Ceftazidime is the third commonly used third-generation cephalosporin; its spectrum differs from that of the other two agents in that it has significant antipseudomonal activity and minimal activity against Gram-positive organisms. It is used predominantly to treat hospital-acquired Gramnegative bacterial infections and documented infections with P. aeruginosa and as empiric therapy for neutropenic

patients with fever. Gram-negative bacteria that have acquired extended-spectrum ␤-lactamase activity are resistant to third-generation cephalosporins. Cefepime is the only currently available fourth-generation cephalosporin. Like ceftriaxone, it is highly active against Enterobacteriaceae, Neisseria, H. influenzae, and Grampositive organisms; additionally, it is as active as ceftazidime against P. aeruginosa. Cefepime is also more resistant to the chromosomally encoded ␤-lactamases of Enterobacter than are third-generation cephalosporins. Unlike ceftazidime, however, cefepime is not approved for treatment of meningitis. An uncommon adverse effect is the development of autoantibodies against red blood cell antigens, typically without significant hemolysis. Ceftaroline and ceftobiprole, are fifth-generation cephalosporins. These drugs are distinct in having antimicrobial activity against multidrug-resistant S. aureus, including methicillin-resistant, vancomycin-intermediate S. aureus and vancomycin-resistant strains, as well as S. pneumoniae and respiratory Gram-negative pathogens such as Moraxella catarrhalis and H. influenzae, including ␤-lactamase-expressing strains. Both compounds must be administered intravenously. Ceftaroline is approved for treatment of community-acquired pneumonia and skin infections; ceftobiprole is under evaluation by the U.S. Food and Drug Administration (FDA). Clinical trials suggest a safety profile similar to that of other cephalosporins. As noted above, cephalosporins can generally be used in patients with non-life-threatening allergy to penicillins. Nevertheless, cephalosporins can cause hypersensitivity reactions themselves and should be avoided in patients with known cephalosporin hypersensitivity. Interestingly, cefotetan and cefoperazone contain an N-methylthiotetrazole (NMTT) side chain that causes two unique adverse effects. The first is an alcohol intolerance syndrome known as the disulfiram-like reaction (disulfiram is a drug that inhibits alcohol metabolism; see Chapter 18, Pharmacology of Drugs of Abuse). The second involves an effect on vitamin K metabolism that results in decreased synthesis of vitamin K-dependent coagulation factors; thus, cefotetan and cefoperazone should be used with caution in patients taking warfarin and in patients with underlying coagulation abnormalities (see Chapter 22, Pharmacology of Hemostasis and Thrombosis). Cefotetan, like most of the cephalosporins, can also cause antibody-mediated hemolysis. Monobactams and Carbapenems

The only available monobactam, aztreonam, is active against most Gram-negative bacteria, including P. aeruginosa, but it has no activity against Gram-positive organisms. Aztreonam is particularly useful in patients with serious penicillin allergy who have infections due to resistant Gram-negative organisms because of its lack of cross-allergenicity with penicillins; however, Gram-negative bacteria with extended-spectrum ␤-lactamases are resistant to the drug. Its use is limited by IV-site phlebitis, and its short half-life necessitates frequent dosing. There are four carbapenems used in clinical practice: imipenem, meropenem, doripenem, and ertapenem. All four are broad-spectrum and cover most Gram-positive, Gram-negative, and anaerobic organisms. None is active against MRSA, VRE, or Legionella; and Gram-negative bacteria with carbapenemases (especially K. pneumoniae)

CHAPTER 34 / Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis 613

The combination of these two agents, which are administered intravenously, is currently in phase 2 clinical trials.

 CONCLUSION AND FUTURE DIRECTIONS The bacterial cell wall presents a number of unique antibacterial targets. This structure consists of a three-dimensional mat of cross-linked peptide-sugar polymers called murein and is synthesized in three stages: (1) synthesis of murein monomers; (2) polymerization of monomers into murein polymers; and (3) cross-linking of polymers to complete the wall. Antibacterial agents act in all three stages of cell wall synthesis: fosfomycin and cycloserine act in the first stage; vancomycin, teicoplanin, telavancin, and bacitracin act in the second stage; and the ␤-lactams, the largest and most important group, act in the third stage. ␤-Lactams—which include the penicillins, cephalosporins, monobactams, and carbapenems—are bactericidal; autolytic cell death most likely results from the unopposed action of wall remodeling proteins called autolysins. Structural and chemical differences among the ␤-lactams determine their spectra of activity against bacteria with different cell wall architectures. Resistance to ␤-lactam antibiotics is generally conferred by plasmid-encoded ␤-lactamases. Pharmacologists have addressed this mechanism of resistance: (1) by developing new ␤-lactam agents, for example, the second- and thirdgeneration cephalosporins that are resistant to degradation by many ␤-lactamases; and (2) by co-administering ␤-lactam “decoys,” such as clavulanic acid and sulbactam, that serve as ␤-lactamase inhibitors. Because ␤-lactamases can be encoded on plasmids, they can spread through bacterial (and human) populations with great speed, making antibiotic development an ongoing “arms race.” Antimycobacterial agents act by blocking various steps in the synthesis of molecules, such as mycolic acid and arabinogalactan, that are unique to the mycobacterial cell wall. Resistance to these agents is typically due to chromosomal mutation, but combination therapy is critically important to avoid the development of mutational resistance. Future innovations will likely include the development of

new agents directed against the additional unique molecular targets that are presented by the biochemistry of the bacterial cell wall.

Acknowledgment We thank Robert R. Rando and Anne G. Kasmar for their valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Brennan PJ. The envelope of mycobacteria. Annu Rev Biochem 1995; 64:29–63. (Reviews the structure, composition, and synthesis of the mycobacterial cell wall.) Bush K. Alarming ␤-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr Opin Microbiol 2010;13:558–564. (Reviews ␤lactam resistance in Gram-negative bacteria, focusing on recent reports of ESBL- and carbapenemase-mediated resistance.) El Zoeiby A, Sanschagrin F, Levesque RC. Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 2003;47:1–12. (Reviews the structure, catalytic action, and inhibition of MurA–MurF.) Gale EF, Cundliffe E, Reynolds PE, et al. The molecular basis of antibiotic action. 2nd ed. London: John Wiley; 1981. (Classic on antibiotics that describes the experiments that led to the determination of many of the mechanisms of action discussed in this chapter.) Howden BP, Davies JK, Johnson PD, et al. Reduced vancomycin susceptibility in Staphylococcus aureus: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev 2010;23:99–139. (Reviews VISA and VRSA, including definitions, risk factors, and mechanisms of resistance.) Jacoby GA, Munoz-Price LS. The new beta-lactamases. N Engl J Med 2005;352:380–391. (Reviews the pharmacology of ␤-lactamases.) Kelkar PS, Li JT. Cephalosporin allergy. N Engl J Med 2001;345:804–809. (Comprehensive literature review of cephalosporin reactions in patients with a history of penicillin allergy.) Ma Z, Lienhardt C, McIlleron H, et al. Global tuberculosis drug development pipeline: the need and the reality. Lancet 2010;375:2100–2109. (Reviews approved and investigational drugs for the treatment of tuberculosis.) Paterson DL, Bonomo DA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 2005;18:657–686. (Reviews the microbiology, transmission, and treatment of extended-spectrum ␤-lactamase-producing organisms.) Rattan A, Kalia A, Ahmad N. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives. Emerg Infect Dis 1998;4:195–209. (Discusses the problem of resistance in tuberculosis.)

35 Pharmacology of Fungal Infections Ali Alikhan, Charles R. Taylor, and April W. Armstrong

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 618-619 BIOCHEMISTRY OF THE FUNGAL MEMBRANE AND CELL WALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618 PATHOPHYSIOLOGY OF FUNGAL INFECTIONS . . . . . . . . . . . 620 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 620 Inhibitor of Fungal Nucleic Acid Synthesis: Flucytosine . . . 620 Inhibitor of Fungal Mitosis: Griseofulvin . . . . . . . . . . . . . . 622

Inhibitors of the Ergosterol Synthesis Pathway . . . . . . . . . 622 Inhibitors of Squalene Epoxidase. . . . . . . . . . . . . . . . . 622 Inhibitors of 14␣-Sterol Demethylase . . . . . . . . . . . . . 622 Inhibitors of Fungal Membrane Stability: Polyenes . . . . . . 624 Inhibitors of Fungal Wall Synthesis: Echinocandins . . . . . . 625 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 625 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

 INTRODUCTION

implications, because prognosis often correlates inversely with the duration of time from clinical presentation to accurate diagnosis. Consequently, one major focus of modern mycology is the development of rapid, nonculture-based methods of early diagnosis. New diagnostic techniques rely on the polymerase chain reaction (PCR), western blot, antigen detection, and identification of fungal metabolites. Because many of these techniques are still investigational, they must be performed in parallel with traditional culturebased methods. The treatment options for opportunistic and systemic fungal infections were once thought to be limited. These options are now expanding, however. Fungal processes that have been exploited in the development of antifungal agents include nucleic acid synthesis, mitosis, and membrane synthesis and stability. Traditional antifungal agents, such as azoles and polyenes, are directed against molecular targets involved in the synthesis and stability of the fungal membrane. The echinocandins, a new class of antifungal agents, target an enzyme complex involved in the synthesis of the fungal cell wall. As the emergence of resistant fungi increases, it will become increasingly important to identify and exploit new molecular targets for antifungal therapy.

Fungi are free-living microorganisms that exist as yeasts (single-cell, round fungi), molds (multicellular filamentous fungi), or a combination of the two (so-called dimorphic fungi). All fungi are eukaryotic organisms. Because of their phylogenetic similarity, fungi and humans have homologous metabolic pathways for energy production, protein synthesis, and cell division. Consequently, there is greater difficulty in developing selective antifungal agents than in developing selective antibacterial agents. The success of many antibacterial agents has resulted from the identification of unique molecular targets in bacteria, emphasizing the necessity for identifying unique fungal targets that can be exploited. Certain patient populations are particularly susceptible to fungal infections (mycoses). These populations include surgical and intensive care unit (ICU) patients, patients with prostheses, and patients with compromised immune defenses. In the past three decades, the extensive use of broadspectrum antibiotics, the wider use of long-term intravenous catheters, and infection with human immunodeficiency virus (HIV) have correlated with an increasing incidence of opportunistic and systemic mycoses. Additionally, the successes of organ transplantation, immunosuppressive therapy, and cancer chemotherapy have contributed to an increasing number of chronically immunosuppressed patients, who are particularly susceptible to fungal infections. Traditionally, the diagnosis of fungal infections has relied on culture-based methods and direct examination of specimens under light microscopy. However, the indolent growth of fungi makes culturing inefficient, while direct microscopic examination may not be reliable or provide definitive speciation. These disadvantages have important clinical 618

 BIOCHEMISTRY OF THE FUNGAL MEMBRANE AND CELL WALL Although fungi have a cellular ultrastructure similar to that of animal cells, there are a number of unique biochemical differences that have been exploited in the development of antifungal drugs. To date, the most important biochemical difference lies in the principal sterol used to maintain

Acetyl CoA

HMG CoA

Mevalonate Allylamines Benzylamines

Squalene Squalene epoxidase

Two representative triazoles: OH N N

HO

N

N

N

F

N

H

Lanosterol F

Imidazoles Triazoles

14α-sterol demethylase

Fluconazole F OH N N

N N

F

F

Voriconazole H

H

HO

Ergosterol

Membrane synthesis

N

CHAPTER 35 / Pharmacology of Fungal Infections 621 Endoplasmic reticulum (inhibit ergosterol synthesis) Allylamines Benzylamines Imidazoles Triazoles

Nucleus

Mitotic spindle Griseofulvin

be demonstrated experimentally. The mechanism of this synergistic interaction appears to involve enhancement of flucytosine uptake by fungal cells due to amphotericininduced damage to the fungal plasma membrane. The spectrum of activity of flucytosine as a single agent is limited to candidiasis, cryptococcosis, and chromomycosis. Combination treatment with amphotericin B is recommended in acute cryptococcal meningitis in HIV-infected adults. A pharmacokinetic advantage of flucytosine is its large volume of distribution, with excellent penetration into the central nervous system (CNS), eyes, and urinary tract. Dose-dependent adverse effects include bone marrow suppression leading to leukopenia and thrombocytopenia, nausea, vomiting, diarrhea, and hepatic dysfunction. Flucytosine is contraindicated during pregnancy.

NH2

Cell wall Echinocandins DNA synthesis Flucytosine

F N

Plasma membrane Polyenes (amphotericin B)

N H

Flucytosine

OH OH O HO

Cytosine permease

OH O

OH

OH

O

OH

OH

O

OH

Cell membrane

H O

O NH2 OH

O

Cytosine deaminase

OH

O Amphotericin B

F

NH

FIGURE 35-2.

Cellular targets of antifungal drugs. The currently available antifungal agents act on distinct molecular targets. Flucytosine inhibits fungal DNA synthesis. Griseofulvin inhibits fungal mitosis by disrupting mitotic spindles. Allylamines, benzylamines, imidazoles, and triazoles inhibit the ergosterol synthesis pathway in the endoplasmic reticulum. Polyenes bind to ergosterol in the fungal membrane and thereby disrupt plasma membrane integrity. Amphotericin B is a representative polyene. Echinocandins inhibit fungal cell wall synthesis.

N H

O

5-Fluorouracil (5-FU)

O F

(5-FU). (5-FU is itself an antimetabolite that is used in cancer chemotherapy; see Chapter 38, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance.) Subsequent reactions convert 5-FU to 5-fluorodeoxyuridylic acid (5-FdUMP), which is a potent inhibitor of thymidylate synthase. Inhibition of thymidylate synthase results in inhibition of DNA synthesis and cell division (Fig. 35-3). Flucytosine appears to be fungistatic under most circumstances. Although mammalian cells lack cytosine-specific permeases and cytosine deaminase, fungi and bacteria in the intestine can convert flucytosine into 5-fluorouracil, which can cause adverse effects in host cells. Flucytosine is typically used in combination with amphotericin B to treat systemic mycoses; when the drug is used as a single agent, resistance emerges rapidly due to mutations in fungal cytosine permease or cytosine deaminase. Although flucytosine has no intrinsic activity against Aspergillus, synergistic killing of Aspergillus by the combination of flucytosine and amphotericin B can

NH N

O -

O

P

O

O

O

OH

H

H

OH

H

H

5-Fluorodeoxyuridylic acid monophosphate (5-FdUMP)

dUMP

Thymidylate synthase

dTMP

FIGURE 35-3. Mechanism of action of flucytosine. Flucytosine enters the fungal cell via a transmembrane cytosine permease. Inside the cell, cytosine deaminase converts flucytosine to 5-fluorouracil (5-FU), which is subsequently converted to 5-fluorodeoxyuridylic acid monophosphate (5-FdUMP). 5-FdUMP inhibits thymidylate synthase and thereby blocks the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP). In the absence of dTMP, DNA synthesis is inhibited.

622 Principles of Chemotherapy

Inhibitor of Fungal Mitosis: Griseofulvin Derived from Penicillium griseofulvum in the 1950s, griseofulvin inhibits fungal mitosis by binding to tubulin and a microtubule-associated protein and thereby disrupting assembly of the mitotic spindle. The drug is also reported to inhibit fungal RNA and DNA synthesis. Griseofulvin accumulates in keratin precursor cells and binds tightly to keratin in differentiated cells. The prolonged and tight association of griseofulvin with keratin allows new growth of skin, hair, or nail to be free of dermatophyte infection. Griseofulvin appears to be fungistatic under most circumstances. The therapeutic use of oral griseofulvin is currently limited, due to the availability of topical antifungal medications as well as other oral antifungal agents with fewer adverse effects. Griseofulvin can be used to treat fungal infection of the skin, hair, and nail due to Trichophyton, Microsporum, and Epidermophyton. The drug is not effective against yeast (such as Pityrosporum) and dimorphic fungi. Doses should be taken at 6-hour intervals because blood levels of griseofulvin can be variable; absorption is enhanced if the drug is taken with a fatty meal. It is important to continue treatment until the infected skin, hair, or nail is completely replaced by normal tissue. Griseofulvin use is not associated with a high incidence of serious adverse effects. A relatively common (up to 15%) adverse effect is headache, which tends to disappear as therapy continues. Other nervous system effects include lethargy, vertigo, and blurred vision; these adverse effects can be exacerbated by the consumption of alcohol. Occasionally, hepatotoxicity or albuminuria without renal insufficiency can be observed. Hematologic adverse effects—including leukopenia, neutropenia, and monocytosis—can occur during the first month of therapy. Serum sickness, angioedema, exfoliative dermatitis, and toxic epidermal necrolysis are extremely rare but potentially life-threatening adverse effects. Chronic use can sometimes result in increased fecal protoporphyrin levels. Concurrent administration with barbiturates decreases the gastrointestinal absorption of griseofulvin. Because griseofulvin induces hepatic cytochrome P450 enzymes, it can increase the metabolism of warfarin and potentially reduce the efficacy of low-estrogen oral contraceptive medications. Griseofulvin should be avoided during pregnancy, since fetal abnormalities have been reported.

Inhibitors of the Ergosterol Synthesis Pathway Inhibitors of Squalene Epoxidase Allylamines and Benzylamines

In the ergosterol synthesis pathway (Fig. 35-1), squalene is converted to lanosterol by the action of squalene epoxidase. Inhibitors of squalene epoxidase prevent the formation of lanosterol, which is a precursor for ergosterol. These drugs also promote accumulation of the toxic metabolite squalene in the fungal cell, making them fungicidal under most circumstances. The antifungal agents that inhibit squalene epoxidase can be divided into allylamines and benzylamines based on their chemical structures: terbinafine and naftifine are allylamines, whereas butenafine is a benzylamine. Terbinafine is available in both oral and topical formulations. When taken orally, the drug is 99% protein-bound in the plasma, and it undergoes first-pass metabolism in the liver. Because of this first-pass metabolism, the oral bioavailability of terbinafine is 40%. The drug’s elimination

half-life is extremely long, approximately 300 hours, because terbinafine accumulates extensively in the skin, nails, and fat. The oral form of terbinafine is used in the treatment of onychomycosis, tinea corporis, tinea cruris, tinea pedis, and tinea capitis. Terbinafine is not recommended in patients with renal or hepatic failure or in pregnant women. Very rarely, the oral form of terbinafine can lead to hepatotoxicity, Stevens–Johnson syndrome, neutropenia, and exacerbation of psoriasis or subacute cutaneous lupus erythematosus. Liver function enzymes should be monitored during the treatment course. Plasma levels of terbinafine are increased by co-administration with cimetidine (a cytochrome P450 inhibitor) and decreased by co-administration with rifampin (a cytochrome P450 inducer). Topical terbinafine is available in cream or spray form and is indicated for tinea pedis, tinea cruris, and tinea corporis. Similar to terbinafine, naftifine is a squalene epoxidase inhibitor that has broad-spectrum antifungal activity. Naftifine is only available topically as a cream or gel; it is effective in tinea corporis, tinea cruris, and tinea pedis. Butenafine, a benzylamine, is a topical antifungal agent with a mechanism of action and spectrum of antifungal activity similar to that of the allylamines. Topical allylamines and benzylamines are more effective than topical azole agents against common dermatophytes, especially those causing tinea pedis. However, topical terbinafine and butenafine are less effective than topical azoles against Candida skin infections (see below). Inhibitors of 14 ␣-Sterol Demethylase Imidazoles and Triazoles

Another important molecular target in the ergosterol synthesis pathway is 14␣-sterol demethylase, a microsomal cytochrome P450 enzyme that converts lanosterol to ergosterol. The azoles are antifungal agents that inhibit fungal 14␣-sterol demethylase. The resulting decrease in ergosterol synthesis and accumulation of 14␣-methyl sterols disrupt the tightly packed acyl chains of the phospholipids in fungal membranes. Destabilization of the fungal membrane leads to dysfunction of membrane-associated enzymes, including those in the electron transport chain, and may ultimately lead to cell death. Azoles are not completely selective for the fungal P450 enzyme, however, and they can also inhibit hepatic P450 enzymes. While the extent of hepatic P450 enzyme inhibition varies among the azoles, drug–drug interactions are an important consideration whenever an azole antifungal agent is prescribed. For example, cyclosporine is an immunosuppressant drug used to prevent graft rejection in recipients of allogeneic kidney, liver, and heart transplants. It is metabolized by hepatic P450 enzymes and excreted in the bile. To minimize the risk of cyclosporine-associated nephrotoxicity and hepatotoxicity, patients concomitantly receiving an azole antifungal agent should be treated with lower doses of cyclosporine. As a group, the azoles have a wide range of antifungal activity and are clinically useful against B. dermatitidis, Cryptococcus neoformans, H. capsulatum, Coccidioides species, P. brasiliensis, dermatophytes, and most Candida species. Azoles have intermediate clinical activity against Fusarium, Sporothrix schenckii, Scedosporium apiospermum, and Aspergillus species. Pathogens mediating zygomycosis (invasive fungal infections caused by Zygomycetes species) and Candida krusei are resistant to azoles. The azoles are

CHAPTER 35 / Pharmacology of Fungal Infections 625

Nystatin, a structural relative of amphotericin B, is a polyene antifungal agent that also acts by binding ergosterol and causing pore formation in fungal cell membranes. The drug is used topically to treat candidiasis involving the skin, vaginal mucosa, and oral mucosa. Nystatin is not absorbed systemically from the skin, vagina, or gastrointestinal tract.

Inhibitors of Fungal Wall Synthesis: Echinocandins The key components of the fungal cell wall are chitin, ␤(1,3)-D-glucan, ␤-(1,6)-D-glucan, and cell wall glycoproteins. Because human cells do not have a cell wall, fungal cell wall components represent unique targets for antifungal therapy, and antifungal agents directed at these targets are likely to be relatively nontoxic. Echinocandins are a class of antifungal agents that target fungal cell wall synthesis by noncompetitively inhibiting the synthesis of ␤-(1,3)-Dglucans. Disruption of cell wall integrity results in osmotic stress, lysis of the fungal cell, and ultimately fungal cell death. The three antifungal agents in the echinocandin class are caspofungin, micafungin, and anidulafungin; all are semisynthetic lipopeptides derived from natural products. The echinocandins have in vitro and in vivo antifungal activity against Candida and Aspergillus species. All three echinocandins are fungicidal against Candida species, including C. krusei and C. glabrata, and fungistatic against Aspergillus species. They have poor activity against zygomycetes. All three agents are currently available only in parenteral form because they are insufficiently bioavailable for oral use. Caspofungin was the first echinocandin to be approved. The drug is used as primary therapy for esophageal candidiasis and candidemia, as salvage therapy for Aspergillus infections, and as empiric therapy for febrile neutropenia. Like the other echinocandins, caspofungin is highly proteinbound (97%) in the plasma; it is metabolized in the liver via peptide bond hydrolysis and N-acetylation; and it penetrates poorly into the CSF (although animal data indicate that the echinocandins do have some activity in the CNS). Caspofungin does not require dose adjustment for renal insufficiency, but dose adjustment is required for patients with moderate hepatic dysfunction. Because co-administration with cyclosporine significantly increases plasma concentrations of caspofungin and elevates liver function enzymes, this drug combination is generally not recommended unless the expected benefits outweigh the risks. Similarly, co-administration with tacrolimus significantly increases plasma concentrations of tacrolimus. To achieve therapeutic plasma concentrations, caspofungin dosing may need to be increased in patients receiving nelfinavir, efavirenz, phenytoin, rifampin, carbamazepine, or dexamethasone. Micafungin is approved for the treatment of esophageal candidiasis and as antifungal prophylaxis for recipients of hematopoietic stem cell transplants. It is also effective against candidemia and pulmonary aspergillosis. Anidulafungin is approved for the treatment of esophageal candidiasis and

candidemia. Several small case series have reported the use of echinocandins in combination with amphotericin B, flucytosine, itraconazole, or voriconazole in patients with refractory fungal infections. Aminocandin is an investigational echinocandin with a spectrum of activity similar to that of the other echinocandins. It has a half-life three- to four-fold greater than that of the other echinocandins, thus permitting less frequent administration. Echinocandins are generally well tolerated; their adverseeffect profile is comparable to that of fluconazole. Because echinocandins contain a peptide backbone, symptoms related to histamine release can be observed (see Suggested Reading). Other adverse effects include headache, fever (more common with caspofungin), rash, abnormal liver function tests, and, rarely, hemolysis.

 CONCLUSION AND FUTURE DIRECTIONS The development of antifungal agents has progressed significantly since the introduction of amphotericin B. As the population of immunocompromised patients increases, opportunistic fungal infections that are resistant to conventional antifungal therapy pose new challenges to researchers and clinicians. For example, new antifungal therapy is greatly needed in the treatment of zygomycosis. Effective topical antifungal agents are eagerly sought for the treatment of nail and hair dermatophytosis, because oral therapies for these superficial fungal infections carry risks such as hepatotoxicity. The development of protease inhibitors and phospholipase inhibitors represent new frontiers in the treatment of Candida and Cryptococcal species, respectively. As novel and unique molecular targets are identified in fungal pathogens, newer antifungal agents will be developed with the goal of minimizing mechanism-based (“on-target”) toxicity while expanding antifungal spectrum of action.

Suggested Reading Gauwerky K, Borelli C, Korting HC. Targeting virulence: a new paradigm for antifungals. Drug Discov Today 2009;14:214–222. (Discusses virulence factors of fungi and their inhibitors, with an emphasis on new options for antifungal development, including inhibitors of the secreted aspartic proteinase of C. albicans.) Mohr J, Jonson M, Cooper T, et al. Current options in antifungal pharmacotherapy. Pharmacotherapy 2008;28:614–645. (Discusses mechanism of action, clinical efficacy, and safety of polyenes, azoles, echinocandins, and investigational antifungal drugs.) Naeger-Murphy N, Pile JC. Clinical indications for newer antifungal agents. J Hosp Med 2008;4:102–111. (Discusses the use of newly available drugs in the echinocandin class and newer generation triazoles in several common and/or important clinical situations.) Patterson TF. Advances and challenges in management of invasive mycosis. Lancet 2005;366:1013–1025. (Focused discussion of fungal pathogens that occur in immunocompromised hosts and management strategies for these opportunistic pathogens.) Ruiz-Herrera J, Victoria Elorza M, Valentin E, et al. Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity. FEMS Yeast Res 2006;6:14–29. (Comprehensive review of the fungal cell wall.)

36 Pharmacology of Parasitic Infections Louise C. Ivers and Edward T. Ryan

INTRODUCTION & CASES 1-2 . . . . . . . . . . . . . . . . . . . 629-630 MALARIAL PLASMODIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 Physiology of Malarial Plasmodia . . . . . . . . . . . . . . . . . . . 629 Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 Heme Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631 Electron Transport Chain . . . . . . . . . . . . . . . . . . . . . . . 631 Pharmacology of Antimalarial Agents . . . . . . . . . . . . . . . . 632 Inhibitors of Heme Metabolism . . . . . . . . . . . . . . . . . . 632 Inhibitors of Electron Transport . . . . . . . . . . . . . . . . . . 634 Inhibitors of Translation . . . . . . . . . . . . . . . . . . . . . . . . 635 Inhibitors of Folate Metabolism . . . . . . . . . . . . . . . . . . 636 Antimalarial Drug Resistance . . . . . . . . . . . . . . . . . . . . . . 636 OTHER PROTOZOA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637 CASE 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637 Physiology of Luminal Protozoa . . . . . . . . . . . . . . . . . . . . 637 Life Cycle of Entamoeba histolytica . . . . . . . . . . . . . . . 637 Fermentation Pathways . . . . . . . . . . . . . . . . . . . . . . . 637

Pharmacology of Antiprotozoal Agents . . . . . . . . . . . . . . . 638 Metronidazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638 Tinidazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639 Nitazoxanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639 Other Antiprotozoal Agents . . . . . . . . . . . . . . . . . . . . . 639 HELMINTHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640 CASE 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640 Physiology of Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . 641 Life Cycle of Onchocerca volvulus . . . . . . . . . . . . . . . . 641 Neuromuscular Activity . . . . . . . . . . . . . . . . . . . . . . . . 641 Pharmacology of Antihelminthic Agents . . . . . . . . . . . . . . 641 Agents That Interrupt Neuromuscular Activity . . . . . . . 641 Other Antihelminthic Agents . . . . . . . . . . . . . . . . . . . . 642 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 643 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643

 INTRODUCTION

on specific fermentation pathways, and the dependence of helminths on neuromuscular activity. These three examples are not all-inclusive, but rather emphasize opportunities to use or design pharmacologic agents to interrupt metabolic requirements specific to parasites.

More than one billion people worldwide are infected with parasites. Parasites of medical importance include protozoa (such as the organisms that cause malaria, toxoplasmosis, giardiasis, amebiasis, leishmaniasis, and trypanosomiasis) and helminths (“worms”). Worms that infect humans include cestodes (“flat worms” or “tape worms,” such as the worm that causes taeniasis), nematodes (“round worms,” which cause filariasis, strongyloidiasis, and ascariasis), and trematodes (“flukes,” such as the worms that cause schistosomiasis). Ideally, antiparasitic drugs should be targeted to structures or biochemical pathways present or accessible only in parasites. Many antiparasitic drugs act by unknown or poorly defined mechanisms of action, however. This chapter focuses on a number of the better-defined agents, including those active against Plasmodia species (which cause malaria), Entamoeba histolytica (which causes amebiasis), and Onchocerca volvulus (which causes onchocerciasis, a filarial infection referred to as “river blindness”). In each of these cases, antiparasitic agents interfere with metabolic requirements of the parasite: the dependence of malarial plasmodia on heme metabolism, the dependence of luminal parasites

 MALARIAL PLASMODIA Each year, approximately 300 million individuals in more than 90 countries develop malaria, and almost one million individuals die of malaria. Malaria is the most important parasitic disease of humans and one of the most important infections of humans. Human malaria is caused by one of five species of plasmodial parasites: Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The most serious type of malaria is that caused by P. falciparum.

Physiology of Malarial Plasmodia Life Cycle The life cycle of malaria involves a parasite, a mosquito vector, and a human host (Fig. 36-1). An Anopheles spp. mosquito can ingest sexual forms of malarial parasites (gametocytes) 629

Infection Sporozoites

Liver

Circulation

Merozoites

Transmission to mosquito

Asexual cycle

Gametocytes

Plasmodium food vacuole

ADP

Hemoglobin ATP

Proteolytic enzymes Plasmepsins Falcipain Falcilysin

Proton ATPase

H+ Chloroquine

Amino acids

+ Ferriprotoporphyrin IX (heme) Protonated chloroquine

Hemozoin (polymerized heme)

PfCRT

632 Principles of Chemotherapy

cytochromes (large protein complexes involved in electron transport and oxidative phosphorylation). These cytochromes, together with a number of mitochondrial-targeted proteins derived from the plasmodial nuclear genome, make up a rudimentary electron transport chain similar in organization to that found in mammals (Fig. 36-3). In this electron transport chain, integral proteins of the mitochondrial inner membrane are reduced and then oxidized as they transport electrons from one intermediate protein to another. The energy liberated by electron transport is used to drive proton pumping across the mitochondrial membrane, and the energy stored in the proton gradient drives ATP synthesis. In this electron transport chain, oxygen is the final electron acceptor, resulting in the reduction of oxygen to water. Plasmodia derive most of their ATP directly from glycolysis and probably do not use mitochondrial electron transport as a significant source of energy. However, plasmodia do rely on electron transport for the oxidation of key enzymes involved in nucleotide synthesis. For example, dihydroorotate dehydrogenase (DHOD), the enzyme that mediates an early step in pyrimidine synthesis (see Chapter 38, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance), catalyzes the oxidation of dihydroorotate to orotate. As part of this reaction, DHOD is reduced, and the enzyme must be reoxidized before it can continue with another cycle of catalysis. Ubiquinone, an integral membrane protein located near the beginning of the electron transport chain, accepts electrons from reduced DHOD, thus regenerating the oxidized form of DHOD necessary for pyrimidine synthesis.

Dihydroorotate

DHOD (reduced) H+

H+

e-

Outside Mitochondrial membrane

Q

e-

e-

Cyt c

e-

Cyt bc1

Cyt c oxidase

Inside

H+ Atovaquone

FIGURE 36-3.

Pharmacology of Antimalarial Agents The currently available antimalarial agents target four physiologic pathways in plasmodia: heme metabolism (chloroquine, quinine, mefloquine, and artemisinin), electron transport (primaquine and atovaquone), protein translation (doxycycline, tetracycline, and clindamycin), and folate metabolism (sulfadoxine-pyrimethamine and proguanil). The following section discusses the pharmacologic agents that target these pathways. Clinically, antimalarials can be classified into agents used for prophylaxis (to prevent malaria in individuals residing in or traveling through a malaria zone), agents used for treating individuals with acute blood-stage malaria, and agents used to eliminate hypnozoite liver-stage malarial infections. Generally, agents used for prophylaxis must be well tolerated and easy to administer. Inhibitors of Heme Metabolism For many centuries, agents that disrupt intraerythrocytic malarial parasites have been the foundation of antimalarial treatment regimens. Most of these compounds are congeners of quinoline and, as a result, are all believed to possess similar mechanisms of action. Artemisinin, discussed at the end of this section, is also thought to act by inhibiting heme metabolism, although its structure is different from that of the quinolines. Chloroquine

Orotate

DHOD (oxidized)

Because plasmodia depend on de novo pyrimidine synthesis for DNA replication, interrupting the ability of ubiquinone to oxidize DHOD can disrupt plasmodial DNA replication (see below).

2e-+ 2H+ + 1/2O2

H+

H2O

The mitochondrial electron transport chain in plasmodia. The electron transport chain consists of a series of oxidation/reduction steps that culminate in the donation of electrons to oxygen, forming water. In plasmodia, the electron transport chain acts as an electron acceptor for reduced dihydroorotate dehydrogenase (DHOD), an enzyme that is essential for plasmodial pyrimidine synthesis. In this cascade, reduced ubiquinone (Q ) transfers electrons to the cytochrome bc1 complex (Cyt bc1), which then passes electrons to cytochrome c (Cyt c) and, finally, to cytochrome c oxidase (Cyt c oxidase). In a 4-electron reduction of molecular oxygen (shown here as the half-reaction), cytochrome c oxidase donates electrons to oxygen to form water. This chain of electron transfers also involves the pumping of protons across the mitochondrial membrane by Cyt bc1 and Cyt c oxidase; the resulting electrochemical gradient of protons is used to generate ATP (not shown). Atovaquone antagonizes the interaction between ubiquinone and the plasmodial cytochrome bc1 complex, thereby disrupting pyrimidine synthesis by preventing the regeneration of oxidized DHOD.

For the past 2,000 years, humans have used the roots of Dichroa febrifuga or the leaves of hydrangea in the treatment of individuals with malaria. More recently, the bark of the cinchona tree was found to be a more effective remedy. In all these plants, a quinoline compound is the pharmacologically active antiplasmodial agent. Chloroquine, a 4-aminoquinoline, was introduced in 1935 for use in the treatment of malaria. Chloroquine is a weak base that, in its neutral form, freely diffuses across the membrane of the parasite’s food vacuole. Once inside the acidic environment of the vacuole, chloroquine is rapidly protonated, making it unable to diffuse out of the vacuole. As a result, protonated chloroquine accumulates to high concentrations inside the parasite’s food vacuole, where it binds to ferriprotoporphyrin IX and inhibits the polymerization of this heme metabolite. Accumulation of unpolymerized ferriprotoporphyrin IX leads to oxidative membrane damage and is toxic to the parasite. Chloroquine thus poisons the parasite by preventing the detoxification of a toxic product of hemoglobin catabolism (Fig. 36-2). Chloroquine is concentrated by as much as 100-fold in parasitized erythrocytes compared to uninfected erythrocytes. In addition, the concentration of chloroquine required to alkalinize lysosomes of mammalian cells is much higher than that needed to raise the pH in malarial food vacuoles. Therefore, chloroquine is relatively nontoxic to humans, although the drug commonly causes pruritus in darkly pigmented individuals, and it can exacerbate psoriasis and porphyria. Taken in supratherapeutic doses, however, chloroquine can cause vomiting, retinopathy, hypotension,

CHAPTER 36 / Pharmacology of Parasitic Infections 633

confusion, and death. In fact, chloroquine is used globally in suicides each year (largely because it is inexpensive, available, and toxic at high doses), and accidental ingestion by children can be fatal. When initially introduced, chloroquine was a first-line drug used against all types of malaria; however, it is now ineffective against most strains of P. falciparum in Africa, Asia, and South America (Fig. 36-4). Hypotheses regarding the mechanisms responsible for chloroquine resistance are based on the finding that chloroquine-resistant plasmodia accumulate less chloroquine inside food vacuoles than chloroquine-sensitive plasmodia do. In the food vacuole, protonated amino acids are generated by the parasite as it degrades hemoglobin. These protonated amino acids exit the lysosome by means of a transmembrane protein called PfCRT, encoded by pfcrt on P. falciparum chromosome 7. A number of mutations in PfCRT have been associated with chloroquine resistance; for example, a substitution of threonine for lysine at position 76 (K76T) is highly correlated with chloroquine resistance. This mutated PfCRT probably pumps protonated chloroquine out of the food vacuole. This altered pump action could also be detrimental to the parasite, perhaps because of altered amino acid export and/or changes in vacuole pH. Many P. falciparum strains with mutations in pfcrt carry a second mutation in the gene pfmdr1 encoding Pgh1, a food vacuole membrane protein involved in pH regulation. It is speculated that this second mutation provides a “corrective” action that allows chloroquineresistant P. falciparum to continue growth in the presence of a pfcrt mutation. Strains of P. vivax with decreased susceptibility to chloroquine are now reported with increasing frequency in areas of Papua New Guinea, Indonesia, and other focal areas of Oceania and Latin America, although the exact mechanism of decreased susceptibility to chloroquine in these strains has not yet been established. Despite concerns regarding increasing resistance, chloroquine remains the drug of choice for treating most individuals with malaria caused by P. vivax,

Resistance to sulfadoxine/ pyrimethamine

Resistance to chloroquine

P. ovale, P. malariae, and P. knowlesi and by chloroquinesensitive strains of P. falciparum. It can also be used prophylactically to prevent malaria caused by sensitive strains of plasmodia. Quinine and Quinidine

Quinine is an alkaloid that consists of a quinoline ring linked by a secondary carbinol to a quinuclidine ring. Its optical isomer, quinidine, has identical pharmacologic actions. Because of quinine’s structural similarity to other antimalarial quinolines, quinine is thought to attack plasmodia by the mechanism described above. Quinine has also been shown to intercalate into DNA through hydrogen bonding, thus inhibiting DNA strand separation, transcription, and translation. The overall effect is a decrease in the growth and replication of the erythrocytic form of plasmodia. Quinine and quinidine have been used to treat individuals with acute blood-stage malaria but are not used prophylactically. Use of quinine can cause cinchonism, a syndrome that includes tinnitus, deafness, headaches, nausea, vomiting, and visual disturbances. Quinine and quinidine can also prolong the cardiac QT interval (see Chapter 23, Pharmacology of Cardiac Rhythm). Mefloquine

Mefloquine is a quinoline compound that is structurally related to other antimalarial agents. Unlike quinine, mefloquine does not bind to DNA. Its exact mechanism of action is unknown, although mefloquine appears to disrupt polymerization of hemozoin in intraerythrocytic malarial parasites. Mefloquine has a number of adverse effects, including nausea, cardiac conduction abnormalities (including bradycardia, prolongation of the QT interval, and arrhythmia), and neuropsychiatric effects, including vivid dreams/ nightmares, insomnia, anxiety, depression, hallucinations, seizures, and, rarely, psychosis. The mechanism(s) responsible for these adverse effects is unknown. Mefloquine can be used both therapeutically and prophylactically. Strains of

Resistance to sulfadoxine/ pyrimethamine

Resistance to sulfadoxine/ pyrimethamine

Resistance to sulfadoxine/ pyrimethamine, mefloquine, and halofantrine

FIGURE 36-4. Geographic distribution of drug-resistant Plasmodium falciparum. Historically, chloroquine has been the drug of choice for prophylaxis and treatment of individuals with P. falciparum malaria. Unfortunately, P. falciparum is now resistant to chloroquine in most areas of the world (blue shading). In many areas, P. falciparum is also resistant to other antimalarial agents, including sulfadoxine–pyrimethamine, mefloquine, and halofantrine. (Halofantrine is associated with potentially lethal cardiac toxicity and is therefore seldom used.)

634 Principles of Chemotherapy

(WHO) strongly recommends against use of artemisinins as monotherapy. Artemisinins should be used as fixed combinations, usually including a rapidly acting artemisinin and a second agent with a longer half-life (referred to as artemisinin combination therapy [ACT]). Combinations include artemether–lumefantrine, artesunate–mefloquine, artesunate– amodiaquine, and dihydroartemisinin–piperazine. Oral, parenteral, and rectal suppository formulations are available. The WHO now recommends that ACT should be used as the first-line treatment for chloroquine-resistant P. falciparum malaria. In comparison to quinine, artesunate is superior and associated with a decreased risk of death, more rapid parasite clearance, and lower incidence of adverse events. In vitro resistance to artemisinin has been associated with mutations in the parasite calcium pump PfATP6 (see above), and there have been recent reports of relative artemisinin resistance in patients in Asia. Overall, artemisinin and its derivatives are better tolerated than most other antimalarial agents. In laboratory animals, intramuscular injection of oil-based formulations of artemisinin has been shown to cause brainstem neuropathy; this potentially lethal effect has not been observed in humans, but some studies have found evidence suggesting that artemisinins may be associated with auditory impairment and other neurotoxic effects in humans. Hypoglycemia occurs less often than with quinine-based therapy. Safety data in pregnancy are lacking.

P. falciparum resistant to both chloroquine and mefloquine have been reported in areas of Southeast Asia. Artemisinin

Artemisinin, from the wormwood plant Artemisia annua, has been used in China (where it is known as qinghao) for centuries in the treatment of individuals with fever. Artemisinin derivatives have now become the first-line drug for treating individuals with falciparum malaria in many parts of the world. The compound is both a sesquiterpene lactone and a cyclic endoperoxide. When activated by free or heme-bound iron, it forms a carbon-centered free-radical compound (Fig. 36-5). This free radical has the ability to alkylate many proteins as well as heme. The mechanism of specificity of the drug for plasmodia-infected erythrocytes is unknown—potential sources of specificity include artemisinin’s requirement for heme for free radical formation and artemisinin’s preferential accumulation in plasmodia. Drug action may relate to free radical production in the food vacuole of the parasite and subsequent inhibition of PfATP6, the parasite Ca2⫹ ATPase that is the ortholog of the mammalian SERCA calcium pump (see Chapter 24, Pharmacology of Cardiac Contractility). Administration of artemisinin and its derivatives (artesunate, artemether, dihydroartemisinin) is associated with a rapid decrease in the level of malaria parasites in the blood of an infected individual and rapid resolution of symptoms in patients with blood-stage malaria. Unlike many of the other antimalarials, artemisinins affect blood-stage gametocytes, and thus can decrease transmission of malaria from an infected human. Artemisinin is not effective as a prophylactic agent against malaria. Due to the short half-life of artemisinins and the subsequent risk of recrudescence of malaria, and to decrease the likelihood of drug resistance, the World Health Organization

Inhibitors of Electron Transport Although the electron transport chain is a ubiquitous feature of eukaryotic cells, two agents have been developed that appear to selectively interrupt the plasmodial electron transport chain. This selectivity is due to different molecular structures of the same biochemical target, rather than the Drug-heme adduct Fe

Fe

Heme

Activation

Artemisinin

Alkylation

Fe (free or heme-bound)

Artemisinin (free radical or electrophillic intermediate)

H O O

Drug-protein adduct

O H

H O

Protein O

FIGURE 36-5.

Proposed mechanism of action of artemisinin. Artemisinin is a cyclic endoperoxide that forms a free radical after activation by iron (Fe). The mechanism of action of artemisinin is not known with certainty, but may involve alkylation of macromolecules such as heme and proteins, resulting in the formation of artemisinin–heme adducts and artemisinin–protein adducts that are toxic to plasmodia. One such adduct may involve PfATP6, a parasite Ca2⫹ATPase (not shown).

636 Principles of Chemotherapy

an increased risk of antibiotic-associated diarrhea and colitis caused by Clostridium difficile. Clindamycin is not used as a malaria chemoprophylactic. Inhibitors of Folate Metabolism Folic acid is a vitamin involved in the transfer of one-carbon units in a variety of biosynthetic pathways, including those of DNA and RNA precursors and certain amino acids (see Chapter 32). In humans, folate is an essential vitamin and must be ingested in the diet. In parasites and bacteria, folate is synthesized de novo, providing a useful target for selective drug action. Inhibition of folate metabolism can result in successful treatment of parasitic infections. In the context of malaria, antifolate drugs act against parasite-specific isoforms of dihydropteroate synthetase and dihydrofolate reductase. Combination therapies that include a sulfonamide and pyrimethamine are used. Two antimalarial formulations are available, sulfadoxine–pyrimethamine and the less frequently used sulfalene–pyrimethamine. Sulfadoxine–Pyrimethamine

Sulfadoxine is a para-aminobenzoic acid (PABA) analogue that competitively inhibits parasite dihydropteroate synthetase, an essential enzyme in the folic acid synthesis pathway. Pyrimethamine is a folate analogue that competitively inhibits parasite dihydrofolate reductase, the enzyme that converts dihydrofolate to tetrahydrofolate (see Figs. 32-6 and 32-7). In combination, sulfadoxine and pyrimethamine act synergistically to inhibit growth of the malarial parasite. Sulfonamide–pyrimethamine combinations are highly effective against blood schizont stages of P. falciparum malaria, but not against gametocytes, and are less effective against other species of malaria. Both drugs are highly protein-bound, resulting in long elimination half-lives. The long half-life of the combination provides selective pressure for the development of drug resistance in areas with highlevel malaria transmission, and increasing resistance to this combination has made it ineffective for treatment and prophylaxis in many parts of the world (Fig. 36-4). Individuals infected with sensitive strains of malaria may be treated with sulfadoxine–pyrimethamine as a convenient single dose. The most serious drug reactions involve hypersensitivity to the sulfonamide component of the combination. Severe skin reactions such as Stevens–Johnson syndrome or erythema multiforme have been reported, but the incidence of these adverse effects is rare after single-dose therapy for malaria. Adverse hematologic effects include megaloblastic anemia, leukopenia, and thrombocytopenia. Sulfonamide– pyrimethamine is not used as a chemoprophylactic agent against malaria. Proguanil

Proguanil is a derivative of pyrimidine and, like pyrimethamine, is an inhibitor of dihydrofolate reductase. Proguanil acts against the hepatic, pre-erythrocytic forms of P. falciparum and P. vivax. Proguanil has been used for prophylaxis in combination with chloroquine in areas of the world where chloroquine resistance is not widespread. However, other prophylactic agents are significantly more effective, and this combination is not recommended. Proguanil may be used in a synergistic combination with atovaquone for both treatment and prevention of malaria (discussed above). Proguanil is usually well tolerated, but it has been associated

with oral ulcerations, pancytopenia, thrombocytopenia, and granulocytopenia.

Antimalarial Drug Resistance Antimalarial drug resistance is a major public health problem and a significant barrier to the effective treatment of individuals with malaria. In association with the collapse of effective prevention efforts, lack of political will, and socioeconomic factors, the waning efficacy of antimalarial drugs has contributed significantly to the increasing burden of malaria morbidity and mortality worldwide. Chloroquine was the standard therapy for treating individuals with malaria for many years after its introduction in 1946. Resistance was first reported in the 1950s and has steadily increased since then; at present, resistance has been reported everywhere in the world except on the island of Hispaniola and in focal parts of Central America, South America, and Asia. The chloroquine-resistance P. falciparum haplotype has recently been detected in Haiti, but clinical resistance has not yet been reported. The risk of therapeutic failure with chloroquine is over 60% in many areas of Sub-Saharan Africa and over 80% in Southeast Asia. Childhood mortality doubled in Eastern and Southern Africa in the 1980s and 1990s as chloroquine and sulfadoxine–pyrimethamine resistance increased; chloroquine resistance was associated with an overall doubling of childhood mortality from malaria, with increases as high as 11-fold in certain areas. Similarly, P. vivax resistance to chloroquine was unknown until 1989 but is now endemic in Indonesia and Papua New Guinea. Reports of chloroquine-resistant P. vivax have also emerged in South America, Brazil, Myanmar, and India. Resistance to sulfadoxine–pyrimethamine was reported after the combination was introduced in 1971 as a second-line therapy for treating individuals with chloroquine-resistant P. falciparum. Resistance to sulfadoxine–pyrimethamine was initially reported in Southeast Asia but is now relatively widespread in South America and prevalent in Africa as well. Strains of P. falciparum resistant to mefloquine were noted in Southeast Asia following the widespread introduction of this agent in the 1980s. Mefloquine resistance has not spread more widely as yet, in large measure due to the fact that the drug is not now routinely used to treat individuals with malaria. Strains of P. falciparum with relative resistance to artemisinin were reported in Cambodia in 2008. Many factors contribute to the development of drug resistance by malaria parasites, including inappropriate and/or unsupervised drug use, inconsistent drug availability, poor adherence to treatment regimens due to adverse effects and other factors, inconsistent quality of drug manufacturing, presence of counterfeit drugs, and prohibitive drug costs. Combining therapies to reduce the development of resistance is a strategy that has long been employed in the treatment of individuals with tuberculosis, leprosy, and HIV infection, and this approach is strongly recommended in the treatment of individuals with malaria. The World Health Organization (WHO) has demanded cessation of production of all standalone artemisinin products and has requested that only twodrug, fixed-combination, artemisinin-containing products be manufactured. Although rapidly acting artemisinins reduce parasite burden by a factor of 104 with every treatment cycle, resulting in rapid clearance of parasites from the bloodstream, the short half-life of artemisinins favors the possibility of

Nucleus

Excretion in feces

Karyosome

Cyst

Ingestion by human of contaminated food or water

Pseudopodium Encystation in colon

Excystation in small intestine

Nucleus

Vacuole

Trophozoite

Asymptomatic colonization

Intestinal amebiasis

Intestinal perforation and/or liver abscess

PFOR-dependent activation Pyruvate

Ferredoxin

Nitroreductase-dependent activation Reduced metronidazole (active)

PFOR Acetyl CoA

Reduced ferredoxin

ADHE

Ethanol

Acetate

Metronidazole (inactive)

NADP+ Nitroreductase NADPH

Stratum corneum Epidermis Dermis

Adult filaria

Subcutaneous nodule

Subcutaneous space

Fat cells

Microfilaria (in tissues)

Ivermectin

Corneal inflammation with sclerosing keratitis

Dermatitis

CHAPTER 36 / Pharmacology of Parasitic Infections 643

 CONCLUSION AND FUTURE DIRECTIONS The development of new antiparasitic agents will rely on continued exploitation of molecular and metabolic differences between parasites and hosts. Recent advances in the application of molecular biological and genetic techniques to study parasitic eukaryotes, and detailed knowledge of parasite, vector, and host genomes, transcriptomes, and proteomes, should facilitate the development of more selective agents effective against many parasitic infections. The development of resistance to antiparasitic agents is of increasing concern, most notably among malarial and leishmanial parasites, and will necessitate both the judicious use of currently available agents and the development of new agents, including antiparasitic vaccines. Despite long-standing efforts to develop effective treatments for malaria, the disease remains a major global cause of morbidity and mortality. Development of an effective malaria vaccine could have a major impact on this global burden. However, the development of an effective vaccine has been hampered by a number of difficult scientific challenges, including the diversity of parasite species and strains, the diversity of parasite life forms, the intracellular location of the parasites, and the ability of P. falciparum to undergo antigenic variation. The situation has been worsened by the lack of meaningful economic incentives for vaccine

development. Unfortunately, the complexity of the parasites and of their intimate relationship with infected hosts suggests that the development of effective antiparasite vaccines (especially against malaria) will be difficult.

Suggested Reading Anderson VR, Curran MP. Nitazoxanide: a review of its use in the treatment of gastrointestinal infections. Drugs 2007;67:1947–1967. (Reviews antiparasitic and antianaerobic bacterial properties of this interesting agent.) Hoerauf A. Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr Opin Infect Dis 2008;6:673–681. (Reports antibacterial treatment targeting endosymbionts for filarial infections.) Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. Artemisinin Resistance in Cambodia 1 (ARC1) Study Consortium. N Engl J Med 2008;359:2619–2620. (Reports disturbing harbinger of resistance to artemisinin derivatives.) Nosten F, White NJ. Artemisinin-based combination treatment of falciparum malaria. Am J Trop Med Hyg 2007;77(6 Suppl):181–192. (Reviews artemisinin combination treatment of malaria.) Omura S. Ivermectin: 25 years and still going strong. Int J Antimicrob Agents 2008;3:91–98. (Reviews a major antiparasitic agent with a number of uses.) Whitty CJ, Chandler C, Ansah E, Leslie T, Staedke SG. Deployment of ACT antimalarials for treatment of malaria: challenges and opportunities. Malar J 2008;7(Suppl 1):S7. (Addresses challenges of delivering artemisinin combination treatment courses.)

37 Pharmacology of Viral Infections Robert W. Yeh and Donald M. Coen

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 649-650 PHYSIOLOGY OF VIRAL REPLICATION . . . . . . . . . . . . . . . . . 649 Viral Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 651 Inhibition of Viral Attachment and Entry . . . . . . . . . . . . . . 651 Maraviroc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651 Enfuvirtide (T-20) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 Inhibition of Viral Uncoating . . . . . . . . . . . . . . . . . . . . . . . 653 Inhibition of Viral Genome Replication . . . . . . . . . . . . . . . 655 Antiherpesvirus Nucleoside and Nucleotide Analogues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655 Anti-HIV and -HBV Nucleoside and Nucleotide Analogues . . . . . . . . . . . . . . . . . . . . . . 659 Nonnucleoside DNA Polymerase Inhibitors . . . . . . . . . 661

Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . 661 HIV Integrase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 661 Inhibition of Viral Maturation . . . . . . . . . . . . . . . . . . . . . . 662 Inhibition of Viral Release . . . . . . . . . . . . . . . . . . . . . . . . . 664 Antiviral Drugs with Unknown Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Fomivirsen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Docosanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667 Ribavirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667 Drugs That Modulate the Immune System . . . . . . . . . . . . 668 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 669 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669

 INTRODUCTION

antiviral drugs exploit differences between the structures and functions of viral and human proteins to achieve selectivity of antiviral action.

Viral infections are among the leading causes of morbidity and mortality worldwide. Although progress has been made on antiviral drug development, public health measures and prophylactic vaccines remain the primary means by which society controls the spread of viral infections. The persistence of the acquired immunodeficiency syndrome (AIDS) epidemic makes this painfully clear. Despite advances in anti-HIV drug therapies, AIDS continues to be a common cause of death, particularly in some African nations, where as many as one person in five is infected with the human immunodeficiency virus (HIV). This enormous prevalence is largely attributable to failures in public health measures and the lack of an effective vaccine against HIV, in a setting where anti-HIV drugs are too expensive. Despite this bleak statistic, the array of drugs available to combat viruses has been instrumental in saving millions of lives each year and in improving the quality of life for countless others with viral illnesses. This chapter describes the physiology of viral replication and the steps in the viral life cycle that are targeted by current antiviral medications. Key concepts for the chapter include: (1) viruses replicate intracellularly by utilizing host cell machinery; (2) despite this mode of replication, a number of potential targets have been exploited for antiviral drug therapy; and (3) most current

 PHYSIOLOGY OF VIRAL REPLICATION Viruses replicate by co-opting the host cell’s metabolic machinery. As a result, there are fewer differences between viruses and their human hosts to exploit for drug development than between bacteria and humans. It is also more difficult to develop agents that are active against a broad spectrum of viruses than it is against bacteria. This difficulty arises because viruses are a heterogeneous group of infectious agents, whereas most bacteria share a common cell wall structure and distinct transcriptional and translational machinery. Despite these obstacles, all viruses encode proteins that are substantially different from their human counterparts. Additionally, certain host proteins are more important for viral replication than they are for human health. In principle, antiviral drugs could target many of these proteins. In practice, however, relatively few viral proteins and even fewer host proteins have thus far served as useful targets for therapy. Nevertheless, it is a testament to the remarkable progress in antiviral drug development that more viral proteins have been exploited for antiviral therapy than the number of bacterial proteins that have been exploited for antibacterial therapy. 649

CHAPTER 37 / Pharmacology of Viral Infections 651

Virus Receptor Attachment and entry inhibitors Maraviroc Enfuvirtide (T-20)

Attachment and entry Host cell

Ion channel blockers Amantadine Rimantadine

Uncoating

Polymerase inhibitors Acyclovir Zidovudine Efavirenz Integrase inhibitors Raltegravir

Genome replication

RNA synthesis Host ribosome

Protein synthesis Assembly and maturation

Protease inhibitors Saquinavir Ritonavir

Egress and release

Neuraminidase inhibitors Zanamivir Oseltamivir

FIGURE 37-1.

Viral life cycle and pharmacologic intervention. The viral life cycle can be divided into a sequence of individual stages, each of which is a potential site for pharmacologic intervention. Shown is a generic replication cycle of viruses in cells, alongside which are listed the names of drug classes and examples of individual agents that block each stage. Many of the currently approved antiviral agents are nucleoside analogues that target genome replication, typically by inhibiting viral DNA polymerase or reverse transcriptase. Several other drug classes target other stages in the viral life cycle, including attachment and entry, uncoating, assembly and maturation, and egress and release. It should be noted that the details of viral replication differ for each type of virus, often presenting unique targets for pharmacologic intervention and drug development.

typically involves cleavage of viral polyproteins by proteases. For some viruses, maturation occurs within the host cell; for others, such as HIV, it occurs outside the host cell. Viruses egress from the cell either by cell lysis or by budding through the cell membrane. For influenza viruses, the newly formed virions require an additional step of release from the extracellular surface of the host cell membrane. In summary, nearly all viruses replicate via the following stages: attachment, entry, uncoating, genome replication, transcription, translation, assembly, and egress. Some viruses have additional stages such as maturation and release. The stages of retrovirus infection occur in a different order from those of most other viruses, and retroviruses have additional stages in their life cycle. For example, replication of HIV includes the additional stage of integration, in which the viral genome is incorporated into the host genome (Fig. 37-2). Specific host and/or viral proteins are involved in each of these stages. Differences between viral and host proteins at any of these stages can be targeted for antiviral therapy. Different viruses have vastly different arrays of genes. Some, such as hepatitis B virus (HBV), have compact genomes that encode only coat proteins and a few proteins used in gene expression and genome replication. Others, such as

herpesviruses, encode scores of proteins that perform many different functions. The viral proteins that have thus far served as the best targets for antiviral drugs are enzymes involved in genome replication or maturation, although other stages in the viral life cycle can also serve as targets for antiviral agents.

 PHARMACOLOGIC CLASSES AND AGENTS Inhibition of Viral Attachment and Entry All viruses must infect cells to replicate. Therefore, inhibiting the initial stages of viral attachment and entry provides a conceptual “preventive” measure against infection and could limit the spread of virus throughout the body. Two anti-HIV drugs, maraviroc and enfuvirtide (T-20), act at these stages. Both drugs have unusual properties for antiviral drugs: maraviroc targets a host protein rather than a viral protein, and enfuvirtide is a peptide. Maraviroc Maraviroc targets the chemokine receptor CCR5. The development of maraviroc stemmed from clinical studies of individuals who had been exposed repeatedly to HIV, yet did not develop AIDS. It was found that some of these individuals

652 Principles of Chemotherapy 1 Attachment

2 Fusion

HIV

ssRNA gp120 gp41 Protease Matrix protein Integrase Core protein Reverse transcriptase

CD4

Chemokine receptor

3 Reverse transcription DNA Integrase

5 Transcription 4 Integration RNA (genomic and mRNA) Protease

6 Translation

Integrase Core protein Reverse transcriptase

7 Virion assembly and budding

8 Maturation (Protease)

Life cycle of HIV. HIV is a retrovirus that infects CD4⫹ cells. 1. Virus attachment is dependent on binding interactions between viral gp41 and gp120 proteins and host cell CD4 and certain chemokine receptors. 2. Fusion of the viral membrane (envelope) with the host cell plasma membrane allows the HIV genome complexed with certain virion proteins to enter the host cell. 3. Uncoating permits the single-stranded RNA (ssRNA) HIV genome to be copied by reverse transcriptase into double-stranded DNA. 4. The HIV DNA is integrated into the host cell genome, in a reaction that depends on HIV-encoded integrase. 5. Gene transcription and posttranscriptional processing by host cell enzymes produce genomic HIV RNA and viral mRNA. 6. The viral mRNA is translated into proteins on host cell ribosomes. 7. The proteins assemble into immature virions that bud from the host cell membrane. 8. The virions undergo proteolytic cleavage, maturing into fully infective virions. Currently approved anti-HIV drugs target viral attachment and fusion, reverse transcription, integration, and maturation. The development of drug resistance can be significantly retarded by using combinations of drugs that target a single stage (e.g., two or more inhibitors of reverse transcription) or more than one stage in the HIV life cycle (e.g., reverse transcriptase inhibitors and protease inhibitors).

FIGURE 37-2.

have a deletion in the CCR5 gene. Absence of the CCR5 gene product prevents infection by the strains of HIV that are most frequently transmitted between individuals. The deletion otherwise has little negative impact on human health. Drug companies then performed screens for compounds that could prevent binding of chemokines to CCR5 and chemically modified the leading candidate compounds to optimize their pharmacodynamic and pharmacokinetic properties. (Such “target-based screens” had earlier achieved success in the development of anti-HIV nonnucleoside reverse transcriptase inhibitors; see Box 37-2.) Maraviroc, the end result of this process, blocks infection of HIV strains that use CCR5 for attachment and entry (Fig. 37-3). However, maraviroc is not active against HIV strains that use the CXCR4

receptor. It is approved for use in combination with other anti-HIV drugs in patients who have continued detectable viral loads or who have multidrug-resistant virus. Enfuvirtide (T-20) Enfuvirtide is a peptide that is structurally similar to a segment of gp41, the HIV protein that mediates membrane fusion. The proposed mechanism for gp41-mediated membrane fusion and T-20 action is illustrated in Figure 37-3. In the native virion, gp41 is held in a conformation that prevents it from fusing membranes or binding T-20. Attachment of HIV to its cellular receptors triggers a conformational change in gp41 that exposes a segment that can insert into membranes (fusion peptide), a heptad repeat region (HR1), and a second heptad

CHAPTER 37 / Pharmacology of Viral Infections 653

G

CCR5

Maraviroc

F

O

F

NH

N N

N N

Maraviroc CCR5 blocked by maraviroc Host cell plasma membrane

A

CD4

Chemokine receptor

B

Fusion peptide

F

Enfuvirtide (T-20)

HR1

gp120

gp41 HR2

gp41

Intermediate

Viral membrane (envelope)

C

D

Trapped intermediate

E

Hemifusion stalk

Fusion pore

FIGURE 37-3. Model for HIV gp41-mediated fusion and maraviroc and enfuvirtide (T-20) action. A. HIV glycoproteins exist in trimeric form in the viral membrane (envelope). Each gp120 molecule is depicted as a ball attached noncovalently to gp41. B. The binding of gp120 to CD4 and certain chemokine receptors in the host cell plasma membrane causes a conformational change in gp41 that exposes the fusion peptide, heptad-repeat region 1 (HR1) and heptad-repeat region 2 (HR2). The fusion peptide inserts into the host cell plasma membrane. C. gp41 undergoes further conformational changes, characterized mainly by unfolding and refolding of the HR2 repeats. D. Completed refolding of the HR regions creates a hemifusion stalk, in which the outer leaflets of the viral and host cell membranes are fused. E. Formation of a complete fusion pore allows viral entry into the host cell. F. Enfuvirtide (T-20) is a synthetic peptide drug that mimics HR2, binds to HR1, and prevents the HR2–HR1 interaction (dashed arrow). Therefore, the drug traps the virus–host cell interaction at the attachment stage, preventing membrane fusion and viral entry. G. Maraviroc is a small-molecule antagonist of the CCR5 chemokine receptor; the drug blocks cellular infection of HIV strains that use CCR5 for attachment and entry (dashed arrow). The structure of maraviroc is shown. repeat region mimicked by T-20 (HR2). The gp41 then refolds, so that the HR2 segments bind directly to the HR1 segments. If the fusion peptide has properly inserted into the host cell membrane, this refolding brings the virion envelope and the cell membrane into close proximity, allowing membrane fusion to occur (by mechanisms that remain poorly understood). When T-20 is present, however, the drug binds to the exposed HR1 segments and prevents the refolding process, thereby preventing fusion of the HIV envelope with the host cell membrane. Enfuvirtide is approved for use in combination with other anti-HIV drugs in patients whose HIV infection has not been controlled by first-line anti-HIV medications. Because

enfuvirtide is a peptide, it must be administered parenterally, typically by twice daily subcutaneous injections.

Inhibition of Viral Uncoating The adamantanes, amantadine and rimantadine (structures in Fig. 37-4) are inhibitors of viral uncoating that are active exclusively against influenza A virus (and not against influenza B or C viruses). A well-supported model for the mechanism of action of these drugs is diagrammed in Figure 37-4. Influenza virions enter cells via receptor-mediated endocytosis and are

654 Principles of Chemotherapy ADP

Viral membrane NA

H+

M2 HA bound to sialic acid on cell receptor

ATP

CH3 CHNH2

Matrix protein

NH2

Internalized cell receptor RNP Endosomal membrane

Amantadine

Rimantadine

Early endosome

Low pH

Low pH + amantadine or rimantadine

ADP

ADP

Amantadine or rimantadine

H+

H+ Acid-induced dissociation of matrix structure

ATP

H+

H+

H+

ATP

H+

H+

H+

H+ H+

H+

H+ H+

Acid-induced structural change in HA triggers membrane fusion

H+ H+ H+ H+

H+

M2 channel opens to permit entry of protons

H+

H+

RNP released from endosome

Late endosome

FIGURE 37-4.

Uncoating of influenza virus and effect of amantadine and rimantadine. The structures of the adamantanes, amantadine and rimantadine, are shown. Influenza virus enters host cells by receptor-mediated endocytosis (not shown) and is contained within an early endosome. The early endosome contains an H⫹-ATPase that acidifies the endosome by pumping protons from the cytosol into the endosome. A low pH-dependent conformational change in the viral envelope hemagglutinin (HA) protein triggers fusion of the viral membrane with the endosomal membrane. Fusion alone is not sufficient to cause viral uncoating, however. In addition, protons from the low-pH endosome must enter the virus through M2, a pH-gated proton channel in the viral envelope that opens in response to acidification. The entry of protons through the viral envelope causes dissociation of matrix protein from the influenza virus ribonucleoprotein (RNP), releasing RNP and thus the genetic material of the virus into the host cell cytosol. Amantadine and rimantadine block M2 ion channel function and thereby inhibit acidification of the interior of the virion, dissociation of matrix protein, and uncoating. Note that the drug is shown as “plugging” the channel (lower right panel, upper channel graphic); however, there is also evidence that the drug may bind to the outside of the channel instead (lower right panel, lower channel graphic). NA, neuraminidase.

internalized into endosomes (see Chapter 1, Drug–Receptor Interactions). As endosomes acidify because of the action of an endosomal proton pump, two events occur. First, the conformation of the viral envelope protein hemagglutinin changes drastically. This conformational change permits fusion of the influenza virus envelope with the endosome membrane (see the above discussion of HIV-mediated membrane fusion). By itself, this action could liberate viral ribonucleoprotein (including the virion’s RNA genome), but that would not be sufficient to permit its transcription: a second pH-dependent event within the virion is also required. This entails the influx of protons through a proton channel called M2 in the viral envelope, which causes dissociation of the virion matrix protein from the rest of the ribonucleoprotein. Amantadine and rimantadine inhibit the influx of protons through M2. Exactly how this inhibition occurs is not clear. As hydrophobic molecules with a positive charge at one end,

these drugs resemble blockers of cellular ion channels (see Chapters 11 and 23). However, the currently available data are controversial regarding the details of how the adamantanes block the M2 channel. One set of studies has been interpreted to support a model in which the adamantanes simply “plug” (physically occlude) the channel. The other set of studies supports a model in which the drugs bind to the outside of the channel and allosterically prevent it from opening. Amantadine can cause lightheadedness and difficulty concentrating; these adverse effects are likely due to its effects on host ion channels. Indeed, the unintended effects of amantadine on host channels likely account for this drug’s other therapeutic use—the treatment of Parkinson’s disease (see Chapter 13, Pharmacology of Dopaminergic Neurotransmission). Rimantadine is an analogue of amantadine that has a similar antiviral mechanism and has gained much wider use than amantadine in

656 Principles of Chemotherapy A Native nucleosides NH2 N HO

O

N

N

HO

N

O

NH

N

N

O

OH

NH2

N

HO

O

Deoxycytidine

Deoxythymidine

O

O

N O

O

N

NH

HO O

NH

N

N

H2N

NH2

N

N

N

H2N

NH2

N

O

OH

Valacyclovir (prodrug)

OH

Ganciclovir

N

O

Valganciclovir (prodrug) NH2

N

O

N

N

NH

HO

N

N

NH2

O

N

N

NH2

NH2

O

Acyclovir

N

O O

O O

NH

O N

N NH

O

OH

OH

Deoxyguanosine

HO

N O

O

B Antiherpesvirus nucleoside and nucleotide analogues

N

NH

N HO

OH

Deoxyadenosine

O

NH2

N

OH

NH2

N

P

O

O

HO

O

O

OH

O

HO

Famciclovir (prodrug)

Penciclovir

Cidofovir

C Anti-HIV nucleoside and nucleotide analogues O NH HO

N

N

O

HO

O

N

O

NH2 F

N

N

NH HO

O

NH2

NH2

O

O

HO

N

N

O

HO

O

N

S

O

S O

O

N3

Zidovudine (AZT)

Stavudine (d4T)

Zalcitabine (ddC)

Lamivudine (3TC)

Emtricitabine (FTC) NH2 N

O

O

N HO

N

NH

N

N

N

P O

N

O

N

HO

N O

N

NH

NH2

O O

N

O

O O

O

O

Didanosine (ddI)

D Anti-hepatitis B nucleoside and nucleotide analogues NH2 N

Tenofovir disoproxil

Abacavir

E Anti-RNA virus nucleoside analogue O

O N

N

N

N

HO

O P

N

CH2

N

N

NH2

NH N

N O

NH2

O

HO

OH

OH

Adefovir

HO

Entecavir

OH

Ribavirin

HO

FIGURE 37-5. Antiviral nucleoside and nucleotide analogues. A. The nucleosides used as precursors for DNA synthesis are depicted in their anti conformations. Each nucleoside consists of a purine (adenine and guanine) or pyrimidine (cytosine and thymidine) base attached to a deoxyribose sugar. These deoxynucleosides are phosphorylated in stepwise fashion to the triphosphate forms (not shown) for use in nucleic acid synthesis. B. Except for cidofovir, the antiherpesvirus nucleoside and nucleotide analogues are structural mimics of deoxyguanosine. For example, acyclovir consists of a guanine base attached to an acyclic sugar. Cidofovir, which mimics the deoxynucleotide deoxycytidine monophosphate, uses a phosphonate (C–P) bond to mimic the physiologic P–O bond of the native nucleotide. Valacyclovir, famciclovir, and valganciclovir are more orally bioavailable prodrugs of acyclovir, penciclovir, and ganciclovir, respectively. C. Anti-HIV nucleoside and nucleotide analogues mimic a variety of endogenous nucleosides and nucleotides and contain variations not only in the sugar but also in base moieties. For example, AZT is a deoxythymidine mimic that has a 3⬘-azido group in place of the native 3⬘-OH. Stavudine, zalcitabine, and lamivudine also contain modified sugar moieties linked to natural base moieties. Tenofovir, which is shown as its prodrug tenofovir disoproxil, is a phosphonate analogue of deoxyadenosine monophosphate. Of the analogues that contain modified base moieties, didanosine mimics deoxyinosine and is converted to dideoxyadenosine, while emtricitabine contains a fluoro-modified cytosine and abacavir contains a cyclopropyl-modified guanine. D. Adefovir is a phosphonate analogue of the endogenous nucleotide deoxyadenosine monophosphate, while entecavir is a deoxyguanosine analogue with an unusual moiety substituting for deoxyribose. These two compounds and lamivudine (see panel C) are approved for use in the treatment of HBV infection. E. Ribavirin, which contains a purine mimic attached to ribose, is approved for use against the RNA viruses HCV and RSV.

CHAPTER 37 / Pharmacology of Viral Infections 657

A

O

O N

N HSV or VZV thymidine kinase

NH

N

N

NH

N

N

OH HO

NH2

NH2

O P

HO O

O

O Acyclovir monophosphate

Acyclovir

Cellular kinase

O N

OH O

N

OH O

N

NH

N OH HO

O

N

NH2

P

P

O

O

O

OH

Cellular kinase

O

P

NH

HO

O

Acyclovir triphosphate (pppACV)

N

OH O

NH2

O

P

P

O

O

O Acyclovir diphosphate

Viral DNA polymerase

B

pppACV

ACV

ACV

pppdG pppdC dC dG

1

dC

2 Binding of pppACV to viral DNA polymerase competes for binding of pppdG.

dC

dG

dG

3 ACV is incorporated into growing DNA chain, blocking further chain growth.

When the next deoxynucleoside triphosphate binds, viral DNA polymerase is "frozen."

FIGURE 37-6.

Mechanism of action of acyclovir. A. Acyclovir is a nucleoside analogue that is selectively phosphorylated by HSV or VZV thymidine kinase to generate acyclovir monophosphate. Host cellular enzymes then sequentially phosphorylate acyclovir monophosphate to its diphosphate and triphosphate (pppACV) forms. B. Acyclovir triphosphate has a three-step mechanism of inhibition of herpesvirus DNA polymerase in vitro: (1) it acts as a competitive inhibitor of dGTP (pppdG) binding; (2) it acts as a substrate and is base-paired with dC in the template strand to become incorporated into the growing DNA chain, causing chain termination; and (3) it traps the polymerase on the ACV-terminated DNA chain when the next deoxyribonucleoside triphosphate (shown here as dCTP, or pppdC ) binds.

Next, ACV triphosphate acts as a substrate and is incorporated into the growing DNA chain opposite a C residue. The polymerase translocates to the next position on the template but cannot add a new deoxyribonucleoside triphosphate because there is no 3⬘-hydroxyl on ACV triphosphate; hence, ACV triphosphate is also a chain terminator. Finally, provided that the next deoxyribonucleoside triphosphate is present, the viral polymerase freezes in a “dead-end complex,” leading to apparent inactivation of the enzyme (Fig. 37-6B). (The mechanism of polymerase “freezing” remains unknown.) Interestingly, cellular DNA polymerase ␣ does not undergo inactivation to the dead-end complex. It is not yet known whether the

inactivating step is important in vivo, or whether ACV incorporation and chain termination are sufficient to inhibit viral replication. Regardless, studies of ACV-resistance mutations in the viral DNA polymerase gene show that the effects of ACV triphosphate on viral polymerase constitute a major component of acyclovir selectivity. All acyclovir-resistant mutants studied to date contain mutations in the thymidine kinase (TK) gene, the DNA polymerase gene, or both. Because TK is not essential for virus replication in cell culture, mutations that completely or partially inactivate the enzyme do not prohibit virus replication. Also, some TK mutations render the enzyme incapable of

658 Principles of Chemotherapy

phosphorylating acyclovir while permitting the phosphorylation of thymidine. Because DNA polymerase is essential for virus replication, resistance mutations do not inactivate but instead only alter this enzyme, so that higher concentrations of ACV triphosphate are required to inhibit the enzyme. Clinically, acyclovir-resistant HSV is mainly a problem in immunocompromised hosts. In animal models of HSV infection, acyclovir-resistant mutants are frequently found to have reduced pathogenicity, but the degree of attenuation depends greatly on the type of mutation. These studies suggest that there are multiple mechanisms by which the virus can mutate to retain both drug resistance and pathogenicity. Valacyclovir is a prodrug form of acyclovir that has approximately fivefold greater oral bioavailability than acyclovir (Fig. 37-5). This compound, which contains an acyclovir structure covalently attached to a valine moiety, is rapidly converted to acyclovir after oral administration.

ganciclovir in infected cells compared to uninfected cells. Ganciclovir triphosphate inhibits CMV DNA polymerase more potently than it does cellular DNA polymerases. Thus, as with acyclovir and HSV, ganciclovir is selective against CMV at two steps: phosphorylation and DNA polymerization. However, the selectivity against CMV at each step is not as great as the selectivity of acyclovir against HSV; accordingly, the drug is more toxic than acyclovir. Toxicity is most commonly manifested as bone marrow suppression, especially neutropenia. Ganciclovir resistance is a clinical problem in a substantial fraction of patients. Valganciclovir is a prodrug form of ganciclovir that has greater oral bioavailability than ganciclovir. Valganciclovir is a valine ester of ganciclovir, making the relationship between valganciclovir and ganciclovir similar to that between valacyclovir and acyclovir (Fig. 37-5).

Famciclovir and Penciclovir

Also known as hydroxyphosphonylmethoxypropylcytosine (HPMPC), this phosphonate-containing acyclic cytosine analogue represents a twist on the mechanism of action of antiherpesvirus nucleoside analogues. Indeed, cidofovir can be considered a nucleotide rather than a nucleoside analogue. With its phosphonate group, cidofovir mimics deoxycytidine monophosphate; thus, in effect, it is already phosphorylated (Fig. 37-5). Therefore, cidofovir does not require viral kinases for its phosphorylation, and, accordingly, it is active against kinase-deficient viral mutants that are resistant to ganciclovir. Although cidofovir structurally resembles a phosphorylated compound, this drug enters cells with reasonable efficiency. It is further phosphorylated (twice) by cellular enzymes to yield an analogue of dCTP, which inhibits herpesvirus DNA polymerases more potently than cellular DNA polymerases. Selectivity has been confirmed by mapping cidofovir-resistance mutations to the DNA polymerase gene in CMV. Cidofovir is approved for use in the treatment of CMV retinitis in patients with HIV/AIDS. Cidofovir diphosphate has a long intracellular half-life. Therefore, its use requires relatively infrequent dosing (only once each week or less). Because of its mechanism of renal clearance, cidofovir must be co-administered with probenecid. (Probenecid inhibits a proximal tubule anion transporter and thereby decreases cidofovir excretion.) Nephrotoxicity is a major problem, and great care must be taken in administering this drug. Two related phosphonate-containing drugs are the acyclic deoxyadenosine monophosphate analogues tenofovir and adefovir (Fig. 37-5). Tenofovir, which was approved as an anti-HIV drug in 2001, can be administered just once each day, an important advantage for HIV-infected individuals who must comply with complex combination chemotherapy regimens. Adefovir was approved as an anti-HBV drug in 2002. The mechanisms of action of these drugs against their respective viruses are similar to that of cidofovir against CMV. (See discussions below of HIV and HBV replication, and of other drugs active against these viruses.)

Cidofovir

Famciclovir (Fig. 37-5) is the diacetyl 6-deoxy analogue of penciclovir, the active form of the drug. Famciclovir is well absorbed orally and subsequently modified by an esterase and an oxidase to yield penciclovir. In humans, this results in approximately 70% oral bioavailability. Like acyclovir, the structure of penciclovir consists of a guanine linked to an acyclic sugar-like molecule that lacks a 2' CH2 moiety. Penciclovir’s mechanism of action is similar to that of acyclovir (Fig. 37-6), with only quantitative differences detected by both biochemical assays and analyses of resistant mutants. Penciclovir is more efficiently activated by HSV and VZV TK than is acyclovir, but penciclovir triphosphate is a less selective inhibitor of the viral DNA polymerases than is ACV triphosphate. Famciclovir is used in the treatment of HSV infections and shingles (which is caused by reactivation of VZV), and penciclovir ointment is used to treat cold sores caused by HSV. Ganciclovir

Human CMV infections are inapparent in most adults, but CMV can cause life-threatening diseases such as pneumonia or sight-threatening retinitis in immunocompromised individuals. CMV is much less sensitive to acyclovir than are HSV and VZV, primarily because much less phosphorylated acyclovir accumulates in CMV-infected cells than in HSVor VZV-infected cells. Ganciclovir is a nucleoside analogue that was originally synthesized as a derivative of acyclovir with the intention of developing another anti-HSV drug, but it proved too toxic for that indication. It turned out, however, that ganciclovir is much more potent than acyclovir against CMV, and ganciclovir was the first antiviral drug approved for use against CMV. Like acyclovir, ganciclovir contains a guanine linked to an acyclic sugar-like molecule that lacks a 2' moiety. However, ganciclovir contains the 3' CHOH group that is missing in acyclovir (Fig. 37-5). Thus, ganciclovir more closely resembles the natural compound, deoxyguanosine, and this resemblance may account for its greater toxicity. (In fact, ganciclovir is so toxic that it should be used only for serious infections.) CMV does not encode a homolog of the HSV TK (which phosphorylates ganciclovir very efficiently). However, genetic studies have revealed the existence of a viral protein kinase called UL97 that phosphorylates ganciclovir, leading to a 30-fold increase in the amount of phosphorylated

Other Antiherpesvirus Nucleoside Analogues

Several other nucleoside analogues with antiherpesvirus activity were developed and approved before the development of acyclovir. These agents have greater toxicity than acyclovir, and so are not widely used, but are listed in the Drug Summary Table.

CHAPTER 37 / Pharmacology of Viral Infections 661

is a potent inhibitor of the HBV polymerase. Two other antiHBV drugs are adefovir and entecavir (Fig. 37-5). Nonnucleoside DNA Polymerase Inhibitors Nucleoside analogues can inhibit cellular as well as viral enzymes. As a result, efforts have been made to discover compounds with different structures that can selectively target viral enzymes. The first such compound to be used clinically was foscarnet (phosphonoformic acid [PFA]; Fig. 37-7). Foscarnet inhibits both DNA and RNA polymerases encoded by a wide variety of viruses. It has a relatively broad spectrum of activity in vitro (including against HIV), but clinically, it is used for certain serious HSV and CMV infections where therapy with acyclovir or ganciclovir has not succeeded (e.g., because of resistance). It should also be noted that some acyclovir-resistant and ganciclovir-resistant polymerase mutants exhibit at least moderate resistance to foscarnet. Mechanistically, foscarnet differs from nucleoside analogues in that it does not require activation by cellular or viral enzymes; rather, foscarnet inhibits viral DNA polymerase directly by mimicking the pyrophosphate product of DNA polymerization. Selectivity results from the increased sensitivity of viral DNA polymerase relative to cellular enzymes; this biochemical result was confirmed by the existence of foscarnet-resistant DNA polymerase mutants. As might be expected of a compound that so closely mimics a natural compound (pyrophosphate), foscarnet’s selectivity is not as high as acyclovir’s; it inhibits cell division at concentrations not much higher than its effective antiherpesvirus concentration. Major drawbacks to foscarnet use include its lack of oral bioavailability and its poor solubility; renal impairment is its major dose-limiting toxicity. Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) The nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz, nevirapine, delavirdine, and etravirine were developed using the rational approach of target-based, highthroughput screening (Box 37-2 and Fig. 37-7). Indeed, the NNRTIs were among the first successes of this now widely used approach. Unlike the nucleoside analogues, these drugs inhibit their target directly, without the need for chemical modification. X-ray crystallographic studies have shown that NNRTIs bind near the catalytic site of RT. NNRTIs permit RT to bind a nucleoside triphosphate and primer template, but inhibit the joining of the two. The NNRTIs are orally bioavailable, and their adverse effects (most commonly, rash) are typically less serious than those of foscarnet and most nucleoside analogues. The main limitation of NNRTI use is that resistance develops rapidly, requiring the use of these drugs in combination with other anti-HIV drugs (Box 37-1). One NNRTI, efavirenz, was the first anti-HIV drug to be taken once a day. In 2006, a single pill combining efavirenz, tenofovir, and FTC was approved by the FDA for once-a-day administration. HIV Integrase Inhibitors Integrase, the enzyme that carries out HIV genome integration, is an essential enzyme for HIV genome replication. Integrase assembles onto sequences at the ends of HIV DNA, cleaves dinucleotides from each 3' strand, transfers these strands to target (cellular) DNA, and covalently ligates the HIV DNA to target DNA (Fig. 37-8). Scientists developed an assay for inhibition of the DNA strand transfer reaction of integrase, and this assay was used to screen for active compounds.

O HN

O P

OH

N

N

N

HO OH O Foscarnet

Nevirapine

NH2

N

Br

N N H

N

O

Etravirine

N

N

N O

H N

HN

N

S O

N H

O

Delavirdine

F3C Cl O

N H

O

Efavirenz

FIGURE 37-7.

Nonnucleoside DNA polymerase and reverse transcriptase inhibitors. Foscarnet is a pyrophosphate analogue that inhibits viral DNA and RNA polymerases. Foscarnet is approved for the treatment of HSV and CMV infections that are resistant to antiherpesvirus nucleoside analogues. The nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz, nevirapine, delavirdine, and etravirine inhibit HIV-1 reverse transcriptase. The NNRTIs are approved in combination with other antiretroviral drugs for the treatment of HIV-1 infection. Note that the structures of the NNRTIs are significantly different from those of the anti-HIV nucleoside and nucleotide analogues (compare with Fig. 37-5).

CHAPTER 37 / Pharmacology of Viral Infections 663

A

HIV DNA CAGT3' GTCA5'

5'ACTG 3'TGAC

5'LTR

3'LTR 3' end processing

CA3' GTCA5'

5'ACTG 3'AC

5'LTR

3'LTR Strand transfer

Raltegravir

Target (cellular) DNA p VWXYZ VWXYZ p

5

'A C TG VWXYZAC

CAVWXYZ GTCA

5'LTR

3'LTR

5'

Repair/ligation

VWXYZTG VWXYZAC

CAVWXYZ GTVWXYZ

5'LTR

3'LTR

B D

N-terminal zincfinger domain

D

E

Core domain

C-terminal domain

O

C

N

O-K+ N

F

N H N

O

H N

N O

O

Raltegravir

FIGURE 37-8.

Integration of HIV DNA into cellular DNA and effect of anti-HIV integrase inhibitor. A. Schematic rendering of the action of HIV integrase. Double-stranded HIV DNA is generated by reverse transcription as a blunt-ended, linear molecule with repeated sequences known as long terminal repeats (LTR) at both ends. The 5⬘ LTR includes the promoter/enhancer for HIV transcription, and the 3⬘ LTR includes the polyadenylation signal. At the termini of both LTRs are identical sequences of four base pairs. In the first step of integration (3⬘ end processing), HIV integrase removes the two terminal nucleotides from the 3⬘ strands from both ends of the viral DNA, resulting in two-base (AC), 5⬘ overhangs. In the second step (strand transfer), integrase creates a staggered cleavage of host DNA, and then catalyzes the attack of the 3⬘ OH ends of the viral DNA on phosphodiester bonds in the host DNA, resulting in the formation of new phosphodiester bonds linking host and viral DNA at both ends of the viral genome. The AC overhang of viral DNA is not joined, and the process also results in single stranded gaps in the host DNA on each side of the viral genome. This leads to the third step (repair/ligation), in which the AC overhangs are removed and the gaps in host DNA filled in, creating a short duplication of host sequences on either side of the integrated viral DNA. Raltegravir inhibits the strand transfer reaction. B. Domain structure of an HIV integrase monomer. Raltegravir binds at the active site in the catalytic core domain and inhibits the strand transfer reaction. The catalytic triad Asp-64, Asp-116, and Glu-152 is shown as D-D-E in the core domain. C. Structure of raltegravir.

664 Principles of Chemotherapy NH2 O O

H

O

HN

O S O

H N

N

N

N H NH2 OH

O

OH

O

O

Amprenavir

HN

N

O

OH

O

Lopinavir

O N

N

S

O

O N H

O

NH

Indinavir

H N

N H

OH

H N

N

O

N H

O

OH

N

O

H N

N H

Saquinavir

N O

H

N

HO

S

O

N H

N

N

OH

H N

O

S

H

OH H

Ritonavir

Nelfinavir

N OH

OH

O H3CO

N H

H N O

O N

N H

O

H N

OCH3

O NH SO2

O N F 3C

Atazanavir

Tipranavir

FIGURE 37-9. Anti-HIV protease inhibitors. Shown are the structures of the anti-HIV protease inhibitors amprenavir, saquinavir, lopinavir, indinavir, ritonavir, nelfinavir, atazanavir, and tipranavir. These compounds mimic peptides (peptidomimetics), and all but tipranavir contain peptide bonds. Two additional anti-HIV protease inhibitors, darunavir and fosamprenavir (a prodrug form of amprenavir), are not shown.

Inhibition of Viral Release Rational design has also led to the development of inhibitors of influenza virus neuraminidases. The rationale for these inhibitors, which block viral release from the host cell, follows from the mechanism of viral attachment and release. Influenza virus attaches to cells via interactions between hemagglutinin, a protein on the viral envelope, and sialic acid moieties, which are present on many cell surface glycoproteins. Upon egress of influenza virus from cells at the end of a round of replication, the hemagglutinin on nascent virions again binds to the sialic acid moieties, thereby tethering the virions to the cell surface and preventing viral release. To overcome this problem, influenza virus encodes an envelope-bound enzyme, called neuraminidase, which cleaves sialic acid from the membrane glycoproteins and thereby permits release of the virus. Without

neuraminidase, the virus remains tethered and cannot spread to other cells. In 1992, the structure of the neuraminidase–sialic acid complex was solved. The structure showed that sialic acid occupies two of three well-formed pockets on the enzyme. Based largely on this structure, a new sialic acid analogue was designed to maximize energetically favorable interactions in all three of the potential binding pockets (Fig. 37-11). This compound, now known as zanamivir, inhibits neuraminidase with a Ki of about 0.1 nM. Zanamivir is active against both influenza A and influenza B, with potencies of about 30 nM. Studies of resistant mutants confirm the mechanism of action described above. However, zanamivir has poor oral bioavailability and must be administered by inhaler. Efforts to improve on zanamivir’s pharmacokinetics resulted in a new drug, oseltamivir (Fig. 37-11), whose oral

666 Principles of Chemotherapy A

O

O

H N Ile

H N

Leu Asn

H N

Leu

N

HO

Asn

Protease attack

H N

O OH

Ile

N

Rotational axis of symmetry P-3 P-2

P-1 (phe)

P1 (pro) P2 Model of transition state on substrate sequence

pol Substrate sequence

B OH

OH H2N

H N

Cbz

NH2

H N

Val

OH

Cbz Val

O

H N

Cbz

Val N H

Val

N H

Cbz

OH

A-74702 Protease IC50 > 200 µM

A-74704 Protease IC50 = 5 nM Antiviral activity < 1 µM

O

A-75925 Protease IC50 < 1 nM Antiviral activity < 1 µM Poor aqueous solubility

OH

O

H N

N N

H N

N H

N

N H O

N

OH

O

A-77003 Protease IC50 < 1 nM Antiviral activity = 0.1 µM Good solubility Poor oral bioavailability

O N S

O H N

N

N H

N H O

OH

O

S N

Ritonavir Protease IC50 < 1 nM Antiviral activity = 25 nM Fair solubility Good oral bioavailability

FIGURE 37-10.

Steps in the evolution of ritonavir. A. The HIV pol gene product has a phenylalanine (Phe)-proline (Pro) sequence that is unusual as a cleavage site for human proteases. HIV protease cleaves this Phe-Pro bond. The transition state of the protease reaction includes a rotational axis of symmetry. B. Structure-based development of a selective HIV protease inhibitor began with a compound (A-74702) that contained two phenylalanine analogues and a CHOH moiety between them. This compound, which had weak inhibitory activity, was then modified to maximize antiprotease activity while also maximizing antiviral activity, aqueous solubility, and oral bioavailability. The maximization of antiprotease activity was measured as a progressive reduction in IC50, the drug concentration required to cause 50% inhibition of the enzyme. See Box 37-3 for details.

CHAPTER 37 / Pharmacology of Viral Infections 667

A

HO

B

HO HO

HO

O

H N

OH

OH

O NH H2N

O

Sialic acid

H2N O

Zanamivir

Glycerol Carboxylate

Oseltamivir

Hydrophobic group

Glycerol Carboxylate

Guanidino

Hydroxyl

Active site of neuraminidase

O

H N

HN O

Sialic acid

O

O

H N

HO

C

COOH

COOH

OH

Zanamivir

Carboxylate

Hydrophobic pocket

GS4071 (active metabolite of prodrug oseltamivir)

FIGURE 37-11. Structure-based design of neuraminidase inhibitors. A. Shown is a model of sialic acid (space-filling structure) bound to the influenza A virus neuraminidase, with the amino acids that bind sialic acid depicted in stick form. This structure was used to design transition state analogues that bind more tightly to neuraminidase than sialic acid does, resulting in potent inhibitors of the enzyme. B. Structures of sialic acid and the neuraminidase inhibitors zanamivir and oseltamivir. C. Schematic diagram of the active site of influenza virus neuraminidase, depicting the binding of sialic acid, zanamivir, and GS4071 to several different features of the active site. (Oseltamivir is the ethyl ester prodrug of GS4071.)

Docosanol n-Docosanol is a 22-carbon saturated alcohol with activity against HSV and certain other enveloped viruses. Although shorter chain saturated alcohols have long been known to inactivate virion infectivity, but also exhibit cytotoxicity, docosanol has been reported to lack significant cytotoxicity. Cell culture studies suggest that docosanol acts, at least in part, between the stage of HSV attachment and the stage of viral protein translation, with some effects on virus entry at certain doses. Cells must be pretreated with docosanol for hours for an antiviral effect to become manifest, and there is evidence that, during this time, docosanol is metabolized and incorporated into host cell membranes. However, it is

not clear what basis, if any, exists for selectivity of antiviral action; no docosanol-resistant mutants have been reported that could shed light on the drug’s mechanism of action. Docosanol is FDA-approved as an over-the-counter topical treatment for recurrent oral–facial HSV episodes (cold sores), although its clinical efficacy is controversial, as is the relationship of any such efficacy to an antiviral effect. Ribavirin Ribavirin has been touted as a “broad-spectrum antiviral” and, indeed, it exhibits activity against many viruses in vitro and efficacy against several in vivo. In patients, however, ribavirin has been approved only in aerosol form (in effect,

668 Principles of Chemotherapy

topical application to the lungs) for severe respiratory syncytial virus (RSV) infection, and only in combination with an interferon for chronic hepatitis C virus (HCV) infection. Structurally, ribavirin differs from the other nucleoside analogues in that it has a natural sugar moiety (ribose) attached to a nonnatural base-like moiety that most resembles purines (adenine or guanine) (Fig. 37-5). Its mechanism of action is still not well understood. Ribavirin is converted to a monophosphate by cellular adenosine kinase and is known to inhibit cellular inosine monophosphate dehydrogenase, thereby lowering cellular GTP pools (see Chapter 38). That this mechanism should confer selective antiviral activity might seem unlikely at first, although there is some support for this notion from studies of certain viral mutants. It is possible that particular viral enzymes, such as the enzyme that adds 7-methylguanosine caps to mRNA, have higher Km values (and thus lower affinities) for GTP than do most cellular enzymes. Hence, lowering intracellular GTP concentrations below the Km values of these viral enzymes could have a selective antiviral effect. Inhibition of viral RNA polymerase could represent a second possible selective mechanism for ribavirin action. Interestingly, both ribavirin diphosphate and ribavirin triphosphate have inhibitory activity against the RNA polymerases of certain viruses. A third possible mechanism also involves viral RNA polymerase. The error-prone nature of this enzyme leads to high mutation rates, and ribavirin has been shown to increase the mutation rates of several viruses (including HCV) when studied in an in vitro replication system. The increased mutation rates are thought to be caused by incorporation of ribavirin into RNA (without chain termination), although ribavirin’s effects on GTP pools could also contribute. The proposed mechanism, called “error catastrophe,” postulates that the increased mutation rate pushes the already high error rate of the polymerase “over the edge” of an “error threshold,” so that few or no functional viral genomes are produced. This concept is interesting but controversial. For example, mutations that cause replication of HCV RNA to become resistant to ribavirin have not been found in the viral RNA polymerase gene. Whether any of the proposed mechanisms of ribavirin action are relevant for the therapeutic effect of the drug on human RSV or HCV infections is not known. Indeed, for HCV, it is possible that some of the therapeutic effects of ribavirin are mediated by the immune system. Learning more about the mechanisms of ribavirin action may lead to improved antiviral therapies.

Drugs That Modulate the Immune System Three classes of drugs that make explicit use of host immune processes are used to treat viral infections. These classes include immunization, interferons, and imiquimod. For background on the immune system, see Chapter 41, Principles of Inflammation and the Immune System. Active and passive immunization inhibit viral infection by providing antibodies against viral envelope proteins; these antibodies then block the attachment and penetration of virions into cells and increase virion clearance. Some antibodies are directly virucidal, causing virions to be destroyed or inactivated before the virus can interact with its

receptor(s) on target cells. There are, of course, many vaccines that are examples of active immunization against viruses (e.g., measles, mumps, rubella, hepatitis B), and most of these vaccines are used prophylactically. One example of a vaccine used therapeutically is rabies vaccine, which can save the lives of individuals who are already infected with rabies virus. Examples of passive immunization are the prophylactic use of either pooled human immune globulins with anti-RSV activity or a humanized monoclonal antibody, palivizumab, to prevent RSV infection in highrisk children. The interferons and imiquimod make use of the innate immune response (see Chapter 41) and do not directly target viral gene products. Interferons were first recognized as proteins that are produced in response to virus infection and that can inhibit replication of the same or other viruses. There are two major types of interferons. Type I interferons include interferon-␣ and interferon-␤, which are produced by many cell types and interact with the same cell-surface receptor. Type II interferons include interferon-␥, which is typically produced by cells of the immune system, especially T cells, and interacts with a different receptor. Interaction of interferons with their receptors induces a series of signaling events that activate and/or induce the expression of proteins that combat viral infections. One relatively well understood example of such a protein is a protein kinase, called PKR, which is activated by double-stranded RNA. (Double-stranded RNA is often produced during viral infections.) PKR phosphorylates a component of the host translational machinery, thereby turning off protein synthesis and thus the production of virus in infected cells. Interferon-␣ is used as a therapeutic agent in the treatment of HCV, HBV, condyloma acuminata (which is caused by certain human papillomaviruses [HPVs]), and Kaposi’s sarcoma (which is caused by Kaposi’s sarcoma-associated herpesvirus [KSHV], also known as human herpesvirus 8). Interferon-␣ is usually administered in a form that has been modified with polyethylene glycol (pegylated) to improve its pharmacokinetic profile following injection. Although the mechanism by which interferons inhibit the replication of certain viruses is reasonably well understood (e.g., by inducing PKR), the mechanisms by which interferons act against HCV, HBV, HPVs, and KSHV remain poorly understood. Interestingly, all of these viruses encode proteins that inhibit interferon action. Understanding the mechanism of this inhibition may aid understanding of the action of interferons in inhibiting viral replication. This is an active area of investigation. Interferon-␣ is also used to treat certain relatively rare malignancies, and interferon-␤ is used to treat multiple sclerosis. Again, the mechanisms by which interferons exert their therapeutic effects in these clinical settings are poorly understood. Imiquimod is approved for the treatment of certain diseases caused by HPVs. Imiquimod interacts with the Tolllike receptors TLR7 and TLR8 to boost innate immunity, including the secretion of interferons. Toll-like receptors are membrane proteins that recognize pathogen-associated molecular patterns. Activation of Toll-like receptors induces intracellular signaling events that are important for defense against pathogens. In the case of imiquimod, it is not clear exactly how this stimulation results in effective treatment of disease caused by HPV.

CHAPTER 37 / Pharmacology of Viral Infections 669

 CONCLUSION AND FUTURE DIRECTIONS The various stages in the viral life cycle provide a basis for understanding the mechanisms of action of currently available antiviral drugs and for developing new antiviral therapies. The vast majority of antiviral drugs available today inhibit viruses at the genome replication stage, by taking advantage of structural and functional differences between viral and host polymerases. In addition, maraviroc and enfuvirtide (T-20) inhibit viral attachment and entry, adamantanes inhibit viral uncoating, protease inhibitors inhibit viral maturation, and neuraminidase inhibitors inhibit viral release. It is important to bear in mind, however, that many of these drugs inhibit only one virus (e.g., HIV), and, in some cases, only one species of that virus (e.g., HIV-1 but not HIV-2). Only a tiny fraction of viruses known to cause human disease can be treated effectively with the antiviral therapies that are currently available. Nevertheless, great strides have been made. At this writing, new specific anti-HCV drugs are under review at the FDA. In the case of Mr. M, the treatment of HIV with a combination of drugs could reduce viral loads to undetectable levels and delay the progression of AIDS for many years. Although antiviral therapies do not yet represent

either prevention or cure for this disease, such therapies have already decreased the morbidity and mortality of HIV/AIDS in millions of individuals.

Suggested Reading Coen DM, Richman DD. Antiviral agents. In: Knipe DM, Howley PN, Griffin DE, et al., eds. Fields virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. (Detailed review of the general and specific aspects of the mechanisms and uses of antiviral drugs.) Hay AJ, Wolstenholme AJ, Skehel JJ, et al. The molecular basis of the specific anti-influenza inhibition of amantadine. EMBO J 1985;4:3021–3024. (This classic paper illustrates how viral genetics can be used to identify a drug target.) LaBranche C, Galasso G, Moore JP, et al. HIV fusion and its inhibition. Antiviral Res 2001;50:95–115. (Summarizes the understanding of HIV fusion and includes a discussion of fusion inhibitors under investigation.) von Itzstein M, Wu WY, Kok GB, et al. Rational design of potent sialidasebased inhibitors of influenza virus replication. Nature 1993;363:418–423. (Describes the structure-based design of zanamivir.) Yazdanpanah Y, Sissoko D, Egger M, et al. Clinical efficacy of antiretroviral combination therapy based on protease inhibitors or non-nucleoside analogue reverse transcriptase inhibitors: indirect comparison of controlled trials. Br Med J 2004;328:249–256. (Reviews combination therapies used in the treatment of HIV.)

38 Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance David A. Barbie and David A. Frank

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 674-675 BIOCHEMISTRY OF GENOME SYNTHESIS, STABILITY, AND MAINTENANCE . . . . . . . . . . . . . . . . . . . . . 675 Nucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675 Purine Ribonucleotide Synthesis . . . . . . . . . . . . . . . . . 675 Pyrimidine Ribonucleotide Synthesis . . . . . . . . . . . . . . 675 Ribonucleotide Reduction and Thymidylate Synthesis . . . 676 Nucleic Acid Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 DNA Repair and Chromosome Maintenance . . . . . . . . . . . 676 Mismatch Repair. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678 Base Excision Repair. . . . . . . . . . . . . . . . . . . . . . . . . . 678 Nucleotide Excision Repair . . . . . . . . . . . . . . . . . . . . . 679 Double-Strand Break Repair . . . . . . . . . . . . . . . . . . . . 679 Telomere Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681 Microtubules and Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . 682 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 683 Inhibitors of Thymidylate Synthase . . . . . . . . . . . . . . . . . . 684 Inhibitors of Purine Metabolism . . . . . . . . . . . . . . . . . . . . 684

Inhibitors of Ribonucleotide Reductase . . . . . . . . . . . . . . . 685 Purine and Pyrimidine Analogues That Are Incorporated into DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 Agents That Directly Modify DNA Structure . . . . . . . . . . . 686 Alkylating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 Platinum Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 689 Bleomycin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690 Topoisomerase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 691 Camptothecins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 Anthracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 Epipodophyllotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . 691 Amsacrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692 Microtubule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 692 Inhibitors of Microtubule Polymerization: Vinca Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692 Inhibitors of Microtubule Depolymerization: Taxanes . . . . 692 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 693 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693

 INTRODUCTION

found to cause bone marrow suppression through wartime exposures—were tested in patients with lymphoma and leukemia and shown to induce remissions. These and other findings have since led to the development of multiple classes of antineoplastic drugs designed to interfere with the building blocks of DNA synthesis and mitosis, or to produce DNA damage and chromosomal instability, and thereby to promote cytotoxicity and programmed cell death (apoptosis). Unfortunately, the therapeutic window of these drugs is narrow because they affect normal cells in tissues such as the gastrointestinal tract and bone marrow that undergo cell division. Use of combination chemotherapy with agents from different classes has helped to enhance efficacy while minimizing overlapping dose-limiting toxicities, but the ability to cure patients with most forms of advanced cancer remains limited. In part, this limited efficacy is due to the development of multiple resistance mechanisms, including the failure of tumor cells to undergo apoptosis in

Cancer therapy has traditionally been based on the principle that tumor cells are traversing the cell cycle frequently and are thus more sensitive than normal cells to interference with DNA synthesis and mitosis. Indeed, the antimetabolites, a class of agents that are analogues of endogenous folates, purines, and pyrimidines, and that function as inhibitors of the enzymes of nucleotide synthesis, were some of the first drugs to be tested as chemotherapeutic agents. In the late 1940s, Sidney Farber and colleagues administered the antifolate compound aminopterin to patients with acute leukemia and observed temporary remissions in more than half of the patients. Because of their rapid growth and division, cancer cells are also thought to be more sensitive than normal cells to the effect of DNA-damaging agents. Also in the late 1940s, nitrogen mustards—derivatives of agents that had been 674

676 Principles of Chemotherapy A

Purine precursors

Pyrimidine precursors Folate

Inosine monophosphate (IMP)

Pyrimidines

Ribonucleotides Deoxyribonucleotides DNA RNA

Protein

B Methotrexate Purine precursors

Pyrimidine precursors Folate

6-Mercaptopurine Thioguanine

IMP 6-Mercaptopurine Thioguanine

Pyrimidines

Ribonucleotides

role in pyrimidine synthesis. UMP is itself a nucleotide component of RNA, as well as the common precursor of the RNA and DNA components cytidylate (CMP), deoxycytidylate (dCMP), and deoxythymidylate (dTMP). CTP is formed by the amination of UTP. Ribonucleotide Reduction and Thymidylate Synthesis The ribonucleotides ATP, GTP, UTP, and CTP, which are required for RNA synthesis, are assembled on a DNA template and linked to form RNA. Alternatively, ribonucleotides can be reduced at the 2⬘ position on ribose to form the deoxyribonucleotides dATP, dGTP, dUTP, and dCTP. The conversion of ribonucleotides to deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase. (In actuality, ribonucleotide reductase uses as substrates the diphosphate forms of the four ribonucleotides to produce dADP, dGDP, dUDP, and dCDP; nucleotides can, however, be readily interconverted among their monophosphate, diphosphate, and triphosphate forms.) Note, in Figures 38-2 through 38-4, that ribonucleotide reductase catalyzes the formation of the DNA precursors dATP, dGTP, and dCTP. The DNA precursor dTTP is not synthesized directly by ribonucleotide reductase, however. Rather, dUMP must be modified to form dTMP. As can be seen in Table 38-1, dTMP is the product of dUMP methylation. The methylation of dUMP to dTMP is catalyzed by thymidylate synthase, with methylenetetrahydrofolate (MTHF) serving as the donor of the methyl group (Fig. 38-4). As MTHF donates its methyl group, it is oxidized to dihydrofolate (DHF). DHF must be reduced to THF by dihydrofolate reductase (DHFR) and then converted to MTHF in order to serve as the cofactor for another cycle of dTMP synthesis. Inhibition of DHFR prevents the regeneration of tetrahydrofolate and thereby inhibits the conversion of dUMP to dTMP, eventually resulting in an insufficient cellular level of dTMP for DNA replication.

Hydroxyurea Deoxyribonucleotides

Fludarabine Cytarabine Cladribine

DNA

RNA

Protein

FIGURE 38-1.

Overview of de novo nucleotide biosynthesis. A. Folate is an essential cofactor in the synthesis of inosine monophosphate (IMP), from which all purine nucleotides are derived. Pyrimidine synthesis does not require folate, although folate is required for the methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) (see Fig. 38-2). Ribonucleotides contain one of the purine or pyrimidine bases linked to ribose phosphate. Subsequent reduction of the ribose at the 2⬘ position produces deoxyribonucleotides. Deoxyribonucleotides are polymerized into DNA, while ribonucleotides are used to form RNA (not shown). The central dogma of molecular biology states that the DNA code determines the sequence of RNA (transcription) and that RNA is then translated into protein. B. Methotrexate inhibits dihydrofolate reductase (DHFR) and thereby prevents the utilization of folate in purine nucleotide and dTMP synthesis. 6-Mercaptopurine and thioguanine inhibit the formation of purine nucleotides. Hydroxyurea inhibits the enzyme that converts ribonucleotides to deoxyribonucleotides. Fludarabine, cytarabine, and cladribine are purine and pyrimidine analogues that inhibit DNA synthesis. 5-Fluorouracil inhibits the enzyme that converts dUMP to dTMP (not shown).

Nucleic Acid Synthesis Provided that sufficient levels of nucleotides are available, DNA and RNA can be synthesized, and protein synthesis, cell growth, and cell division can occur. Many drugs, including the antimetabolites discussed in this chapter, can inhibit both DNA and RNA synthesis. To avoid repetition, a detailed discussion of DNA and RNA synthesis is provided in Chapter 33, Pharmacology of Bacterial Infections: DNA Replication, Transcription, and Translation. For the purposes of this chapter, the reader should be aware that RNA and DNA are formed by polymerization of ribonucleotides and deoxyribonucleotides, respectively. RNA polymers are elongated by the enzyme RNA polymerase, and DNA is elongated by DNA polymerase. Although antimetabolites primarily inhibit the enzymes that mediate nucleotide synthesis, some antimetabolites also inhibit DNA and RNA polymerases (see below).

DNA Repair and Chromosome Maintenance Mutations and other DNA lesions can arise spontaneously or as a result of exposure to DNA-damaging chemical agents or radiation. Several general pathways exist for repair of these lesions, including mismatch repair (MMR) for DNA replication errors, base excision repair (BER) for small base modifications and single-strand breaks, nucleotide excision repair (NER) for removal of bulky adducts, and homologous

Base

Ribonucleoside

Ribonucleotide

NH2 N NH2

N

HO

N

-

N

N H

O

N

H

Adenine (A)

H

H

OH

H OH

N

N

N

O

N

N

NH2 N

N N

P

O

O

H

Adenosine

-

O

-

O H

H

H

OH

H OH

H

O

P

H

OH

H

O

N

O

OH

H

Deoxyadenosine

Adenylate (AMP)

N

N

O

HO O

Deoxyribonucleotide

NH2

NH2 N

N

N

Deoxyribonucleoside

H

H

OH

H

H

Deoxyadenylate (dAMP)

Purines O N O

N

N

N

NH2

HO

NH

N H

N

-

O NH2

H

H

H

OH

H OH

O

P

O

N

OH

H

H

OH

H OH

N H

O

P

H H OH

H H OH

O

O

H

Uracil (U)

H

H

OH

H OH

P

H

Uridine

H

H H OH

H

NH2 N

N

O

H

H

OH

H

N

O -

O

P

O

OH

H

H

OH

H

H

Deoxycytidylate (dCMP)

Deoxcytidine

O

O

NH

NH

O

N

O -

H

Uridylate (UMP)

H

H

OH

H

N

O O

P

O

OH

H

H

H

OH

H

O

NH N

NH

O

N

O

NONE

NONE

HO

-

O N H

O

H

Purine synthesis

PRPP

Pyrimidine synthesis

Folate

PRPP

Amino acids

IMP

Ribonucleotides

AMP

GMP

UMP

CMP

dUMP

Deoxyribonucleotides dAMP

H H

dGMP

dTMP

DNA

dCMP

O

P

O

O

O

O-

H

Deoxythymidine

Thymine (T)

Amino acids

H OH

H

Deoxyuridylate (dUMP)

Deoxyuridine

O

O

O

O

NH

O

O

H

O

OH

H

N

O

O-

H

Deoxyguanylate (dGMP)

HO

O

H OH

NH2

H

N

O -

O N H

H

NH

HO

NH

O-

O

NH2

N

O

H

O NH O

O

Deoxyguanosine

Cytidylate (CMP)

N

P

O

H OH

O

Pyrimidines

H

O H

Cytidine

O

H

HO

O

O-

H OH

Cytosine (C)

N

O -

H

H OH

NH

N

O O

N O

NH2 -

NH2

O

O

N

HO

Guanylate (GMP)

HO

N

N

H

N N

N

NH

O

NH2

NH2

NH2

O

Guanosine

Guanine (G)

N

NH

N

O

O

O

O N

NH

H

H

H

OH

H

H

Deoxythymidylate (dTMP)

678 Principles of Chemotherapy 6-Mercaptopurine Thioguanine

GTP

GMP Ribonucleotide reductase

XMP

6-Mercaptopurine

Inosine monophosphate IMP (IMP) dehydrogenase

Hydroxyurea

(IMPDH)

ATP

Adenylosuccinate Hydroxyurea

AMP Ribonucleotide reductase dAMP

dGMP

dATP

dGTP Fludarabine Cladribine DNA

DNA

FIGURE 38-3.

Details of purine synthesis. Inosine monophosphate, or IMP, occupies a central position in the synthesis of purine nucleotides. IMP is oxidized by IMP dehydrogenase (IMPDH) to xanthylate (XMP), which is converted to guanosine monophosphate (GMP). GMP can be incorporated into DNA or RNA as deoxyguanosine triphosphate (dGTP) or guanosine triphosphate (GTP), respectively. Alternatively, IMP can be aminated to adenosine monophosphate (AMP) through an adenylosuccinate intermediate. AMP can be incorporated into DNA or RNA as deoxyadenosine triphosphate (dATP) or adenosine triphosphate (ATP), respectively. 6-Mercaptopurine and thioguanine inhibit IMPDH and thus interrupt GMP synthesis. 6-Mercaptopurine also inhibits the conversion of IMP to adenylosuccinate and thus interrupts AMP synthesis. Hydroxyurea inhibits ribonucleotide reductase and thus inhibits formation of the deoxyribonucleotides required for DNA synthesis. Fludarabine and cladribine are halogenated adenosine analogues that inhibit DNA synthesis.

Aspartate Carbamoyl phosphate

Orotate PRPP

UMP Ribonucleotide reductase

MTHF

DHFR

Hydroxyurea

DHF dTMP

CTP Ribonucleotide reductase

dCTP

dUMP

Thymidylate synthase

THF

UTP

Cytarabine

5-Fluorouracil

DNA

Methotrexate

dTTP

DNA

FIGURE 38-4.

recombination or nonhomologous end-joining for doublestrand breaks (Fig. 38-5). DNA repair pathways are important not only because they can alter the efficacy of chemotherapy, but also because loss of these pathways frequently contributes to tumor development via impairment of genomic integrity and facilitation of mutations in oncogenes and tumor suppressor genes. Telomeres, the repeat sequences that cap the ends of chromosomes, also play an important role in genome stability and prevention of chromosome fusions. The enzyme telomerase, which prevents telomere shortening in cancer cells, represents a key component in the process of immortalization and oncogenic transformation.

Details of pyrimidine synthesis. Aspartate (an amino acid) and carbamoyl phosphate combine to form orotate, which then combines with phosphoribosylpyrophosphate (PRPP) to form uridylate (UMP). UMP occupies a central position in the synthesis of pyrimidine nucleotides. UMP can be sequentially phosphorylated to uridine triphosphate (UTP). UTP is incorporated into RNA (not shown) or aminated to form cytidine triphosphate (CTP). CTP is incorporated into RNA (not shown) or reduced by ribonucleotide reductase to deoxycytidine triphosphate (dCTP), which is incorporated into DNA. Alternatively, UMP can be reduced to deoxyuridylate (dUMP). Thymidylate synthase converts dUMP to deoxythymidylate (dTMP), in a reaction that depends on folate. dTMP is phosphorylated to deoxythymidine triphosphate (dTTP), which is incorporated into DNA. Hydroxyurea inhibits the formation of deoxyribonucleotides and thereby inhibits DNA synthesis. Cytarabine, a cytidine analogue, inhibits the incorporation of dCTP into DNA. 5-Fluorouracil inhibits dTMP synthesis by inhibiting thymidylate synthase. Methotrexate inhibits dihydrofolate reductase (DHFR), the enzyme responsible for regenerating tetrahydrofolate (THF) from DHF. By inhibiting DHF reductase, this drug inhibits the formation of methylenetetrahydrofolate (MTHF), which is the folate compound that is required for dTMP synthesis.

Mismatch Repair During DNA replication, errors such as single-base mismatches and insertions or deletions of microsatellite repeat sequences (microsatellite instability) are recognized and repaired by proteins of the mismatch repair (MMR) system. For single-base mismatches, recognition involves a heterodimer between the MSH2 protein and MSH6, while for insertion/deletion loops, MSH2 can also partner with MSH3 (Fig. 38-6). These complexes recruit the proteins MLH1 and PMS2 (as well as MLH3 for insertion/deletion loops), which, in turn, recruit exonucleases and components of the DNA replication machinery for excision and repair of the lesion. Germline mutations in MLH1, PMS2, MSH2, or MSH6 are associated with 70–80% of cases of hereditary nonpolyposis colon cancer. In addition, microsatellite instability, a hallmark of defective MMR, is observed in 15–25% of sporadic colorectal cancers. Base Excision Repair DNA single-strand breaks (SSB), which may be formed directly by ionizing radiation or indirectly due to enzymatic excision of a modified base by a DNA glycosylase, activate the enzyme poly(ADP-ribose) polymerase 1 (PARP1) (Fig. 38-7). At the site of the breakage, PARP1 transfers ADP-ribose moieties from NAD to itself and to a number of other proteins involved in DNA and chromatin metabolism.

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 679

Replication errors

Oxygen radicals Ionizing radiation Chemicals (nitrosamines) Chemotherapeutic agents (alkylating agents, temozolomide)

UV radiation Chemicals (2-AAF, benzo(a)pyrene) Chemotherapeutic agents (platinum agents)

Ionizing radiation Chemicals (bioflavonoids, radiomimetic chemicals) Chemotherapeutic agents (bleomycin, topoisomerase I and II inhibitors)

Base pair mismatches Insertion/deletion loops

Abasic sites Base modifications Single-strand breaks

Bulky adducts

Double-strand breaks

Mismatch repair

Base excision repair

Nucleotide excision repair

Double-strand break repair

FIGURE 38-5. Mechanisms of DNA damage and repair. In response to DNA damage, there are several general pathways that mediate repair of DNA lesions. Replication errors typically result in base pair mismatches or insertion/deletion loops in regions of microsatellite DNA repeats; these lesions are repaired by the mismatch repair (MMR) pathway. Oxygen radicals, ionizing radiation, and various chemicals and chemotherapeutic agents can cause abasic site formation, base modifications, and single-strand breaks, which are repaired by the base excision repair (BER) pathway. Ultraviolet (UV) irradiation and certain DNA-modifying chemicals and chemotherapeutic agents can cause the formation of bulky adducts that are excised and repaired by the nucleotide excision repair (NER) pathway. Ionizing radiation, radiomimetic chemicals, bleomycin, and natural (bioflavonoids) and chemotherapeutic (camptothecins, anthracyclines, epipodophyllotoxins) topoisomerase inhibitors can induce double-strand DNA breaks that trigger repair by the double-strand break repair (DSBR) pathway. 2-AAF, 2-acetylaminoflourene.

Single-base mismatch

MSH2 MSH6 A T TGC T T AGGC T A ACGCA T CCG

MLH1

Insertion/deletion loop

MSH2

T A T A T

A T A T A

MSH3/6

A T A T A T A T A T A T A T A T A T A T A T A T

PMS2

MLH1

MSH2 MSH6

MSH2

T A T A T

A T A T A

PMS2/ MLH3 MSH3/6

A T TGC T T AGGC T A ACGCA T CCG

A T A T A T A T A T A T A T A T A T A T A T A T

A T TGC T T AGGC T A ACGA A T CCG

A T A T A T A T A T A T A T A T A T A T A T A T A T

FIGURE 38-6. Mismatch repair pathway. Replication errors can result in single base pair mismatches or insertion/deletion loops in microsatellite repeat regions as a result of intrastrand complementary base-pairing. Single-base mismatches are recognized by an MSH2/MSH6 heterodimer, and insertion/deletion loops are recognized by an MSH2/MSH3 or MSH2/MSH6 heterodimer. Additional components of the mismatch repair machinery are then recruited, including MLH1/ PMS2 for single-base mismatches or MLH1/PMS2 or MLH1/MLH3 for insertion/ deletion loops. Exonucleases and components of the DNA replication machinery are subsequently recruited for excision and repair of the lesions.

The covalent addition of negatively charged ADP-ribose oligomers alters the interactions of these proteins with DNA and with other proteins. PARP1 recruits the BER protein XRCC1; together with DNA polymerase ␤ and DNA ligase III, XRCC1 facilitates repair of the lesion. PARP1 has also been implicated in the recognition of DNA double-strand breaks (DSB) and in the recruitment of DNA-dependent protein kinase in DSB repair (see below), as well as in cell death pathways, modification of chromatin structure, transcriptional regulation, and mitotic apparatus function. Nucleotide Excision Repair In response to the formation of bulky adducts that distort the DNA double helix, such as those induced by ultraviolet radiation and DNA-damaging chemotherapeutic agents, a complex set of proteins recognizes and initiates repair of the lesion via a process termed nucleotide excision repair (NER). Repair involves local opening of the double helix around the site of the damage, incision of the damaged strand on both sides of the lesion, excision of the oligonucleotide containing the lesion, and, finally, DNA repair synthesis and ligation. The endonuclease ERCC1 plays an important role in targeted excision of the DNA lesion. The genes involved in nucleotide excision repair were in part identified from study of the clinical syndromes xeroderma pigmentosa and Cockayne syndrome, which are rare photosensitivity disorders that exhibit defects in NER. Double-Strand Break Repair In response to a double-strand break, activation of the ataxia telangiectasia mutated (ATM) kinase results in generation of the phosphorylated histone gamma-H2AX at the site of the break. Together with the protein MDC1, gamma-H2AX recruits to the locus of DNA damage a complex (MRN) containing the proteins Mre11, Rad50, and Nijmegen breakage syndrome gene 1 (NBS1) (Fig. 38-8). The breast and ovarian

680 Principles of Chemotherapy PARP1 Histone

Sister chromatids

A T TGC T AGGC T A ACGA A T CCG

DSB

NAD Nicotinamide ADPr ADPr ADPr ADPr

ADPr ADPr ADPr ADPr

Histone modification

ATM H2AX

P

PARP1 Histone A T TGC T AGGC T A ACGA A T CCG MDC1 MRN complex recruitment ADPr ADPr ADPr ADPr

P

MRE11 NBS1

RAD50

ADPr ADPr ADPr ADPr

XRCC1 PARP1 Histone

A T TGC T AGGC T A ACGA A T CCG

Nuclease-mediated resection

A T TGC T T AGGC T A ACGA A T CCG

BRCA1-P RAD51-BRCA2 RAD54

FIGURE 38-7.

Base excision repair pathway. The enzyme poly(ADP-ribose) polymerase 1 (PARP1) is recruited to single-strand break sites resulting from ionizing radiation or base lesion excision. PARP1 poly-ADP ribosylates (ADPr) a variety of targets at the site of injury, including itself and histones. The ADPr-modified proteins then recruit additional proteins, such as XRCC1, which, in turn, recruit DNA polymerase ␤ and DNA ligase III to repair the lesion.

RAD52

BRCA2 Strand invasion

RAD51

DNA synthesis; branch migration

DNA polymerase DNA ligase

FIGURE 38-8.

Double-strand break repair pathway. The ataxia telangiectasia mutated (ATM) kinase recognizes and binds to double-strand DNA break sites. Upon activation, the ATM kinase marks the site by generating the phosphorylated histone gamma-H2AX. Gamma-H2AX and the protein MDC1 recruit the Mre11/ Rad50/Nijmegen breakage syndrome gene 1 (NBS1) complex (MRN) to the site of injury. After RAD52 is recruited and nucleases mediate DNA resection, BRCA1 is recruited to the site and phosphorylated by ATM, ATR, and CHK2 kinases. Together with RAD51 and BRCA2, phosphorylated BRCA1 facilitates repair of the doublestrand break by homologous recombination (depicted in the figure) or nonhomologous end-joining (NHEJ; not shown).

Ligation; junction resolution

Accurately repaired DNA

ATM ATR, CHK2

BRCA1

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 681

[TTAGGG]n

2-30 kb

3' 5'

[AATCCC]n

cancer susceptibility gene product BRCA1 is also phosphorylated by the kinases ATM, ATR, and CHK2 in response to the double-strand break, and phosphorylated BRCA1, RAD51, and BRCA2 are also recruited to the break site. Subsequent repair is mediated either by homologous recombination, with formation and resolution of a Holliday junction (Fig. 38-8), or by nonhomologous end-joining (NHEJ), in which DNA-dependent protein kinase and a complex of proteins, including XRCC4, catalyze nucleolytic processes that allow end-joining by DNA ligase IV. The DNA repair effected by homologous recombination is more accurate than that mediated by NHEJ.

Unknown nuclease

ss [TTAGGG]n 3' 5'

50-300 nt

TRF1 Folding + other factors

3' 5'

TRF2 Strand invasion + other factors

ds t-loop 5'

ss

3' D loop

FIGURE 38-9.

Telomere structure. Human telomeres are 2 to 30 kilobases (kb) in length and consist of the simple sequence repeats TTAGGG. A 3⬘-terminal 50- to 300-nucleotide (nt) single-stranded overhang is generated by an as yet unidentified nuclease. The telomere binding proteins TRF1, TRF2, and other factors facilitate folding and proximal invasion of double-stranded telomeric DNA by the single-stranded overhang to generate a stable “t-loop” structure. This structure plays an important role in capping and protecting the ends of chromosomes.

Telomere Biology Human telomeres consist of the simple repeat sequence TTAGGG. These repeats are shaped, folded, and bound by a complex of proteins to form a unique structure termed a “t-loop” (Fig. 38-9). In the t-loop structure, a long singlestranded overhang at the 3⬘ end of the DNA invades the proximal double-stranded DNA component; this process is facilitated by TRF1, TRF2, and other protein factors. The t-loop and its associated complex of proteins are thought to play important roles in capping and protecting the chromosome end, as well as protecting telomeres from recognition by the DNA damage checkpoint machinery. Because DNA polymerase is unable to replicate the ends of linear chromosomes completely, telomeres shorten with each division in normal cells. Telomere shortening ultimately results in disruption of the telomeric caps, activation of a DNA damage checkpoint, and a state of cycle arrest termed cellular senescence (Fig. 38-10). When cells are able to bypass this checkpoint via inactivation of the tumor suppressor protein p53, which normally regulates

Telomerase activation

p53 loss, pRB loss

Early population doubling

Late population doubling

p53 activation, pRB activation

Crisis

Immortalization

Senescence

Telomeres

Long

Medium

Short

Short

Long

Proliferation

Yes

Yes

No

Yes

Yes

Cell death

No

No

No

Yes

No

FIGURE 38-10. Chromosome maintenance and its relationship to immortalization. As primary cells undergo successive population doublings, telomeres progressively shorten due to the inability of DNA polymerase to replicate the ends of linear chromosomes. Ultimately, a checkpoint is triggered, mediated by the proteins p53 and pRB, which results in a state of growth arrest termed cellular senescence. Senescence can be bypassed by inactivation of p53 and pRB; ultimately, however, the critically short telomeres cause the cells to enter a state termed crisis and to die. Activation of telomerase allows cells to maintain adequate telomere length and divide indefinitely, resulting in immortalization. Notably, exogenous expression of telomerase alone in primary cells is sufficient for these cells to bypass senescence and become immortalized.

682 Principles of Chemotherapy

cell cycle arrest or apoptosis in response to DNA damage, chromosome fusions are observed. It is thought that the progressive shortening of telomeres with age promotes genomic instability and contributes to oncogenesis. However, cells also continue to die under these conditions. Activation of the enzyme telomerase, which is a reverse transcriptase that uses an RNA template to synthesize TTAGGG repeats, allows cells to restore telomere length and divide indefinitely. Telomerase activation is observed in normal germline cells and some stem cell populations and has been shown to maintain the presence of the 3⬘ overhang in normal cells. The immortalization process associated with telomerase activation is also essential for tumor formation and maintenance. In a minority of tumors, an alternative lengthening of telomeres (ALT) pathway is activated.

Subunit

β-tubulin

24 nm

(GTPase activity)

GDP GTP

α-tubulin

GTP GTP

Microtubules and Mitosis Once a cell has replicated its DNA, it is prepared to undergo mitosis. In this process, chromosomes condense and are segregated into two identical daughter cells. The cell-cycle transitions from DNA replication (S phase) to G2 phase and then to mitosis (M phase) are complex and depend on the coordinated action of a number of so-called cyclin-dependent kinases (CDKs; see Chapter 32). Progression through mitosis is also facilitated by enzymes that include aurora kinases and polo-like kinases. Many cancer cells exhibit dysregulation of cell-cycle timing and abnormalities in mitosis. Thus, pharmacologic inhibition of these regulatory kinases is an active area of cancer research. Currently, however, the microtubule machinery represents the primary target of agents that act in mitosis. Microtubules are cylindrical, hollow fibers composed of polymers of tubulin, which is a heterodimeric protein consisting of ␣-tubulin and ␤-tubulin subunits (Fig. 38-11). ␣-Tubulin and ␤-tubulin are encoded by separate genes, but they have similar three-dimensional structures. Both ␣- and ␤-tubulin bind GTP; in addition, ␤-tubulin (but not ␣-tubulin) can hydrolyze GTP to GDP. Microtubules originate from a central microtubule organizing center (the centrosome, which includes two centrioles and associated proteins), where ␥-tubulin (a protein with homology to ␣-tubulin and ␤-tubulin) nucleates tubulin polymerization. Nascent microtubules assemble into protofilaments, which are longitudinal polymers of tubulin subunits. Each protofilament interacts laterally with two other protofilaments to form a hollow-core tube, 24 nm in diameter, which consists of 13 protofilaments arranged concentrically. Because tubulin is a heterodimer, this tube has inherent asymmetry; the end of a microtubule nearest the centrosome is bordered by ␣-tubulin and is called the (⫺) (“minus”) end, while the end of a microtubule extending from the centrosome is bordered by ␤-tubulin and is called the (⫹) (“plus”) end (Fig. 38-11). Tubulin units are added at different rates to the (⫺) and (⫹) ends; the (⫹) end grows (adds tubulin) twice as fast as the (⫺) end. Microtubules are not static structures. Rather, they possess an inherent property known as dynamic instability (Fig. 38-12). Tubulin heterodimers add to the end of the microtubule with GTP bound to both ␣-tubulin and ␤-tubulin subunits. As the microtubule grows, the ␤-tubulin of each tubulin heterodimer hydrolyzes its GTP to GDP. The hydrolysis of GTP to GDP introduces a conformational change in tubulin that destabilizes the microtubule. The exact

FIGURE 38-11.

Microtubule structure. Microtubules are hollow cylindrical tubes that polymerize from tubulin subunits. Each tubulin subunit is a heterodimer composed of ␣-tubulin (shades of purple) and ␤-tubulin (shades of blue). Both ␣-tubulin and ␤-tubulin bind GTP (dark shades of purple and blue); ␤-tubulin hydrolyzes GTP to GDP after the tubulin subunit is added to the end of a microtubule (lighter shades of purple and blue). Microtubules are dynamic structures that grow and shrink lengthwise; the cylindrical tubes are composed of 13 subunits arranged concentrically, resulting in a diameter of 24 nm. Note that microtubules have an inherent structural asymmetry. One end of a microtubule is limited by ␣tubulin and is referred to as the (⫺) (“minus”) end; the opposite end is limited by ␤-tubulin and is referred to as the (⫹) (“plus”) end.

mechanism of this destabilization is unknown, but it may be related to a decrease in the strength of lateral protofilament interactions or an increase in the tendency for protofilaments to “curve” away from the straight microtubule. Therefore, microtubule stability is determined by the rate of microtubule polymerization relative to the rate of GTP hydrolysis by ␤-tubulin. If a microtubule polymerizes tubulin faster than ␤-tubulin hydrolyzes GTP to GDP, then, in the steady state, there is a cap of GTP-bound ␤-tubulin at the (⫹) end of the microtubule. This GTP cap provides stability to the microtubule structure, allowing further polymerization of the microtubule. Conversely, if tubulin polymerization proceeds more slowly than the hydrolysis of GTP to GDP by ␤-tubulin, then, in the steady state, the (⫹) end of the microtubule is enriched with GDP-bound ␤-tubulin. This GDP-bound tubulin conformation is unstable and causes rapid depolymerization of the microtubule. The ability of microtubules to assemble and disassemble rapidly is important for their many physiologic roles. Pharmacologic agents can disrupt microtubule function either by preventing the assembly of tubulin into microtubules or by stabilizing existing microtubules (and thereby preventing microtubule disassembly). Microtubules have important physiologic roles in mitosis, intracellular protein trafficking, vesicular movement, and cell structure and shape. Mitosis is the physiologic role that is targeted pharmacologically; the other physiologic roles, however, predict many of the adverse effects of drugs that interrupt microtubule function. Recall that microtubules nucleate from centrosomes, which consist of centrioles and other associated proteins. In

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 683

GTP-bound tubulin cap

A Preexisting microtubule

High concentrations of GTP-bound tubulin

Low concentrations of GTP-bound tubulin

β-tubulin α-tubulin

B

Lengthened microtubule Rate of GTP hydrolysis = Rate of polymerization GTP cap preserved

C

Rate of GTP hydrolysis > Rate of polymerization GTP cap shrinks

D

Loss of GTP cap Unstable microtubule; depolymerization

FIGURE 38-12. Dynamic instability of microtubules. A. A preexisting microtubule is characterized by tubulin subunits that have predominantly hydrolyzed the GTP on ␤-tubulin to GDP (light purple and light blue). However, ␤-tubulin subunits that have recently been added to the microtubule have not yet hydrolyzed GTP (dark purple and dark blue). The GTP-bound tubulin subunits form a GTP-bound tubulin cap at the (⫹) end of the microtubule. B. In the presence of a high concentration of GTP-bound free tubulin subunits, new GTP-bound tubulin is added to the (⫹) end of the microtubule at a rate that equals or exceeds the rate of GTP hydrolysis by ␤-tubulin. Maintenance of a GTP-bound tubulin cap results in a stable microtubule. C. In the presence of a low concentration of GTP-bound free tubulin subunits, new GTP-bound tubulin is added to the (⫹) end of the microtubule at a rate less than the rate of GTP hydrolysis by ␤-tubulin. This results in shrinkage of the GTP-bound tubulin cap. D. A microtubule that lacks a GTP-bound tubulin cap is unstable and undergoes depolymerization.

mitosis, the two centrosomes align at opposite ends of the cell. Microtubules are extremely dynamic during M phase; they grow and shrink during M phase at rates much greater than during other phases of the cell cycle. This increased dynamic instability during M phase allows microtubules to locate and attach to the chromosomes. The microtubules emanating from each centrosome bind to kinetochores, which are proteins that attach to the centromere of a chromosome. Once the kinetochore of each chromosome is attached to a microtubule, microtubule-associated proteins act as motors to align the kinetochore-bound chromosomes at the equator of the cell (defined by the midpoint between the two centrosomes). When every chromosome has aligned at the equator, the microtubules shorten, separating a diploid pair of chromosomes into each half of the cell. Finally, cytokinesis (division of the cytoplasm) occurs, and two daughter cells are formed. Although many other proteins are involved in the regulation of mitosis, microtubules have a critical role in the process. Disruption of microtubule function freezes cells in M phase, leading eventually to the activation of programmed cell death (apoptosis).

 PHARMACOLOGIC CLASSES AND AGENTS Traditional antineoplastic chemotherapy can be subdivided into several classes of agents. The antimetabolite drugs are compounds that either inhibit the enzymes involved in nucleotide synthesis and metabolism or are incorporated as analogues into DNA and result in chain termination or strand breaks. These drugs act primarily during the S phase of the cell cycle, when cells are undergoing DNA replication. Another broad class of agents, which induce cytotoxicity by modification of DNA structure and generation of DNA damage, includes alkylating agents, platinum compounds, bleomycin, and topoisomerase inhibitors. These drugs exert their effects during multiple phases of the cell cycle. The final category of agents inhibits microtubule assembly or depolymerization, disrupting the mitotic spindle and interfering with mitosis. For a summary of the major classes of chemotherapeutic agents, their cell cycle specificity, and major toxicities, see Table 40-2 (Chapter 40, Principles of Combination Chemotherapy).

684 Principles of Chemotherapy

Inhibitors of Thymidylate Synthase Thymidylate (dTMP) is synthesized by the methylation of 2⬘-deoxyuridylate (dUMP). This reaction, which is catalyzed by thymidylate synthase, requires MTHF as a cofactor (Fig. 38-4). 5-Fluorouracil (5-FU; Fig. 38-13) inhibits DNA synthesis, primarily by interfering with the biosynthesis of thymidylate. 5-FU is first converted to 5-fluoro-2⬘deoxyuridylate (FdUMP) by the same pathways that convert uracil to dUMP. FdUMP then inhibits thymidylate synthase by forming, together with MTHF, a stable, covalent ternary enzyme–substrate–cofactor complex. Cells deprived of dTMP for a sufficient period of time undergo so-called “thymineless death.” 5-FU can also be metabolized to floxuridine triphosphate (FUTP), which can be incorporated into mRNA in place of uridylate and can thereby interfere with RNA processing. Either inhibition of thymidylate synthase by FdUMP or interference with RNA processing by FUTP, or a combination of the two mechanisms, could explain the toxic effect of 5-FU on cells. However, certain 5-FU congeners that inhibit thymidylate synthase but are not incorporated into RNA show antitumor efficacy similar to that of 5-FU. This finding points to thymidylate synthase inhibition as the dominant mechanism of 5-FU action. 5-FU is used as an antineoplastic agent, especially in the treatment of carcinomas of the breast and gastrointestinal tract. 5-FU has also been used in the topical treatment of premalignant keratoses of the skin and of multiple superficial basal cell carcinomas. Because 5-FU depletes thymidylate from normal cells as well as cancer cells, this agent is highly toxic and must be used with care. Capecitabine is an orally bioavailable prodrug of 5-FU. It is absorbed across the gastrointestinal mucosa and converted by a series of three enzymatic reactions to 5-FU. Capecitabine is approved for the treatment of metastatic colorectal cancer and as second-line therapy in metastatic breast cancer. Clinical trials have demonstrated that the efficacy of oral capecitabine is similar to that of intravenous 5-FU. Elucidation of the mechanism of action of 5-FU has led to the use of a 5-FU/folinic acid (leucovorin) combination as first-line chemotherapy for colorectal cancer. Because 5-FU inhibits thymidylate synthase by forming a ternary complex involving the enzyme (thymidylate synthase), substrate (5-FdUMP), and cofactor MTHF, it was hypothesized that increasing the levels of MTHF would potentiate the activity of 5-FU. Clinical trials proved this hypothesis to be correct, by showing that the efficacy of the combined regimen is greater than that of 5-FU alone. This is an important example

of the use of mechanistic knowledge to improve the clinical effectiveness of a drug. Pemetrexed is a folate analogue that, similar to endogenous folate and the dihydrofolate reductase (DHFR) inhibitor methotrexate (see Chapter 32), is transported into cells by the reduced folate carrier and polyglutamated by the intracellular enzyme folylpolyglutamate synthase. Polyglutamated pemetrexed is a potent inhibitor of thymidylate synthase and a much weaker inhibitor of DHFR; similar to 5-FU, its cytotoxic effect is likely due to the induction of “thymineless” cell death. (Note that the 5-FU derivative 5-FdUMP inhibits thymidylate synthase by binding to the dUMP [substrate] site on the enzyme, whereas pemetrexed inhibits thymidylate synthase by binding to the MTHF [cofactor] site on the enzyme.) Pemetrexed is approved for the treatment of all subtypes of non-small cell lung cancer except for the squamous subtype, due to lack of efficacy of the drug in this subtype. Pemetrexed is also used in combination with cisplatin (see below) in the treatment of malignant pleural mesothelioma. To reduce toxicity to normal cells, patients treated with pemetrexed are also given folic acid and vitamin B12 supplementation.

Inhibitors of Purine Metabolism 6-Mercaptopurine (6-MP) and azathioprine (AZA), a prodrug that is nonenzymatically converted to 6-MP in tissues, are inosine analogues that inhibit interconversions among purine nucleotides (Fig. 38-14). 6-Mercaptopurine contains a sulfur atom in place of the keto group at C-6 of the purine ring. After its entry into cells, mercaptopurine

O N

HN N

H2N

HN

HN

NH O

O

Uracil

FIGURE 38-13.

F

5-Fluorouracil (5-FU)

Structures of uracil and 5-fluorouracil. Note the structural similarity between uracil and 5-fluorouracil (5-FU). Uracil is the base in dUMP, the endogenous substrate for thymidylate synthase (see Fig. 38-4), and 5-FU is metabolized to FdUMP, an irreversible inhibitor of thymidylate synthase.

N H

N

H2N

Thioguanine

N

S

S N N H

Azathioprine (prodrug)

NH

N

HN

N O2

N

N

O

N H

Guanine

N O

S

FIGURE 38-14.

N

HN N

N H

Mercaptopurine

Structures of guanine, thioguanine, azathioprine, and mercaptopurine. Thioguanine, azathioprine, and mercaptopurine are structural analogues of purines. Thioguanine resembles guanine and can be ribosylated and phosphorylated in parallel with endogenous nucleotides. The nucleotide forms of thioguanine irreversibly inhibit IMPDH (see Fig. 38-3) and, upon incorporation into DNA, inhibit DNA replication. Azathioprine is a prodrug form of mercaptopurine; azathioprine reacts with sulfhydryl compounds in the liver (e.g., glutathione) to release mercaptopurine. The nucleotide form of mercaptopurine, thioinosine monophosphate (T-IMP), inhibits the enzymes that convert IMP to AMP and GMP (see Fig. 38-3). T-IMP also inhibits the first committed step in purine nucleotide synthesis.

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 685

is converted by the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT; see Chapter 48) to the nucleotide form, 6-thioinosine-5⬘-monophosphate (T-IMP). T-IMP is thought to inhibit purine nucleotide synthesis by several mechanisms. First, T-IMP inhibits the enzymes that convert IMP to AMP and GMP, including inosine monophosphate dehydrogenase (IMPDH) (Fig. 38-3). Second, T-IMP (as with AMP and GMP) is a “feedback” inhibitor of the enzyme that synthesizes phosphoribosylamine, which is the first step in purine nucleotide synthesis. Both of these mechanisms lead to marked decreases in the cellular levels of AMP and GMP, which are essential metabolites for DNA synthesis, RNA synthesis, energy storage, cell signaling, and other functions. 6-MP may also inhibit DNA and RNA synthesis by less well-characterized mechanisms. The major clinical application of 6-MP is in acute lymphoblastic leukemia (ALL), especially in the maintenance phase of a prolonged combination chemotherapy regimen. 6-MP is also active against normal lymphocytes and can be used as an immunosuppressive agent. For unknown reasons, the prodrug AZA is a superior immunosuppressant compared to 6-MP and is typically the drug of choice for this application. AZA is discussed in detail in Chapter 45, Pharmacology of Immunosuppression. Both the effectiveness and the toxicity of 6-MP are potentiated by allopurinol. Allopurinol inhibits xanthine oxidase, thereby preventing the oxidation of 6-MP to its inactive metabolite 6-thiouric acid. (In fact, allopurinol was discovered in an effort to inhibit the metabolism of 6-MP by xanthine oxidase.) Co-administration of allopurinol with 6-MP allows the dose of 6-MP to be reduced by two-thirds (although toxicity is proportionally increased as well). Allopurinol is often used as a single agent to prevent the hyperuricemia that could result from the destruction of cancer cells by chemotherapeutic agents (tumor lysis syndrome). The use of allopurinol in the treatment of gout is presented in Chapter 48, Integrative Inflammation Pharmacology: Gout. Pentostatin (Fig. 38-15) is a selective inhibitor of adenosine deaminase. The drug is a structural analogue of the intermediate in the reaction catalyzed by ADA and binds to the enzyme with high affinity. The resulting inhibition of ADA causes an increase in intracellular adenosine and 2⬘-deoxyadenosine levels. The increased adenosine and 2⬘deoxyadenosine have multiple effects on purine nucleotide metabolism. In particular, 2⬘-deoxyadenosine irreversibly inhibits S-adenosylhomocysteine hydrolase, and the resulting increase in intracellular S-adenosylhomocysteine is toxic to lymphocytes. This action may account for the effectiveness of pentostatin against some leukemias and lymphomas. Pentostatin is especially effective against hairy cell leukemia.

Inhibitors of Ribonucleotide Reductase Hydroxyurea inhibits ribonucleotide reductase by scavenging a tyrosyl radical at the active site of the enzyme. In the absence of this free radical, ribonucleotide reductase is unable to convert nucleotides to deoxynucleotides, and DNA synthesis is thereby inhibited. Hydroxyurea is approved for use in the treatment of adult sickle cell disease and certain neoplastic diseases. The mechanism of action of hydroxyurea in the treatment of sickle cell disease may or may not be related to inhibition of ribonucleotide reductase. As an alternative to this mechanism,

A

NH2 N

N OH

N

N

N

HO

NH

O H

H

H

OH

H OH

N HO

N

O

Adenosine H

H

H

OH

H

H

Pentostatin (2'-Deoxycoformycin)

B NH2 N

N

NH2 N

N

Cl

N

HO

N

O H H

OH

H H

N

O H

Cladribine

N

F

P HO

O

O OH

H H

OH

HO H

H

Fludarabine-5'-phosphate

FIGURE 38-15.

Structures of adenosine, pentostatin, cladribine, and fludarabine. A. Pentostatin inhibits adenosine deaminase (ADA), the enzyme that converts adenosine and 2⬘-deoxyadenosine to inosine and 2⬘-deoxyinosine, respectively. Pentostatin binds to ADA with very high affinity (Kd ⫽ 2.5 ⫻ 10⫺12 M) because it structurally resembles the intermediate (transition state) in this enzymatic reaction. B. Cladribine and fludarabine-5⬘-phosphate are also adenosine analogues. Cladribine is a chlorinated purine analogue that is incorporated into DNA and causes DNA strand breaks. Fludarabine phosphate is a fluorinated purine analogue that is incorporated into DNA and RNA; this drug also inhibits DNA polymerase and ribonucleotide reductase.

hydroxyurea has been shown to increase the expression of the fetal isoform of hemoglobin (HbF), which inhibits the polymerization of sickle hemoglobin (HbS) and thereby decreases red blood cell sickling under conditions of hypoxia. Hydroxyurea significantly decreases the incidence of painful (vaso-occlusive) crisis in patients with sickle cell disease. The mechanism by which hydroxyurea increases HbF production is unknown. The role of hydroxyurea in the treatment of sickle cell disease is discussed further in Chapter 44, Pharmacology of Hematopoiesis and Immunomodulation. Hydroxyurea is most commonly used in the treatment of myeloproliferative disorders such as polycythemia vera and essential thrombocytosis, or for palliative control of blood counts in acute myelogenous leukemia. In myeloproliferative disorders, hydroxyurea can be used as a single agent or in combination with other agents to inhibit the excessive growth of myeloid cells in the bone marrow. The applications of hydroxyurea for these indications have been limited somewhat by concerns that long-term hydroxyurea use may be leukemogenic; this is an example of the phenomenon that certain antitumor agents can also cause cancer.

686 Principles of Chemotherapy

Purine and Pyrimidine Analogues That Are Incorporated into DNA A number of antimetabolites exert their major therapeutic effect by acting as “rogue” nucleotides. These drugs are substrates for the various pathways of nucleotide metabolism, including ribosylation, ribonucleotide reduction, and nucleoside and nucleotide phosphorylation. The sugar triphosphate forms of these drugs can then be incorporated into DNA. Once incorporated into DNA, these compounds disrupt the structure of DNA, resulting in DNA chain termination, DNA strand breakage, and inhibition of cell growth. Thioguanine is a guanine analogue in which a sulfur atom replaces the oxygen atom at C-6 of the purine ring (Fig. 38-14). As with mercaptopurine, thioguanine is converted by HGPRT to its nucleotide form, 6-thioguanosine-5⬘-monophosphate (6-thioGMP). Unlike T-IMP, the nucleotide form of mercaptopurine, 6-thioGMP is a good substrate for guanylyl kinase, the enzyme that catalyzes the conversion of GMP to GTP. By this mechanism, 6-thioGMP is converted to 6-thioGTP, which is incorporated into DNA. Within the structure of DNA, 6-thioGTP interferes with RNA transcription and DNA replication, resulting in cell death. 6-ThioGMP also irreversibly inhibits IMPDH and thereby depletes cellular pools of GMP (Fig. 38-3). Thioguanine is used in the treatment of acute myelogenous leukemia. Major adverse effects of thioguanine include bone marrow suppression and gastrointestinal injury. Fludarabine phosphate (Fig. 38-15) is a fluorinated purine nucleotide analogue that is structurally related to the antiviral agent vidarabine (see Chapter 37, Pharmacology of Viral Infections). The triphosphate form of fludarabine is incorporated into DNA and RNA, causing DNA chain termination. Fludarabine triphosphate also inhibits DNA polymerase and ribonucleotide reductase and thereby decreases nucleotide and nucleic acid synthesis in cells. The relative importance of these actions in mediating the cellular toxicity of the drug remains to be elucidated. Fludarabine phosphate is used in the treatment of lymphoproliferative disorders, especially chronic lymphocytic leukemia (CLL) and lowgrade B-cell lymphomas. Cladribine is a chlorinated purine analogue that is structurally related to fludarabine phosphate (Fig. 38-15). Cladribine triphosphate is incorporated into DNA, causing strand breaks. Cladribine also depletes intracellular pools of the essential purine metabolites NAD and ATP. Cladribine is approved for use in the treatment of hairy cell leukemia and has been used experimentally in the treatment of other types of leukemia and lymphoma. Cytarabine (araC) is a cytidine analogue that is metabolized to araCTP (Fig. 38-16). AraCTP competes with CTP for DNA polymerase, and incorporation of araCTP into DNA results in chain termination and cell death (Fig. 38-4). Synergism between cytarabine and cyclophosphamide has been noted, presumably because of the reduced DNA repair caused by cytarabine’s inhibition of DNA polymerase. Cytarabine is used to induce and maintain remission in acute myelogenous leukemia; it is especially effective for this indication when combined with an anthracycline. 5-Azacytidine is a cytidine analogue whose triphosphate metabolite is incorporated into DNA and RNA (Fig. 38-16). Once incorporated into DNA, azacytidine interferes with cytosine methylation, altering gene expression and promoting

NH2

NH2

N N

N

N

O

N

HO

O

HO

O H

O

H

H

OH

H OH

H

Cytidine

H

H

OH

H OH

5-Azacytidine NH2 N N

O

HO O H H

OH

HO H

H

Cytosine arabinoside (cytarabine, AraC)

FIGURE 38-16. Structures of cytidine, cytarabine, and azacytidine. Cytarabine and azacytidine are both analogues of the nucleoside cytidine. Cytarabine has an arabinose sugar in place of ribose (note the chirality of the hydroxyl group highlighted in blue). The incorporation of cytarabine triphosphate (araCTP) into DNA inhibits further nucleic acid synthesis, because the replacement of 2⬘deoxyribose by arabinose interrupts strand elongation. Azacytidine has an azide group (highlighted in blue) within the pyrimidine ring; this drug is incorporated into nucleic acids and interferes with the methylation of cytosine bases.

cell differentiation. Azacytidine and its 2⬘-deoxy derivative decitabine (5-aza-2⬘-deoxycytidine) are approved for the treatment of myelodysplastic disease. Gemcitabine is a fluorinated cytidine analogue in which the hydrogen atoms on the 2⬘ carbon of deoxycytidine are replaced by fluorine atoms. The diphosphate form of gemcitabine inhibits ribonucleotide reductase; the triphosphate form of gemcitabine is incorporated into DNA, interfering with DNA replication and resulting in cell death. Gemcitabine is active in several solid tumors, including pancreatic, breast, bladder, and non-small cell lung cancer, and is also incorporated into regimens for hematologic malignancies such as Hodgkin’s disease.

Agents That Directly Modify DNA Structure Alkylating Agents The advent of modern chemotherapy dates to the 1940s, when highly reactive alkylating agents were first noted to induce remissions in otherwise untreatable malignancies. The clinical use of these agents was sparked by observations that nitrogen mustards, derivatives of wartime agents that caused dramatic suppression of hematopoietic cells, could have therapeutic utility in blood-derived malignancies such as leukemias and lymphomas. Soon thereafter, it was suggested that alkylating agents could also be useful in treating epithelial tumors, mesenchymal tumors, carcinomas,

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 687

and sarcomas; in fact, alkylating agents are commonly used against all of these diseases today. Alkylating agents—such as cyclophosphamide, mechlorethamine, melphalan, chlorambucil, and thiotepa—are electrophilic molecules that are attacked by nucleophilic sites on DNA, resulting in the covalent attachment of an alkyl group to the nucleophilic site. Depending on the particular agent, alkylation can take place on nitrogen or oxygen atoms of the base, the phosphate backbone, or a DNA-associated protein. The N-7 and O-6 atoms of guanine bases are particularly susceptible to alkylation. Alkylating agents typically have two strong leaving groups (Fig. 38-17). This structure confers the ability to bis-alkylate (perform two alkylating reactions), enabling the agent to cross-link the DNA molecule either to itself—by linking two guanine residues, for example—or to proteins. Bis-alkylation (crosslinking) seems to be the major mechanism of cytotoxicity (Fig. 38-18A). Alkylation of guanine residues can also result in cleavage of the guanine imidazole ring, abnormal base-pairing between the alkylated guanine and thymine, or depurination (i.e., excision of the guanine residue) (Fig. 32-18B–D). Ring cleavage disrupts the molecular structure of DNA; anomalous DNA base-pairing causes miscoding and mutation; and depurination leads to scission of the sugar–phosphate DNA backbone. Importantly, the mutations caused by these processes can increase the risk of developing new cancers. Although all nitrogen mustards are relatively reactive, the individual agents vary in the speed with which they react with nucleophiles; this fact has significant impact on their clinical use. Highly unstable compounds, such as mechlorethamine, cannot be administered orally because such agents alkylate target molecules within seconds to minutes. Because of this high reactivity, these molecules are powerful vesicants (causing blisters) and can severely damage skin and soft tissue if they leak outside of blood vessels. The rapid reactivity of alkylating agents can be exploited by infusing the drug directly into the site of a tumor. For example, thiotepa can be instilled into the bladder to treat superficial bladder cancers. In contrast to mechlorethamine and thiotepa, chlorambucil and melphalan are much less reactive and can be administered orally. Cyclophosphamide is particularly useful because it is a nonreactive prodrug that requires activation by the hepatic cytochrome P450 system; this agent can be administered either orally or intravenously (Fig. 38-19).

Cl H O N P

O Cl

N

Cl N

O O

Cl Cyclophosphamide

FIGURE 38-17.

N H

N

BCNU (Carmustine, a nitrosourea)

Structures of cyclophosphamide and BCNU. Cyclophosphamide and BCNU (carmustine) each have two chloride leaving groups (blue). The presence of two leaving groups allows these alkylating agents to bis-alkylate and thereby cross-link macromolecules such as DNA. The ability to cross-link DNA is crucial to the DNA damage caused by these agents.

Nitrosoureas, such as BCNU (carmustine; Fig. 38-17), target DNA in much the same way as do cyclophosphamide and other alkylating agents. Like cyclophosphamide, these compounds require bioactivation. Unlike most alkylating agents, however, nitrosoureas also attach carbamoyl groups to their DNA-associated targets. It is not clear whether carbamoylation contributes significantly to the activity of nitrosoureas. Some alkylating agents are better than others at targeting specific tumors. For example, nitrosoureas are useful in the treatment of brain tumors, because their high lipid solubility enables them to cross the blood–brain barrier. Similarly, the alkylating antibiotic mitomycin targets hypoxic tumor cells, such as those at the center of a solid tumor, because it requires bioreductive activation, which occurs more readily in low-oxygen environments. Several nonclassical alkylating agents also deserve mention as clinically useful drugs. The first is dacarbazine, a synthetic molecule that is a component of a potentially curative combination chemotherapy regimen for Hodgkin’s disease. Dacarbazine also has some activity in treating melanoma and sarcomas. Procarbazine is an orally active drug that is used against Hodgkin’s disease. A metabolite of procarbazine functions as a monoamine oxidase inhibitor, and toxicity related to this activity—such as tyramine sensitivity, hypotension, and dry mouth—can occur. Temozolomide, an oral alkylating agent, is an imidazotetrazine derivative of dacarbazine. Temozolomide is widely used in the treatment of gliomas and of glioblastoma multiforme in particular. Its action is synergistic with radiation, and it enhances survival in glioblastoma when used in combination with radiotherapy for this disease. Finally, altretamine is useful for treating refractory ovarian cancer. Although it is structurally related to alkylating agents of the triethylenemelamine class (such as thiotepa), whether the mechanism of action of this drug involves DNA alkylation remains controversial. Through natural selection, tumor cells can develop resistance to a single alkylating agent as well as cross-resistance to other drugs in the same class. Several mechanisms for resistance have been reported. Highly reactive drugs can be deactivated by intracellular nucleophiles such as glutathione. Alternatively, cells can become resistant by reducing uptake of the drug or accelerating DNA repair. One enzyme, O6-methylguanine-DNA methyltransferase (MGMT), prevents permanent DNA damage by removing alkyl adducts to the O6 position of guanine before DNA cross-links are formed. Increased expression of this enzyme in neoplastic cells is associated with resistance to alkylating agents. Conversely, MGMT gene silencing predicts clinical benefit from temozolomide in glioblastoma. Alkylating agent toxicity is dose-dependent and can be severe. As a rule, adverse effects result from damage to DNA of normal cells. Three cell types are preferentially affected by alkylating agents. First, toxicity typically manifests in rapidly proliferating tissues, such as bone marrow, gastrointestinal and genitourinary tract epithelium, and hair follicles. This results in myelosuppression, gastrointestinal distress, and alopecia (hair loss). Second, organ-specific toxicity can result from low activity of a DNA damage repair pathway in that tissue. Third, a tissue can be preferentially affected because the toxic compound accumulates in that tissue; for example, acrolein (a byproduct of the activation of cyclophosphamide or its analogue ifosfamide) can produce hemorrhagic cystitis

688 Principles of Chemotherapy

O N Cl

Cl

N

N

NH N

NH2

DNA chain Mechlorethamine

Guanine

Cl

OH

N N N

N N

NH2

DNA chain

OH

H2N

N

N

N N

Alkylated guanine

A

OH

N

D

Cl

OH

N N

N

B

N

N

DNA chain

DNA chain

N

C

N

N N NH2 Guanine excision from DNA

NH2

Crosslinked DNA

O H

Cl

Cl

OH

N

N

NH2

DNA chain Ring cleavage

N

N DNA chain

N

N

O

N H

N

N HN

O

N

O H

N

DNA chain

N H

Abnormal base pairing (alkylated guanine hydrogen-bonded to thymine)

FIGURE 38-18. Biochemical outcomes of guanine alkylation. In reactions such as those exemplified here with mechlorethamine, guanine alkylation can cause several types of DNA damage. The nitrogen of mechlorethamine performs a nucleophilic attack on one of its own ␤-carbons, resulting in an unstable intermediate that is highly electrophilic (not shown). The nucleophilic N-7 of guanine reacts with this unstable intermediate, resulting in an alkylated guanine. There are four potential outcomes that can result from this initial alkylation, all of which cause structural damage to DNA. A. The process of alkylation can be repeated, with a second guanine acting as a nucleophile. The resulting cross-linking of DNA appears to be a major mechanism by which alkylating agents damage DNA. B. Cleavage of the imidazole ring disrupts the structure of the guanine base. C. The alkylated guanine can hydrogen-bond to thymine rather than cytosine, leading to a mutation in the DNA. D. Excision of the alkylated guanine residue results in a depurinated DNA strand. because of accumulation and concentration in the bladder (Fig. 38-19). This toxicity can be treated by using the sulfhydryl-containing molecule mesna, which is also concentrated in the urine and rapidly inactivates the acrolein. The immune response requires rapid proliferation of lymphocytes; this makes lymphocytes especially vulnerable to damage by alkylating agents. Thus, in addition to their anticancer activity, alkylating agents such as cyclophosphamide are also effective at immunosuppression. This “toxicity” has been put to clinical use: administered at doses lower than those needed for antineoplastic therapy, alkylating agents are used to treat autoimmune diseases and organ rejection (see Chapter 45, Pharmacology of Immunosuppression).

One approach to limiting toxicity has been to develop alkylating agents that accumulate preferentially inside tumor cells. An example of one such agent is melphalan, or phenylalanine mustard; this agent was designed to target melanoma cells, which accumulate phenylalanine for the biosynthesis of melanin. Another example is estramustine, in which the mustard component is conjugated to estrogen; this agent was designed to target breast cancer cells that express the estrogen receptor. Interestingly, neither melphalan nor estramustine works as intended, although they both have clinical utility; through mechanisms that are still poorly understood, melphalan is active against multiple myeloma, and estramustine is used to treat prostate cancer.

Cl H O N P

N

O Cl

Cyclophosphamide Prodrug (inactive) Liver cytochrome P450 oxidase Cl H O N P

HO

Cl

O

N

H O N P

O

N

O

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Cl

4-Hydroxycyclophosphamide (active)

4-Ketocyclophosphamide (inactive)

O

O P H2N

H

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H2N

O

O

H

O

Cl

P

Cl

N OH

+ Cl

Cl

Aldophosphamide (active)

Acrolein (cytotoxic)

Phosphoramide mustard (cytotoxic)

Aldehyde oxidase

O P H2N

HO

Cl N

O

O Cl

Carboxyphosphamide (inactive)

O H3N H3N

Pt

Cl Cl

Cisplatin

H3N H3N

Pt

O O

O Carboplatin

690 Principles of Chemotherapy A

B

C

FIGURE 38-21. Interactions of bleomycin, platinum compounds, and anthracyclines with DNA. A. Bleomycin (highlighted in orange) binds to the DNA double helix and thereby exposes nucleotides in the DNA to the iron (II) atom (large red ball ) that is complexed to bleomycin. In the presence of molecular oxygen, the iron– bleomycin complex generates activated oxygen species that cause single-stranded and double-stranded breaks in the DNA by a free radical mechanism. B. Platinum complexes (highlighted in orange) cross-link N-7 atoms on adjacent guanine residues, forming intrastrand DNA cross-links. C. Daunorubicin, an anthracycline (highlighted in orange), intercalates into DNA structure (see expanded view on right ) and thereby prevents the strand passage and religation steps that are part of the catalytic cycle of type II topoisomerase (see Fig. 33-4). Anthracyclines may also damage DNA by a free radical mechanism. nephrotoxicity without diminishing its antitumor effects. Carboplatin, a cisplatin analogue associated with less nephrotoxicity, has replaced cisplatin in many chemotherapy regimens. Oxaliplatin, a third platinum compound, has activity in the treatment of colorectal cancer. Like cisplatin, oxaliplatin causes cumulative neurotoxicity; oxaliplatin also induces a unique acute neurotoxicity that is exacerbated by exposure to cold temperatures. In non-small cell lung cancer, it has been suggested that levels of the nucleotide excision repair protein ERCC1 predict responsiveness to adjuvant platinum-based chemotherapy.

As described earlier, the NER pathway functions to remove bulky DNA adducts formed by agents such as cisplatin. In fact, lower levels of ERCC1 have been associated with greater benefit from platinum-based therapy, presumably since cells with dysfunctional NER cannot repair platinuminduced DNA damage. Bleomycin The bleomycins, a family of natural glycopeptides synthesized by a species of Streptomyces, have prominent cytotoxic activity. A mixture of several of these glycopeptides,

Vinca alkaloids Exchangeable GTP binding site

V

Taxanes

β-tubulin

T

Nonexchangeable GTP binding site

C

α-tubulin

CHAPTER 38 / Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance 693

important adverse effects. An acute hypersensitivity reaction occurs commonly in response to paclitaxel, or more likely to the vehicle in which paclitaxel is solubilized; this effect can be obviated by administration of dexamethasone (a glucocorticoid receptor agonist) and a histamine H1 receptor antagonist before treatment with paclitaxel. Many patients experience myalgias and myelosuppression from paclitaxel, and high doses of the drug can cause pulmonary toxicity. Peripheral neuropathy, typically manifesting as a “stocking and glove” sensory deficit in the extremities, can limit the cumulative amount of drug that can be administered safely. Abraxane® is an albumin-bound form of paclitaxel with a mean particle size of 130 nanometers. The albumin-bound paclitaxel nanoparticles do not cause a hypersensitivity reaction, do not require premedication, and cause less myelosuppression than traditional, solvent-based paclitaxel. Abraxane is currently approved for the treatment of metastatic breast cancer and is being tested for activity in other tumor types. Docetaxel is most commonly used in the treatment of breast cancer and non-small cell lung cancer. As with paclitaxel, docetaxel causes an acute hypersensitivity reaction that can be obviated by preadministration of glucocorticoids. Docetaxel occasionally exhibits the drug-specific adverse effect of fluid retention, which likely arises from increased capillary permeability. Docetaxel does not cause neuropathy as frequently as paclitaxel. The myelosuppression associated with docetaxel is profound, however, and is usually dose-limiting.

 CONCLUSION AND FUTURE DIRECTIONS The antineoplastic agents described in this chapter exert their effects on the genome by preventing efficient DNA replication, inducing DNA damage, and interfering with mitosis. Because many normal cells as well as cancer cells are transiting through the cell cycle, these agents are associated with multiple dose-limiting toxicities. In addition, although cancer cells are susceptible to DNA damage, in some instances, mutations in key checkpoint proteins such as p53 can prevent the apoptosis that would otherwise be induced by these agents. Novel approaches are being developed to target DNA damage more specifically. For example, it has been shown that mice deficient in PARP1 are able to overcome the defect in single-strand break repair by converting single-strand breaks to double-strand breaks and then repairing the DNA by the DSB repair pathway. Furthermore, normal human cells treated in culture with PARP1 inhibitors are capable

of undergoing normal cell division, although these cells do manifest increased susceptibility to DNA damage as a consequence of defective single-strand break repair. In contrast, cells deficient in BRCA1 or BRCA2, which are involved in DSB repair, are killed in response to treatment with PARP1 inhibitors; compared to normal cells, BRCA1⫺ or BRCA2⫺ cells are up to 1,000-fold more sensitive to the action of PARP1 inhibitors. Presumably, the BRCA1⫺ and BRCA2⫺ cells are more sensitive due to impairment of both single-strand break and DSB repair pathways, resulting in lethal accumulation of DNA damage. Based on these findings, PARP1 inhibitors are currently in clinical trials for the treatment of BRCA-deficient breast or ovarian cancer and may be effective in other tumors in which the DNA damage response is compromised. The observation that telomerase is expressed in most cancer cells and is a key component of the process of immortalization highlights this enzyme as an important target in future cancer therapy. Although telomerase is expressed to some degree in stem cells and in normally cycling cells, most normal cells lack telomerase expression. Therefore, the dependency of tumor cells on the immortalized state could provide telomerase inhibitors with a favorable therapeutic index. However, effective agents have yet to be discovered, and one concern is that multiple cell divisions may be required for telomere length to shorten to a level that is critical for cell survival. Combinations of telomerase inhibitors with traditional cytotoxic agents or newer molecularly targeted therapies could yield synergistic effects. Such strategies, as well as those described in Chapter 39, Pharmacology of Cancer: Signal Transduction, will help to advance cancer therapy by moving beyond general cytotoxic approaches and focusing treatment instead on the molecular abnormalities responsible for driving oncogenesis.

Suggested Reading Brody LC. Treating cancer by targeting a weakness. N Engl J Med 2005;353:949–950. (Advances in targeted cancer therapy.) Gazdar A. DNA repair and survival in lung cancer. N Engl J Med 2007;356:771–773. (DNA repair pathway status in relationship to survival and chemotherapy responsiveness.) Hahn WC. Role of telomeres and telomerase in the pathogenesis of human cancer. J Clin Oncol 2003;21:2034–2043. (Possible therapeutic applications of telomerase inhibitors.) Peltomaki P. Role of DNA mismatch repair defects in the pathogenesis of human cancer. J Clin Oncol 2003;21:1174–1179. (Insights into pathophysiology of DNA repair mechanisms.) Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 2002;108:171–182. (Pathophysiology of BRCA1 and BRCA2.)

39 Pharmacology of Cancer: Signal Transduction David A. Barbie and David A. Frank

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 699-700 BIOCHEMISTRY OF INTERCELLULAR AND INTRACELLULAR SIGNAL TRANSDUCTION . . . . . . . . . 699 Growth Factors and Growth Factor Receptors . . . . . . . . . 699 Intracellular Signal Transduction Pathways. . . . . . . . . . . . 700 Proteasome Structure and Function . . . . . . . . . . . . . . . . . 701 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 707 Growth Factor Receptor and Signal Transduction Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . 707 EGF Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . 707 BCR-ABL/C-KIT/PDGFR Inhibition . . . . . . . . . . . . . . . . 708

FLT3 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708 JAK2 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709 RAS/MAP Kinase Pathway Inhibition . . . . . . . . . . . . . . 709 mTOR Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709 Proteasome Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 710 Angiogenesis Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . 710 Anti-VEGF Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 710 VEGFR Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711 Thalidomide and Lenalidomide . . . . . . . . . . . . . . . . . . 711 Tumor-Specific Monoclonal Antibodies . . . . . . . . . . . . . . . 711 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 712 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712

 INTRODUCTION

 BIOCHEMISTRY OF INTERCELLULAR AND INTRACELLULAR SIGNAL TRANSDUCTION

Traditional antineoplastic therapy has consisted of agents directed against DNA replication and cell division. These drugs exhibit some degree of selectivity against cancer cells, which tend to have a higher growth fraction and, in some cases, an increased susceptibility to DNA damage compared to normal cells. However, the therapeutic window of these drugs is narrow, resulting in toxicity to normal stem cells and in hematologic and gastrointestinal adverse effects. With the impressive advances in basic tumor cell biology over the last several decades and the identification of numerous oncogenes and tumor suppressor genes, the potential exists for development of agents that are targeted more specifically at the molecular circuitry responsible for the dysregulated proliferation of cancer cells. An early example of such a drug is the selective estrogen receptor modulator tamoxifen (see Chapter 29, Pharmacology of Reproduction), which has been one of the most active agents in the treatment of hormone receptor-positive breast cancer, with a relatively modest adverse effect profile. More recently, the remarkable success of imatinib mesylate in the treatment of chronic myelogenous leukemia has suggested that, in some cases, tumor cells are dependent on oncogenes such as BCR-ABL for their survival. This chapter highlights basic principles of targeted cancer therapy, detailing recent advances and directions for the future.

Growth Factors and Growth Factor Receptors Stimulation of cell growth and proliferation by external signals is mediated by the interaction of growth factors with specific cell surface receptors. Growth factor receptors typically contain an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic tail that has either intrinsic tyrosine kinase activity or an associated protein tyrosine kinase (Fig. 39-1A, B). In general, binding of the growth factor ligand results in receptor oligomerization, a conformational change in the cytoplasmic domain of the receptor, and tyrosine kinase activation. Intracellular targets are subsequently phosphorylated, propagating a signal that culminates in progression through the cell cycle and cellular proliferation. One example of a receptor tyrosine kinase is the epidermal growth factor receptor (EGFR), which possesses intrinsic tyrosine kinase activity and is a member of the broader ErbB family of proteins, including EGFR (ErbB1), HER2/neu (ErbB2), ErbB3, and ErbB4. Binding of epidermal growth factor (EGF) or transforming growth factor-␣ (TGF-␣) to EGFR results in receptor homodimerization and propagation of a growth signal. In addition, heterodimerization between 699

A

Ligand (e.g., EGF)

Ligandbinding domain Transmembrane domain Tyr

Tyrosine kinase domain

Tyr

P Tyr

Tyrosine kinase activity Tyr

B

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P Tyr

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Ligand (e.g., EPO)

Ligandbinding domain Transmembrane domain Tyr

Inactive protein tyrosine kinase (e.g., JAK2)

Tyr

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Tyr P

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P Tyr

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702 Principles of Chemotherapy A

B

Cetuximab (anti-ErbB1) Trastuzumab (anti-ErbB2)

EGFR

IGF1

EGF

IGF1R

P Tyr

P

Gefitinib Erlotinib

Tyr P

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P P Tyr

Tyr P

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ABL

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Synthesis of proteins needed for cell growth and cell cycle progression

C EPO

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P Tyr

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STAT STAT P

FIGURE 39-2. Intracellular signaling pathways. A. The RAS-MAP kinase pathway is activated by multiple growth factor receptors (here exemplified by the EGF receptor, EGFR) as well as several intracellular tyrosine kinases such as SRC and ABL. RAS is recruited to the plasma membrane by farnesylation and activated by binding to GTP. Activated RAS stimulates a sequence of phosphorylation events mediated by RAF, MEK, and ERK (MAP) kinases. Activated MAP kinase (MAPK) translocates to the nucleus and activates proteins such as MYC, JUN, and FOS that promote the transcription of genes involved in cell cycle progression. Cetuximab and trastuzumab act as antagonists at the EGF receptor (ErbB1) and HER2 receptor (ErbB2), respectively. Gefitinib and erlotinib inhibit the receptor tyrosine kinase. Farnesyltransferase inhibitors prevent RAS activation. Imatinib and dasatinib inhibit ABL kinase; sorafenib inhibits RAF kinase; and several agents under development (see text) inhibit MEK kinase. B. The PI3 kinase (PI3K) pathway is activated by RAS and by a number of growth factor receptors (here exemplified by the insulin-like growth factor receptor 1 [IGF1R] and the epidermal growth factor receptor [EGFR]). Activated PI3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), which activates phosphoinositide dependent kinase-1 (PDK). In turn, PDK phosphorylates AKT. PTEN is an endogenous inhibitor of AKT activation. Phosphorylated AKT transduces multiple downstream signals, including activation of the mammalian target of rapamycin (mTOR) and inhibition of the FOXO family of transcription factors. mTOR activation promotes the synthesis of proteins required for cell growth and cell cycle progression. Because the FOXO family of transcription factors activates the expression of genes involved in cell cycle arrest, stress resistance, and apoptosis, inhibition of FOXO promotes cell proliferation and resistance to apoptosis. Rapamycin (sirolimus) and its derivatives are mTOR inhibitors that inhibit cell cycle progression and promote apoptosis. C. The STAT pathway is activated by SRC and by a number of growth factor receptors (here exemplified by the erythropoietin receptor [EPOR], which signals to STAT proteins through JAK2 kinase, and by the EGF receptor [EGFR], which signals to STAT proteins indirectly). Phosphorylation of STAT induces SH2 domain-mediated homodimerization, and phosphorylated STAT homodimers translocate to the nucleus and activate transcription. JAK2 inhibitors are under development for the treatment of polycythemia vera and other myeloproliferative disorders, many of which share a common activating mutation in JAK2 (V617F).

CHAPTER 39 / Pharmacology of Cancer: Signal Transduction 703

MAPK

Cyclin D

p16

CDK4/6

Cyclin D CDK4/6

P

RB

P

P

P

RB S phase genes

S phase genes

E2F

E2F

G1 phase

S phase

FIGURE 39-3. Regulation of the G1–S cell cycle transition. Activation of MAP kinase results in increased expression of the D-type cyclins. Cyclin D binds to its catalytic partners cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), which phosphorylate the retinoblastoma protein (RB). Phosphorylation of RB releases its transcriptional repression of S-phase genes, allowing the transcription factor E2F to activate the transcription of genes needed for entry into S phase. These genes include cyclin E as well as DNA polymerase and the enzymes involved in nucleotide synthesis. Cyclin E binds to its catalytic partner CDK2, which further phosphorylates RB, creating a positive feedback loop that drives cells into S phase (not shown). The CDK2/CDK4/CDK6 system is counterbalanced by cyclin-dependent kinase inhibitors (CDKIs) such as p16, which inhibits CDK4/6, and p21 and p27, which inhibit CDK2 (not shown).

B

specificity. The E3 ubiquitin ligase component is largely responsible for target protein specificity. The RING family of E3 ligases contains a characteristic RING finger domain with conserved histidine and cysteine residues complexed with two central Zn2⫹ ions. RING E3 ligases can be subdivided into single-subunit E3 ligases and multisubunit complexes such as the Skp1-Cullin-F-box protein family (SCF) E3 ligases. In the latter complexes, the RING finger component, Rbx, is distinct from the specificity component, the F-box protein, which is so named because of a characteristic motif first identified in cyclin F. Once proteins are selectively ubiquitinated, they are targeted for degradation by the 26S proteasome, which is a cylindrical particle present in both the cytoplasm and nucleus. The core 20S subunit is the catalytic component with multiple proteolytic sites, while the 19S regulatory component mediates binding to ubiquitin-conjugated proteins and has multiple ATPases involved in substrate unfolding and delivery to the central 20S chamber. Substrates are cleaved progressively, with one protein being completely degraded before the next protein enters. Short peptide segments, on average 6 to 10 amino acids in length, are extruded and subsequently hydrolyzed to individual amino acids in the cytosol. Regulation of protein degradation occurs largely at the level of the E3 ubiquitin ligase and governs key aspects of cell cycle control, apoptosis, and other important cellular processes (Fig. 39-4B). For example, CBL is a single-subunit RING E3 ubiquitin ligase that targets phosphorylated EGFR family members for degradation. In addition, both cyclins and cyclin-dependent kinase inhibitors are major targets for

Single-subunit RING E3 ligases

Multisubunit RING E3 ligases

Ub Ub

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FIGURE 39-4. The ubiquitin–proteasome pathway. A. Ubiquitin (Ub) is activated by ATP-dependent conjugation to E1, the first enzyme in the pathway. Activated ubiquitin is then passed from the active-site cysteine of E1 to the active-site cysteine of the ubiquitin-conjugating enzyme E2, which functions coordinately with the ubiquitin ligase E3 to attach ubiquitin to protein targets. Polyubiquitination of target proteins results in their recognition by the 26S proteasome, which consists of a 19S outer regulatory subunit and a 20S internal core chamber. The proteasome mediates proteolytic degradation of the target protein into short peptide fragments. Bortezomib is a proteasome inhibitor that has been approved for use in multiple myeloma and is under investigation for use in other malignancies. B. The RING family of E3 ubiquitin ligases consists of single-subunit enzymes (left) and multisubunit protein complexes (right). Single-subunit ligases include CBL, which targets EGFR for degradation, and MDM2, which targets p53 for degradation. Multisubunit RING E3 ligase complexes include SCF and SCF-like family members, which are named for their Skp1, Cullin, and F-box protein subunits. The F-box protein component mediates target protein specificity; for example, SKP2 targets p27 and FOXO for degradation, Fbw7 targets cyclin E for degradation, and ␤TrCP targets ␤-catenin and I␬B␣ for degradation. SCF-like ligase complexes include the anaphase promoting complex, which targets cyclin B for degradation, and VHL, which targets the ␣ subunit of hypoxia-inducible factor-1␣ (HIF-1␣) for degradation.

704 Principles of Chemotherapy

ubiquitin-mediated proteasomal degradation. The anaphasepromoting complex is a multiprotein RING-containing E3 ligase that is activated by phosphorylation late in mitosis, triggering degradation of cyclin B and progression through mitosis. Regulation of the G1–S cell cycle transition is in part mediated by the cyclin-dependent kinase inhibitor p27, which inhibits cyclin E–CDK2 and cyclin A–CDK2 complexes. Degradation of p27 is regulated by another SCF E3 ligase, which binds p27 via its F-box specificity component Skp2. Thus, overexpression of Skp2, which is found in a number of tumor types, can promote cell cycle progression by degrading p27. Degradation of FOXO by Skp2 is a second mechanism by which overexpression of Skp2 may promote tumorigenesis. Yet another SCF E3 ligase complex regulates cyclin E activity by targeting it for degradation via the F-box protein Fbw7. Loss of Fbw7 has been implicated in tumor progression due to high levels of cyclin E. Another example of an E3 ligase with a critical role in the regulation of apoptosis and cell cycle regulation is MDM2,

a single-subunit RING finger E3 ligase that targets p53 for degradation. Activation of MDM2 is linked to impairment of apoptosis and promotion of tumorigenesis via loss of p53. MDM2 is inhibited by the p14ARF protein, which shares the same genomic locus as the CDK4/6 inhibitor p16. Disruption of this locus, which is one of the most common events in cancer, leads ultimately to both p53 and pRB inactivation. Other key cellular pathways regulated by ubiquitinmediated proteasomal degradation include the WNT signaling and nuclear factor-kappa B (NF␬B) pathways. Both pathways are targeted by the common F-box protein ␤TrCP, which recognizes phosphorylated substrates (Fig. 39-5). Activation of WNT signaling prevents phosphorylation of ␤-catenin, which allows it to escape recognition by ␤TrCP and ubiquitin ligation by SCF E3 ligase. Unphosphorylated ␤-catenin then translocates to the nucleus with its partners TCF/LEF and activates transcription of genes such as myc and cyclin D1. This pathway is also regulated by the adenomatous polyposis coli (APC) gene, which forms part Wnt

A

Frizzled

Absence of Wnt

APC complex (active)

β-catenin

β-catenin

βTrCP complex

P

Disheveled

P

APC complex (inactive)

β-catenin

Ub Ub Ub Ub Ub

β-catenin

P

Nucleus

26S proteasome β-catenin TCF/ LEF

Transcription of genes promoting cell cycle progression

Ub Ub

β-catenin fragments WNT signaling and NF␬B pathways. A. In the absence of WNT signaling, ␤-catenin is phosphorylated by the adenomatous polyposis coli (APC) protein complex. Phosphorylated ␤-catenin is recognized by ␤TrCP and thereby targeted for ubiquitin-mediated proteasomal degradation. Activation of WNT signaling inhibits APC function, allowing ␤-catenin to accumulate and translocate to the nucleus. In the nucleus, ␤-catenin complexes with its partners TCF/LEF and activates the transcription of genes promoting cell cycle progression. Hereditary or acquired loss of APC allows accumulation of ␤-catenin, contributing to oncogenesis in colon cancer. (continued)

FIGURE 39-5.

CHAPTER 39 / Pharmacology of Cancer: Signal Transduction 705

B

Absence of stimuli

IκB kinase (inactive)

IκB

Multiple stimuli

IκB kinase (active)

NFκB

Inactive complex

IκB

IκB

NFκB

P

βTrCP complex

Ub Ub Ub Ub Ub

IκB

P

Nucleus

26S proteasome NFκB

Transcription of genes involved in proliferation and inflammation

Ub Ub

IκB fragments

FIGURE 39-5.

(continued) B. Similarly, the I␬B protein is targeted for ubiquitin-mediated proteasomal degradation as a result of phosphorylation by I␬B kinase and recognition by ␤TrCP. In the absence of stimuli, I␬B binds to and inhibits NF␬B. In the presence of stimuli, proteasomal degradation of I␬B allows NF␬B to translocate to the nucleus and activate the transcription of genes involved in proliferation and inflammation.

of the complex that promotes phosphorylation and subsequent destruction of ␤-catenin. Loss of APC in colorectal cells prevents phosphorylation of ␤-catenin, leading to its accumulation and promotion of cancer. The F-box protein ␤TrCP also regulates signaling through NF␬B, which is inhibited by its association with the inhibitor of NF␬B (I␬B). Phosphorylation of I␬B by a family of I␬B kinases (IKKs) allows ␤TrCP to bind to I␬B and activate its proteasome-mediated destruction. The release of I␬B inhibition allows NF␬B to translocate to the nucleus and activate transcription of genes involved in inflammation, proliferation, and survival. Specific IKKs may be aberrantly activated in cancer cells and thereby generate an environment that favors tumor cell survival.

Angiogenesis Solid tumors require development of a neovasculature in order to sustain growth and survive conditions of hypoxia. Tumor angiogenesis is a complex process involving a number of different pro- and antiangiogenic factors. The vascular endothelial growth factor (VEGF) family of proteins and receptors has emerged as a key regulator of this process. The VEGF family consists of seven ligands, including VEGF-A, -B, -C, -D, and -E and placenta growth factor (PlGF)-1 and -2 (Table

39-2). These ligands have varying affinities for the major VEGF receptors, VEGFR1 (also known as Flt-1), VEGFR2 (Flk-1/KDR), and VEGFR3 (Flt-4). The VEGF receptors are receptor tyrosine kinases. Neuropilins (NRP-1 and -2) are coreceptors that lack an intracellular signaling domain and enhance the binding of ligand to VEGFR1 and VEGFR2. VEGFR1 and VEGFR2 are expressed on the vascular endothelium and play key roles in angiogenic signaling, while signaling through VEGFR3 appears to play a major role in lymphangiogenesis (i.e., development of new lymphatic vessels). VEGFR2, which appears to be the major proangiogenic receptor that is targeted by VEGF-A, has been shown to signal via both a RAF/MAP kinase pathway to promote proliferation of endothelial cells and a PI3K/AKT pathway to promote endothelial cell survival. VEGF also potently induces vascular permeability, utilizing similar signaling pathways both to promote the formation of transendothelial vesicular organelles and to open interendothelial junctions. Invasion and migration of endothelial cells is promoted by activation of matrix metalloproteinases and serine proteases and by reorganization of intracellular actin. Activation of VEGF is mediated by stimuli such as hypoxia, by cytokines and growth factors, and by a variety of oncogenes and tumor suppressor genes. Regulation of the response to hypoxia is mediated by von Hippel-Lindau

Normal or high O2

Low O2

HIF-1α

HIF-1α PHD

PHD O2

HIF-1α

OH

VHL complex

Ub Ub Ub Ub Ub

HIF-1α

OH

Nucleus

26S proteasome HIF-1α HIF-1β Ub Ub

HIF-1α fragments

Transcription of VEGF, PDGF-β, TGF-α, EPO genes

708 Principles of Chemotherapy

Trastuzumab, another chimeric mouse/human IgG monoclonal antibody, is directed against ErbB2 (HER2) (Fig. 39-2A). Approximately 25–30% of breast cancers are associated with amplification and overexpression of Her2/neu; these cancers also display more aggressive behavior. HER2 amplifies the signal generated by other ErbB family members via the formation of heterodimers. Trastuzumab down-regulates HER2 and thereby disrupts this signaling. In vivo, trastuzumab also appears to induce antibody-dependent cellular cytotoxicity and inhibit angiogenesis. Trastuzumab has significant activity in breast cancers with high levels of HER2 amplification. In addition to this intrinsic activity of trastuzumab in the advanced and metastatic breast cancer settings, treatment of HER2-amplified breast cancers with trastuzumab in the adjuvant setting (i.e., after surgical resection of the tumor) enhances the efficacy of chemotherapy and reduces rates of cancer recurrence by 50%. The principal adverse effect of trastuzumab is cardiotoxicity, particularly when used in combination with anthracyclines. Trastuzumab does not cross the blood–brain barrier, and thus CNS relapse of breast cancer may occur. Lapatinib, a small-molecule dual EGFR/HER2 inhibitor, has also been approved for the treatment of metastatic breast cancer with HER2 overexpression. Lapatinib crosses the blood–brain barrier and shows activity against brain metastases. BCR-ABL/C-KIT/PDGFR Inhibition Imatinib

Imatinib is a small-molecule tyrosine kinase inhibitor that was initially developed as a 2-phenylaminopyrimidine derivative specific for PDGFR. Imatinib was subsequently found to be a potent inhibitor of ABL kinases, including the BCRABL fusion protein generated as a result of the t(9;22) chromosomal translocation (Philadelphia chromosome) found in chronic myelogenous leukemia (CML), and was also found to inhibit the receptor tyrosine kinase C-KIT (Fig. 39-2A). Imatinib is the canonical example of a targeted therapeutic agent, because BCR-ABL is uniquely expressed by leukemic cells and is essential for their survival. Initial in vitro studies demonstrated that imatinib potently and specifically inhibits the growth of cells expressing BCRABL. Subsequent evaluation of an oral formulation in mice demonstrated suppression of growth of human BCR-ABLpositive tumors with minimal adverse effects. Early studies of imatinib in patients with chronic-phase CML yielded impressive results, with normalization of blood counts (a hematologic response) in 95% of patients and significant reduction in cells expressing the Philadelphia chromosome (a cytogenetic response) in 41% of patients. In a phase III study, imatinib was superior to standard treatment with interferon and cytarabine in patients with chronic-phase CML, with a hematologic response rate of 95% and a complete cytogenetic response in 76% of patients. Treatment of accelerated or blast-phase CML with imatinib is less effective but is associated with some responses. Imatinib is relatively well tolerated; its principal adverse effects are myelosuppression, superficial edema, nausea, muscle cramps, skin rash, and diarrhea. Given the relatively recent development of imatinib, longer-term follow-up is needed to determine how sustained the responses will be over time. Indeed, a significant fraction of patients still show evidence of the BCR-ABL transcript when sensitive tests such as reverse transcriptase polymerase

chain reaction (RT-PCR) are used for detection, even in cases where a complete cytogenetic response is observed. Mutation of C-KIT, the receptor for stem cell factor (SCF), is found frequently in gastrointestinal stromal tumor (GIST) and in the myeloproliferative disorder systemic mastocytosis. In GIST, mutations and in-frame deletions of C-KIT are typically found in the juxtamembrane domain, resulting in constitutive activation of the tyrosine kinase in the absence of ligand. In contrast, in systemic mastocytosis, the characteristic D816V activating C-KIT mutation is within the tyrosine kinase domain itself. Imatinib has shown significant activity in advanced gastrointestinal stromal tumor, but it has proven largely ineffective in the treatment of systemic mastocytosis. Indeed, biochemical studies show that the drug is not effective at targeting C-KIT kinases with the D816V mutation. Both the idiopathic hypereosinophilic syndrome and a variant of systemic mastocytosis with eosinophilia are characterized by the expression of the FIPL1-PDGFRA fusion protein. This protein, which is generated by an interstitial chromosomal deletion, causes constitutive signaling through PDGFRA. Inhibition of PDGFRA by imatinib has been a successful therapeutic approach in both conditions. Dasatinib and Nilotinib

Crystallographic studies show that imatinib targets the ATPbinding site of ABL only when the activation loop of the kinase is closed, thereby stabilizing the protein in an inactive conformation (see Fig. 1-2). Clinical resistance to imatinib has been recognized in some patients with CML, occasionally due to amplification of BCR-ABL, but more commonly due to the acquisition of resistance mutations. Only a fraction of these mutations directly interfere with drug binding; instead, most mutations affect the ability of ABL to adopt the closed conformation to which imatinib binds. A second class of tyrosine kinase inhibitors, the dual SRC–ABL inhibitors, can bind to the ATP-binding site in ABL irrespective of the conformational status of the activation loop. One of these drugs, dasatinib, has significantly greater efficacy than imatinib against wild-type BCR-ABL, and it inhibits the activity of most clinically relevant imatinib-resistant BCR-ABL isoforms, with the exception of the T315I mutation (Fig. 39-2A). Another structure-based approach to improve the efficacy of imatinib has been to substitute alternative binding groups for the N-methylpiperazine group, resulting in the development of nilotinib. Similar to dasatinib, the affinity of nilotinib for wild-type BCR-ABL is significantly higher than that of imatinib, and nilotinib inhibits most imatinib-resistant mutants with the exception of T315I. Both dasatinib and nilotinib have shown activity in patients with CML who have developed resistance to imatinib, and both are undergoing further clinical testing. Both drugs also inhibit C-KIT kinases with the D816V mutation in vitro and are being tested in patients with systemic mastocytosis. FLT3 Inhibitors One of the most common mutations in acute myelogenous leukemia (AML), occurring in approximately 25–30% of patients, involves internal tandem duplication within the juxtamembrane domain of the FLT3 receptor tyrosine kinase. This mutation results in ligand-independent dimerization and activation of signaling via the RAS/MAPK and STAT

CHAPTER 39 / Pharmacology of Cancer: Signal Transduction 709

pathways. A number of FLT3 inhibitors have been developed and demonstrate anti-leukemia cell activity in vitro. Several experimental agents, such as PKC412, have demonstrated single-agent activity in patients with relapsed/refractory AML carrying FLT3 mutations. Studies are under way to examine whether FLT3 inhibitors may improve outcomes in AML in combination with standard chemotherapy. JAK2 Inhibitors Despite the success of imatinib in the treatment of CML, the genetic basis of the other major myeloproliferative disorders (polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis) has, until recently, remained obscure. It is now apparent that a common activating mutation in JAK2 (V617F) underlies the aberrant signaling and proliferation in most cases, although how one mutation leads to this spectrum of disorders remains unclear (see Fig. 39-2C). The V617F mutation is found in the pseudokinase domain of JAK2, and disruption of this autoinhibitory region leads to unchecked activity of the kinase. In vitro, selective JAK2 inhibitors cause cells containing the JAK2 V617F mutation to be growth inhibited and undergo apoptosis; and in animal models, JAK2 inhibitors demonstrate therapeutic efficacy against JAK2(V617F)-induced hematologic disease. Thus, JAK2 inhibitors are under development for the treatment of polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. RAS/MAP Kinase Pathway Inhibition Oncogenic mutation of ras is one of the most common events in malignancy, occurring in approximately 30% of human cancers. K-ras mutations are frequently observed in non-small cell lung cancer, colorectal cancer, and pancreatic carcinoma, while H-ras mutations are found in kidney, bladder, and thyroid cancers, and N-ras mutations occur in melanoma, hepatocellular carcinoma, and hematologic malignancies. However, despite the frequency of these mutations, inhibition of RAS has thus far been difficult to achieve and has yielded minimal clinical success. Most efforts have been focused on targeting farnesylation of RAS and inhibiting downstream effectors. Farnesylation of RAS is essential for its association with the plasma membrane and subsequent activation. A number of farnesyltransferase inhibitors (FTIs) have been developed that inhibit RAS farnesylation (Fig. 39-2A). While these inhibitors demonstrate activity against RAS in vitro, some RAS mutants exhibit resistance. Moreover, there are many other targets of farnesylation that could be inhibited by FTIs and are likely responsible for the cytotoxic effects of these drugs. FTIs that have been tested clinically include tipifarnib and lonafarnib. Tipifarnib has demonstrated activity in relapsed/refractory AML, although responses appear to be independent of ras mutations. Clinical testing of FTIs in solid tumors has not yet met with success. Immediately downstream of RAS is the serine/threonine kinase RAF, which phosphorylates MEK, which in turn phosphorylates MAP kinase, leading to transcription factor activation (Fig. 39-2A). There are three RAF family members—A-RAF, B-RAF, and C-RAF. Activating mutations in B-RAF have been found in a significant proportion of malignant melanomas and are also observed at a lower frequency in lung, colorectal, ovarian, and thyroid cancers. Sorafenib was initially designed as a C-RAF inhibitor, but it

also demonstrates high inhibitory activity on both wild-type and mutant B-RAF. Sorafenib has shown significant activity against melanoma cell lines that contain activating B-RAF mutations, and the drug is currently in clinical testing for use in melanoma. Sorafenib also inhibits the tyrosine kinase activity of VEGFR-2 and PDGFR-␤ and has demonstrated clinical efficacy in the treatment of advanced renal cell carcinoma and hepatocellular carcinoma. There are two MEK homologues, MEK1 and MEK2, both of which have dual serine-threonine and tyrosine kinase activity, phosphorylating and activating ERK1 and ERK2. CI-1040 is a highly active inhibitor of both MEK1 and MEK2 (Fig. 39-2A). Early clinical testing of CI-1040 in patients with solid tumors has shown some activity but unfavorable pharmacokinetic characteristics. More potent and bioavailable second-generation MEK inhibitors have been developed and are in clinical trials. An important emerging concept is the need to identify specific subsets of cancers that are susceptible to specific targeted agents, exemplified by the sensitivity of EGFR-mutant NSCLC to gefitinib and erlotinib. One current approach is to identify gene expression profiles that are markers of oncogene activation. For example, a specific gene expression profile has been characterized for RAS activation, and this profile correlates with RAS mutation and RAS pathway activation in cell lines and tumor specimens. Only cell lines displaying gene expression profiles concordant with RAS activation respond to FTIs in vitro. Thus, selection of patients for clinical trials based on such an approach may enrich for clinical activity of agents such as FTIs. Another approach has been to identify subsets of RAS pathway activation profiles that predict responsiveness to downstream inhibition of targets such as MEK. Comparison of cell lines with activating N-RAS mutations and those with activating B-RAF mutations has shown that only the latter cell lines exhibit high sensitivity to the MEK inhibitor CI-1040, possibly because MEK is more immediately downstream of RAF. Therefore, selection of patients with tumors containing B-RAF mutations for clinical trials of MEK inhibitors will potentially yield greater efficacy. Finally, it is possible that unanticipated pathways will represent particularly important targets in the presence of oncogenes such as K-RAS. The advent of genetic screening in mammalian cells using RNA interference (RNAi) provides an opportunity to identify particular signaling pathways upon which a tumor may be dependent. mTOR Inhibitors Signaling via the PI3K/AKT pathway leads to downstream activation of the mammalian target of rapamycin (mTOR) (Fig. 39-2B). mTOR is a serine-threonine kinase that regulates multiple cellular functions, including cell growth and proliferation, via activation of protein synthesis. mTOR regulation is accomplished in part by activation of the 40S ribosomal protein S6 kinase (p70S6k) and inactivation of the 4E-binding protein (4E-BP1), which regulates translation of certain mRNAs. Dysregulated mTOR activity is seen in a wide variety of malignancies in which the PI3K pathway is activated or PTEN is lost. In addition, hamartoma syndromes such as tuberous sclerosis result in activation of mTOR. The tuberous sclerosis protein complex (TSC1/2) acts as an intermediary between AKT and mTOR: native TSC1/2 inhibits mTOR, and activation of AKT results in phosphorylation of TSC1/2 and subsequent de-repression of mTOR.

712 Principles of Chemotherapy

found almost exclusively on mature B cells. The anti-CD20 IgG1 monoclonal antibody rituximab has demonstrated significant single-agent activity and enhancement of the effects of chemotherapy in B-cell non-Hodgkin’s lymphoma (NHL), and is now routinely incorporated in the therapy of this disorder. Principal adverse effects include immunosuppression due to the targeting of normal mature B cells and hypersensitivity reactions related to the chimeric nature of the antibody. Conjugation of radioactive isotopes to anti-CD20 antibodies, such as iodine-131 (131I) tositumomab and yttrium-90 (90Y) ibritumomab tiuxetan, has allowed targeted radioimmunotherapy of B-cell NHL. These agents are being incorporated into treatment regimens of patients with refractory disease and as induction therapy for stem cell transplantation. Alemtuzumab is a humanized monoclonal antibody directed against the pan-leukocyte antigen CD52. This agent has been used in the treatment of chronic lymphocytic leukemia (CLL) and as a component of conditioning regimens for stem cell transplantation. Because alemtuzumab induces lysis of both T-cell and B-cell populations, its principal adverse effect is significant immunosuppression, including increased risk for Pneumocystis jiroveci pneumonia and for fungal, cytomegalovirus, and herpesvirus infections. Therefore, prophylaxis for opportunistic infections is required. Two additional examples of antibody conjugates are denileukin diftitox and gemtuzumab ozogamicin. Denileukin diftitox, a recombinant fusion protein composed of fragments of diphtheria toxin and human IL-2, targets the CD25 component of the IL-2 receptor and has demonstrated activity in T-cell NHL. Gemtuzumab ozogamicin is a conjugate between the antitumor antibiotic calicheamicin and a monoclonal antibody directed against CD33, which is found on the surface of leukemic blasts in more than 80% of patients with AML.

 CONCLUSION AND FUTURE DIRECTIONS Elucidation of the molecular and biochemical circuitry that regulates normal cell proliferation and identification of the key mutations that promote oncogenesis have provided the ability to target specific pathways that are dysregulated in tumors. The success of imatinib in the treatment of CML demonstrates that cancers can become dependent on oncogenes such as BCR-ABL, requiring oncoprotein signaling for continued proliferation and survival. Although inhibitors of receptor tyrosine kinases and intracellular kinases have a greater therapeutic index than traditional antineoplastic therapies and have had some success in certain tumors, in many cases, responses are neither durable nor complete. The

identification of subsets of tumors in which specific pathways are activated, such as the EFGR mutation in NSCLC, will guide therapy and improve response rates. Oncogenic microarray signatures and correlations between specific mutations and sensitivity to targeted agents will facilitate the design of clinical trials focusing on subsets of patients with the highest likelihood of response. Efficacy will also be improved with second- and third-generation drugs that have higher specificity for targets and the ability to overcome resistance mutations. It is clear, however, that multiple factors contribute to tumor development, including downstream mutations in pathways regulating cell cycle progression, apoptosis, proteasomal degradation, and angiogenesis. The biology of these processes and of tumor cell invasion and acquisition of metastatic potential will likely provide novel targets for directed therapy. As with combination chemotherapy, the successful targeted therapies of the future will likely involve inhibition of multiple pathways using a combination of agents directed at the defects found in individual tumors. Furthermore, systematic approaches involving RNAi and chemical screens may identify previously unanticipated vulnerabilities associated with specific cancer genotypes, a concept derived from “synthetic lethal” screening in yeast. The higher degree of specificity inherent in such strategies will likely give them a superior therapeutic index compared to traditional combination antineoplastic chemotherapy and will hopefully be met with a greater degree of clinical success.

Suggested Reading Bartlett JB, Dredge K, Dagleish AG. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat Rev Cancer 2004;4:314–322. (Historic and scientific overview of thalidomide and its derivatives.) Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70. (Seminal overview of the characteristic genetic changes leading to oncogenesis.) Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005;23:1011–1027. (Overview of VEGF pathways.) Kaelin WG. The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 2005;5:689–698. (Novel approaches to cancer genotype-guided drug development.) Krause DS, van Etten RA. Tyrosine kinases as targets for cancer therapy. N Engl J Med 2005;353:172–187. (Advances in tyrosine kinase inhibition.) Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005;23:4776–4789. (Biochemical details of ubiquitin pathways.) Wullchleger S, Loewith R, Hall M. TOR signaling in growth and metabolism. Cell 2006;124:471–484. (Possible applications of mTOR inhibitors.)

40 Principles of Combination Chemotherapy Quentin J. Baca, Donald M. Coen, and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 716-717 ANTIMICROBIAL COMBINATION THERAPY . . . . . . . . . . . . . 716 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration . . . . . . . . . . . . . . . . . . . . . . . . 716 Types of Drug Interactions—Synergy, Additivity, and Antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718 Examples of Antimicrobial Combination Therapy . . . . . . . 719 Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719 Synergistic Combinations . . . . . . . . . . . . . . . . . . . . . . 720 Co-administration of Penicillins with ␤-Lactamase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . 721 Polymicrobial and Life-Threatening Infections . . . . . . . 721 Unfavorable Drug Combinations . . . . . . . . . . . . . . . . . . . . 721

ANTIVIRAL COMBINATION THERAPY: HIV . . . . . . . . . . . . . . 721 ANTINEOPLASTIC COMBINATION CHEMOTHERAPY . . . . . . 722 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . 722 Rationale for Combination Chemotherapy . . . . . . . . . . . . 724 Examples of Antineoplastic Combination Chemotherapy. . 725 Hodgkin’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 725 Testicular Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726 Treatment of Refractory or Recurrent Disease . . . . . . . . . 726 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 726 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727

 INTRODUCTION

(2) to enhance the activity (efficacy) of the drug therapy against a specific infection (synergy); (3) to reduce toxicity to the host; (4) to treat multiple simultaneous infections (sometimes called polymicrobial infections); and (5) to treat a life-threatening infection empirically before the microorganism causing the infection has been identified. Because microbes are genetically distant from humans, antimicrobial drug combinations can target several different molecules that are specific to the microbe(s), potentially without a concomitant increase in adverse effects. In contrast, antineoplastic drug combinations are often limited by adverse effects (see below). The following section provides a conceptual framework for the different types of antimicrobial drug interactions and discusses specific examples of antimicrobial combination therapy.

Many infections and some cancers can be successfully treated with single-drug therapies. Such regimens often fail, however, when pathogens or tumors develop resistance to chemotherapeutic agents, when multiple pathogens with different drug susceptibilities are simultaneously present, or when the dose of the therapeutic agent is limited by toxicity. Under these circumstances, combination chemotherapy may offer decisive advantages. The drugs in a multidrug regimen can interact synergistically to enhance the antimicrobial or antineoplastic effectiveness of the combination and can decrease the likelihood that resistance will emerge. Combinations are frequently used when treatment must be initiated before the definitive identification of the pathogen, and synergistic combinations can be used to reduce toxicity when the individual drugs in the combination have low therapeutic indices. Although combination chemotherapy opens new avenues for the expedient elimination of a pathogen or tumor, it also introduces the potential for multiple adverse effects and drug interactions. The goal of any combination drug regimen should be to efficiently remove the offending pathogen or tumor without incurring unacceptable host toxicity.

 ANTIMICROBIAL COMBINATION THERAPY In the treatment of microbial infections, drug combinations are used (1) to prevent the emergence of drug resistance; 716

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Antimicrobial agents with activity against a pathogenic bacterial, protozoal, or fungal microorganism can be characterized by the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for the drug–pathogen pair. The MIC is defined as the lowest concentration of drug that inhibits growth of the microorganism after 18–24 hours of incubation in vitro. The MBC is defined as the lowest concentration of drug that kills 99.9%

718 Principles of Chemotherapy

Remove drug

Number of live bacteria

Add drug

Bacteriostatic drug

Bactericidal drug

Time

FIGURE 40-1. Comparison of the effects of bacteriostatic and bactericidal drugs on bacterial growth kinetics in vitro. In the absence of drug, bacteria grow with exponential (first-order) kinetics. A bactericidal drug kills the target organism, as demonstrated by the time-dependent decrease in the number of live bacteria. A bacteriostatic drug prevents microbial growth without killing the bacteria. Removal of a bacteriostatic drug is followed by an exponential increase in bacterial number as the previously inhibited bacteria resume growth. Bacteriostatic drugs eradicate infections by limiting the growth of the infecting organism for a long enough period of time to allow the host immune system to kill the bacteria.

Microbial killing rate

concentration-dependent (Fig. 40-2). Time-dependent bactericidal agents exhibit a constant rate of killing that is independent of drug concentration, provided that the drug concentration is greater than the minimum bactericidal concentration (MBC). Thus, the overriding consideration for the clinical use of such agents is not the absolute drug concentration that is achieved, but for how long the drug concentration remains in the therapeutic range (which is defined as [drug] ⬎ MBC). In contrast, concentration-dependent

Concentration-dependent drug

Time-dependent drug

MBC

Drug concentration

FIGURE 40-2.

Relationship between rate of microbial killing and drug concentration for time-dependent and concentration-dependent bactericidal drugs. Time-dependent bactericidal agents exhibit a constant rate of microbial killing at concentrations of drug greater than the minimum bactericidal concentration (MBC) (solid line). In contrast, concentration-dependent bactericidal agents show increased killing with increasing drug concentration (dashed line). Note that the efficacy of concentration-dependent bactericidal agents eventually plateaus, because the effective concentration of the drug becomes limited by the rate of drug diffusion to its molecular target

bactericidal agents have a rate of killing that increases with drug concentration for [drug] ⬎ MBC. For such agents, a single very large dose may be sufficient to eliminate the infection.

Types of Drug Interactions—Synergy, Additivity, and Antagonism The discussion has thus far considered the general properties of drugs used as single agents to treat a microbial infection. When such drugs are used in combination with other agents, these effects can be modified (either enhanced or diminished). In fact, drugs that have little or no activity against an organism when used as single agents can show high activity when used in combination with another agent. One example of this concept involves the treatment of Enterococcus faecalis, a Gram-positive organism that exhibits little susceptibility to aminoglycosides. Recall that aminoglycosides are thought to kill bacteria by inducing misreading of the genetic code and translation of defective proteins, which cause further cellular damage (see Chapter 33). In the case of E. faecalis, aminoglycosides are unable to penetrate the organism’s thick cell wall to reach their target, the 30S ribosomal subunit. However, when used in combination with a cell wall synthesis inhibitor such as vancomycin or a ␤-lactam antibiotic, aminoglycosides are able to reach the bacterial ribosomes and effectively kill the bacteria (see Chapter 34). The potentiating effect of the cell wall synthesis inhibitor on the activity of the aminoglycoside is an example of the important pharmacologic concept of synergy. From this example, one could ask whether combining two drugs with individual activity against a particular microbe always results in a more efficacious drug combination. Surprisingly, for many combinations, this turns out not to be the case. In fact, when two drugs with activity against the same pathogen are combined, the drugs can interact to enhance the efficacy of the combination (synergy) or to diminish the efficacy (antagonism). Alternatively, the drugs may not interact, and the effect of the combination is simply the sum of the effects of each drug used individually (additivity). The interaction between two antimicrobial drugs is often quantified by selecting a particular endpoint (e.g., inhibition of bacterial growth) and then measuring the effect of various combinations of the two drugs that reach this endpoint. When such data are plotted, additional information can be obtained (Fig. 40-3). The x- and y-intercepts correspond to the MICs of the two drugs, and the concavity of the curve indicates the nature of the interaction between the two drugs—concave-up is synergistic; concave-down is antagonistic; linear is additive. The following discussion provides a mathematical rationale for these relationships. Suppose that drugs A and B inhibit a particular enzyme required for bacterial growth. In this case, the ratio [A]/MICA would represent the fraction of bacterial growth inhibition that can be attributed to the presence of drug A. This is known as the fractional inhibitory concentration of A (FICA). Similarly, FICB ⫽ [B]/MICB is the fraction of growth inhibition that can be attributed to drug B. Now, suppose that the concentration of A is decreased by a small amount, ⫺d[A]. To compensate for this loss of growth inhibition (dFICA ⫽ ⫺d[A]/MICA), the concentration of B must be increased by an amount ⫹d[B]. For additive drugs, the ratio ⫺d[A]/d[B] (which is the same as the slope of the curve in Fig. 40-3) is a constant because one

A0

MIC of drug A

Antagonistic

Additive

Synergistic

0 0

B0

MIC of drug B

CHAPTER 40 / Principles of Combination Chemotherapy 721

infections caused by Gram-negative enteric organisms. An analogous combination, sulfadoxine and pyrimethamine, is used in the treatment of malaria, toxoplasmosis, and other protozoal infections. These combinations illustrate a second mechanism whereby drugs can exert a synergistic effect. The mechanism of synergy is based on the inhibition of two steps in folic acid biosynthesis affecting the cellular concentration of the same critical metabolite, dihydrofolate (see Chapter 32). The reduced form of this metabolite, tetrahydrofolate, is a required substrate for purine biosynthesis and for many one-carbon transfer reactions and is thus necessary for DNA replication and cell division (see Fig. 32-7). Co-administration of Penicillins with ␤-Lactamase Inhibitors The combination of a ␤-lactam antibiotic and a ␤-lactamase inhibitor (e.g., clavulanic acid, sulbactam, tazobactam) illustrates a mechanism of drug interaction that is not technically synergistic (because the ␤-lactamase inhibitor has no antibacterial activity of its own) but that shares a functional similarity with the drug combinations discussed above. Clavulanic acid is an inhibitor of ␤-lactamase, an enzyme used by many ␤-lactam-resistant Gram-positive and Gramnegative bacteria to inactivate penicillins (see Chapter 34). By preventing the hydrolysis and inactivation of penicillins, clavulanic acid (and other ␤-lactamase inhibitors) greatly increases the potency of penicillins (and other ␤-lactams) against bacteria that express ␤-lactamase. This combination has been effective in the treatment of infections due to penicillin-resistant Streptococcus pneumoniae, which is a common cause of otitis media in infants. Such organisms have typically acquired resistance to penicillins through a plasmid-encoded ␤-lactamase. Polymicrobial and Life-Threatening Infections Combinations of antimicrobial drugs are used not only to prevent the emergence of resistance and to act synergistically against a specific, known pathogen, but also to treat polymicrobial infections and infections in which treatment must be initiated before the microbe causing the infection is identified. Consider, for example, the case of a ruptured appendix or colonic diverticulum that has leaked bacteria into the peritoneal cavity. Such an intra-abdominal abscess is likely to contain a wide spectrum of microorganisms—much too broad to be targeted effectively by a single antibiotic. After draining the abscess, treatment with a combination of antibacterial agents such as an aminoglycoside—to kill aerobic Gram-negative Enterobacteriaceae (e.g., Escherichia coli)— and clindamycin or metronidazole—to kill anaerobes (e.g., Bacteroides fragilis; see Chapter 36)—often results in clearance of the infection. (Note that it may sometimes be necessary to treat with antagonistic drug combinations in order to cover the spectrum of microorganisms that are likely to be present.) In cases where presumptive treatment is indicated before the causative microorganism is identified, body fluids such as blood, sputum, urine, and cerebrospinal fluid (CSF) should be submitted for culture before initiating therapy. A combination of drugs with activity against the microbes that are most likely to be involved in the infection (or that could result in the most serious outcome) is then administered until a positive bacteriologic identification is made and drug susceptibility results are obtained. At that point, it may be possible to discontinue unnecessary drugs and to implement specific monotherapy.

Unfavorable Drug Combinations Antagonism can sometimes result from combination chemotherapy, although this situation is to be avoided if possible. Antagonism is most commonly observed when static drugs are used in combination with cidal drugs. For example, tetracyclines are bacteriostatic antimicrobials that antagonize the bactericidal activity of penicillins (see Chapter 33). Recall that the bactericidal activity of penicillins depends on cell growth. By inhibiting the transpeptidation reaction involved in bacterial cell wall cross-linking, the penicillins create an imbalance between cell wall synthesis and autolysinmediated cell wall degradation. If the bacterial cell continues to grow, this leads to spheroplast formation and eventually to osmotic lysis. A protein synthesis inhibitor such as tetracycline, which arrests cell growth, would therefore antagonize the effect of a ␤-lactam. Similarly, imidazoles and triazoles are fungistatic agents that antagonize the fungicidal activity of amphotericin B (see Chapter 35). The mechanism of antagonism can be appreciated by noting that amphotericin B acts by binding ergosterol and forming pores in the fungal membrane, whereas imidazoles and triazoles inhibit a microsomal cytochrome P450-dependent enzyme, 14␣-sterol demethylase, which is involved in ergosterol biosynthesis. Thus, the imidazoles and triazoles oppose the action of amphotericin B by decreasing the concentration of the target for the latter drug. Despite these considerations, static and cidal antimicrobial drugs are sometimes used clinically in combination when no good alternatives exist. In such cases, it may be required to increase the dose of one or both drugs to overcome the antagonistic drug–drug interaction. The resulting increase in the therapeutic drug concentration(s) can also increase the likelihood of adverse effects.

 ANTIVIRAL COMBINATION THERAPY: HIV As discussed in Chapter 37, Pharmacology of Viral Infections, no anti-HIV drug shows long-term suppressive benefit when used as a single agent. This is due largely to the development of drug resistance. The viral life cycle is central to understanding the reason that monotherapy for HIV fails to suppress long-term viral replication (see Chapter 37; Fig. 37-2). After virus attachment and fusion, the viral enzyme reverse transcriptase (RT) synthesizes double-stranded DNA from the single-stranded viral RNA genome. The DNA is then integrated into the host cell genome and transcribed over and over using the host cell’s transcription machinery. These complete genomic transcripts are eventually packaged into virions that infect new cells. However, HIV RT is relatively unfaithful, so replication error rates are quite high. In addition, transcription of the integrated DNA into RNA is also error-prone. As a result, on average, every new HIV particle contains one mutation relative to its parental virus. Although the resulting error rate is not so high as to be intolerable to the virus, it is sufficiently high that, after repeated cycles of infection, reverse transcription, and transcription, a substantial number of viruses encode altered targets of anti-HIV therapy and thereby acquire resistance, even prior to treatment. In the setting of high mutation rates, combination chemotherapy is beneficial. Combinations of RT inhibitors (e.g., AZT and 3TC) are more effective than one RT inhibitor alone, in part because resistance to one nucleoside analogue does not

722 Principles of Chemotherapy

necessarily confer resistance to another. The current standard of care for treatment of HIV infection is “triple therapy.” Triple therapy can use many combinations—for example, two nucleoside analogue RT inhibitors and a nonnucleoside reverse transcriptase inhibitor (NNRTI), a nucleoside analogue in combination with an NNRTI and a protease inhibitor, or two nucleoside analogues and a protease inhibitor. Clinical trials have shown that such combinations are able to reduce viral RNA plasma levels below the limit of detection (typically, 50 copies/mL). At such low levels of viral replication, the probability of resistance emerging to any one of the drugs is greatly reduced. Thus, for example, it has been shown that combinations remain effective for much longer periods of time than does any single agent. However, the complicated administration schedules and adverse effects of some combinations can reduce adherence. Although combination formulations of common anti-HIV therapies have reduced “pill burden,” simplified treatment, and increased adherence, the optimal time to begin therapy remains unclear. Early treatment for symptomatic patients with low CD4 T-cell counts (⬍350 cells/␮L) does reduce HIV-associated morbidity and mortality, but the relative risks and benefits of aggressive treatment for asymptomatic patients with high CD4 counts have not been well established.

 ANTINEOPLASTIC COMBINATION CHEMOTHERAPY Antineoplastic chemotherapy faces several intrinsic difficulties. Cancer cells can be thought of as “altered self” cells that maintain a number of similarities to normal, noncancerous cells, making it difficult to target the cancer cells specifically. Also, many of the currently available cancer chemotherapeutic agents have serious adverse effects that limit their dose and frequency of administration. Despite these hurdles, combination chemotherapy has led to remarkable advances in the treatment of cancer, including the examples of Hodgkin’s disease and testicular cancer discussed at the end of this section. Table 40-2 provides an overview of the major antineoplastic drug classes, including their mechanisms of action, cell cycle specificities, major resistance mechanisms, and dose-limiting toxicities. Note that all of these drug classes have been discussed in previous chapters; the following discussion integrates relevant information about the individual drugs in a clinical context.

General Considerations To appreciate the challenges that must be faced in treating cancer with drug therapies, it is useful to examine the current model for oncogenic transformation. Normal somatic cells undergo differentiation as they mature from a small regenerating stem cell population. Because cells lose the ability to divide as they progress along their differentiation pathway, malignancies tend to arise in populations of immature or undifferentiated cells (perhaps even stem cells). At the molecular level, the process of malignant transformation involves multiple steps, including the loss of tumor suppressor gene products (e.g., p53 and Rb) and the activation of proto-oncogenes (e.g., ras and c-myc) through such processes as somatic mutation, DNA translocation, and gene amplification. Acquired alterations in genes that regulate the progression of cells through the cell cycle confer a growth advantage on malignant cells, which proliferate in the absence of normal

growth regulatory signals. Some of the most aggressive transformed cells multiply at a rate of about two divisions a day. At this rate, a single such cell could give rise to a clinically detectable mass of 1 g (109 cells) in just 15 days, and a tumor burden of 1 kg (1012 cells), which is often incompatible with life, could be achieved in 20 days. Fortunately, oncogenesis usually occurs much more slowly than this—a fact that supports the concept of screening for many types of cancer (e.g., cervical, prostate, and colon). A malignant cell can give rise to a small colony of cells (106 cells) rather quickly, but further growth is held in check by the limited availability of oxygen and nutrients. Because oxygen can diffuse passively in tissues over a distance of only 2–3 mm, cells in the center of the growing tumor mass become hypoxic and enter the G0 (resting) phase. Accordingly, the percentage of cells that are actively dividing (i.e., the growth fraction of the tumor) decreases as tumor size increases. Moreover, the continued proliferation of cells at the tumor margins causes a further decrease in the pO2 in the center of the tumor, and hypoxic tumor cells begin to die (central necrosis). The tumor continues to grow, albeit at a slower rate, because the rate of cell division at the margins exceeds the rate of central necrosis. At some point, hypoxic tumor cells can express or induce the stromal expression of angiogenic factors (e.g., vascular endothelial growth factor [VEGF]) that induce vascularization of the tumor. Vascularization can be accompanied by a sudden increase in the growth fraction, as cells are pulled out of G0 phase and into the cell cycle. Because a single malignant cell can expand clonally to give rise to a tumor, it is thought that every malignant cell must be destroyed to effect a cure of the cancer. This hypothesis, together with the “log-kill” hypothesis for tumor cell killing (see Chapter 32), suggests that multiple cycles of chemotherapy must be administered at the highest tolerable doses and the most frequent tolerable intervals to achieve a cure. Antineoplastic chemotherapy usually follows first-order kinetics (i.e., a constant fraction of tumor cells is killed with each cycle of chemotherapy). These kinetics of tumor cell killing are unlike the time-dependent killing characteristic of many antimicrobial drugs, which follows zero-order kinetics (i.e., a fixed number of microbes is killed per unit time). Adding to the difficulty of successful cancer treatment is the phenomenon of tumor progression, in which a clonally derived population of malignant cells becomes heterogeneous through the accumulation of multiple genetic and epigenetic alterations. When subjected to selective pressure by immune surveillance or the administration of an antineoplastic agent, subclones of the tumor with relatively nonantigenic or drugresistant phenotypes are selected for. Mutations that confer drug resistance are of particular concern, because many transformed cells, having lost the ability to repair DNA damage, are characterized by genomic instability. Thus, deletions, gene amplifications, translocations, and point mutations are not infrequent events and can result in antineoplastic drug resistance through any of the mechanisms shown in Table 40-3. With the possible exception of more recently developed classes of therapies based on molecular targets selectively expressed by a malignant clone of cells (e.g., a monoclonal antibody directed against a tumor cell antigen or an enzyme inhibitor directed against a mutated signal transduction molecule; see Chapter 1, Drug–Receptor Interactions; Chapter 39, Pharmacology of Cancer: Signal Transduction; and Chapter 53,

726 Principles of Chemotherapy

Before the introduction of alkylating agents in the mid1960s, single-agent chemotherapy for advanced HD resulted in a median survival of 1 year. With the development of MOPP (mechlorethamine, vincristine, procarbazine, and prednisone), the first successful antineoplastic drug combination, half of these patients were cured of their disease. Treatment remained limited by significant toxicity, however, including early gastrointestinal and neurological complications as well as late sterility and secondary malignancies (myelodysplastic syndrome, acute nonlymphocytic leukemia, and non-Hodgkin’s lymphoma). Further investigation led to the development of the ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) combination, which is less toxic and more effective than MOPP. ABVD is the current standard of care for advanced HD, although trials of novel combination therapies are under way. The rationale for the ABVD drug combination comes from the knowledge that it combines both cell cycle selective and nonselective agents as well as drugs with different dose-limiting toxicities. Compared to MOPP, ABVD is associated with significantly fewer hematological and gonadal complications and secondary malignancies. Testicular Cancer The principles of antineoplastic combination chemotherapy are also exemplified in the treatment of testicular cancer. This tumor arises from the spermatogenic epithelium of the testis and is usually detected as a testicular mass on physical examination. The tumor metastasizes through lymphatic channels to pelvic and periaortic lymph nodes before disseminating widely through hematogenous routes. Treatment of local disease (without evidence of metastasis) involves surgical removal of the affected testis with or without pelvic radiation. Advanced disease requires systemic treatment with combination chemotherapy. The standard of care is BEP (Fig. 40-4). Of the three drugs in this regimen (bleomycin, etoposide, and cisplatin), cisplatin is the cell cycle nonspecific drug that may draw nondividing tumor cells into the actively cycling pool, where they are susceptible to the synergistic action of

Drug Bleomycin

Day 2

Etoposide

Day 1-5

Cisplatin

Day 1-5

1

Day 9

8

Day 16

15

22

Time (days)

FIGURE 40-4.

The bleomycin-etoposide-platinum (BEP) combination chemotherapy regimen for testicular cancer. The BEP regimen used to treat testicular cancer consists of a combination of bleomycin, etoposide, and cisplatin. Cisplatin is a cell cycle nonspecific agent; this drug may draw nondividing cells into the cell cycle, where they can be killed by the G2-phase specific agent bleomycin and the S/G2-phase specific agent etoposide. The intermittent dosing schedule limits drug toxicity and allows time for the bone marrow to recover from drug-induced myelosuppression. The 3-week cycle shown is typically administered four times in succession (12 weeks total).

the cell cycle specific agents bleomycin and etoposide. The drugs in this combination have different molecular targets, act on different phases of the cell cycle, and have different dose-limiting toxicities. Intermittent dosing allows each affected organ (lung, bone marrow, and kidney, respectively) time to recover between cycles. After surgical removal of the primary tumor, such a regimen usually results in a cure.

Treatment of Refractory or Recurrent Disease Despite the fact that combination chemotherapy has resulted in vastly improved survival for some cancers, many cancers become refractory to standard combination chemotherapy. If a standard chemotherapy regimen fails, options include experimental drug therapies, palliative care, or novel drugs approved for use after treatment failure. Many patients choose to enroll in experimental clinical trials. This decision may be based on the hope that an investigational agent could prove efficacious, but with the understanding that the true benefit may be realized only by future patients. Palliative and hospice care are alternatives to continued drug treatment in cases of advanced metastatic disease. An increasing number of agents with novel mechanisms of action are becoming available for disease that is otherwise refractory to treatment. Many of these agents selectively target tumor-specific antigens and signal transduction pathways, as discussed in Chapters 39 and 53. Optimizing combinations of these and other antineoplastic agents for efficacy and safety will be an important challenge for the future.

 CONCLUSION AND FUTURE DIRECTIONS The principles of combination chemotherapy highlight the importance of combination drug treatment in a variety of clinical situations. The use of drug combinations has greatly enhanced the effectiveness of treatment of both infectious and neoplastic diseases. The advantages offered by multidrug regimens over individual drug therapy (monotherapy) include increased antimicrobial, antiviral, and antineoplastic efficacy, decreased overall drug resistance, decreased host toxicity, and broader coverage of suspected pathogenic organisms. These advantages are illustrated in the rational use of drug combinations to treat infections with Mycobacterium tuberculosis and HIV, as well as neoplastic disorders such as Hodgkin’s disease and testicular cancer. Treatment of multidrug-resistant microorganisms such as MDR-TB and MDR-HIV remains a special challenge, as does treatment of genetically heterogeneous cancers with low growth fractions such as lung, colon, breast, and prostate cancers. Continued refinement of combination chemotherapy regimens will rely on increased understanding of molecular targets and metabolic pathways used by microorganisms and cancer cells.

Acknowledgment The authors thank Shreya Kangovi and Gia Landry for initial drafts of the case of Mr. M and the discussion in the chapter related to his case. We thank Ryan L. Albritton for his valuable contributions to this chapter in the First and Second Editions of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

CHAPTER 40 / Principles of Combination Chemotherapy 727

Suggested Reading Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 2008;8:592–603. (Reviews adaptive and intrinsic mechanisms that may explain cancer resistance to bevacizumab and other antiangiogenic therapies.) Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy of advanced Hodgkin’s disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med 1992;327:1478–1484. (These antineoplastic drug combinations remain the standard of care for advanced Hodgkin’s disease.) Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs— worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 2006;55:301–305. (Surveys international network of tuberculosis [TB] laboratories for incidence and prevalence of multidrug-resistant [MDR] and extensively drugresistant [XDR] TB isolates.) Chou R, Huffman LH, Fu R, et al. Screening for HIV: a review of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med 2005;143:55–73. (Compares benefits and risks of screening for HIV and reviews efficacy of highly active antiretroviral therapy [HAART] for patients with advanced HIV infection.) Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55. (Detailed analysis of models for synergistic, antagonistic, and additive drug combinations.)

Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621–681. (Detailed analysis of models for synergistic, antagonistic, and additive drug combinations.) Dancey JE, Chen HX. Strategies for optimizing combinations of molecular targeted anticancer agents. Nat Rev Drug Discov 2006;5:649–659. (Discusses principles for determining combinations of antineoplastic agents that could be most promising to test in preclinical and clinical trials.) Harvey RJ. Synergism in the folate pathway. Rev Infect Dis 1982;4:255– 260. (Describes the kinetics of synergism between trimethoprim and the sulfonamides.) Luo J, Solimini NL, Elledge SJ. Principles of cancer therapy: oncogene and non-oncogene addiction. Cell 2009;136:823–837. (Reviews antineoplastic therapies targeting the 12 hallmarks of cancer and proposes principles for developing new antineoplastic therapies and combinations.) Ormerod LP. Multidrug-resistant tuberculosis (MDR-TB): epidemiology, prevention and treatment. Br Med Bull 2005;73/74:17–24. (Reviews epidemiology, prevention, and treatment of multidrug-resistant tuberculosis.) Yazdanpanah Y, Sissoko D, Egger M, et al. Clinical efficacy of antiretroviral combination therapy based on protease inhibitors or non-nucleoside analogue reverse transcriptase inhibitors: indirect comparison of controlled trials. Br Med J 2004;328:249–256. (Reviews combination therapies used in the treatment of HIV.)

VI Principles of Inflammation and Immune Pharmacology

41 Principles of Inflammation and the Immune System Ehrin J. Armstrong and Lloyd B. Klickstein

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 729-730 OVERVIEW OF THE IMMUNE SYSTEM . . . . . . . . . . . . . . . . . 730 Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730 Antigen-Presenting Cells. . . . . . . . . . . . . . . . . . . . . . . 731 Activation of the Innate Immune Response . . . . . . . . . 732 Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732 Major Histocompatibility Complex . . . . . . . . . . . . . . . . 732 Immune Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733 Humoral and Cellular Immunity . . . . . . . . . . . . . . . . . . 733 Tolerance and Costimulation . . . . . . . . . . . . . . . . . . . . 734 CHEMICAL MEDIATORS OF INFLAMMATION . . . . . . . . . . . . 736 Histamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736 Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

Eicosanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Cytokines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Other Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 THE INFLAMMATORY RESPONSE . . . . . . . . . . . . . . . . . . . . 737 Dilation of Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Recruitment of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 CHRONIC INFLAMMATION . . . . . . . . . . . . . . . . . . . . . . . . . . 738 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 738 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739

 INTRODUCTION

cells of the immune system (Chapter 44, Pharmacology of Hematopoiesis and Immunomodulation, and Chapter 45, Pharmacology of Immunosuppression). This approach (because it is dependent on understanding the molecular events in the relevant pathways) is still in its infancy but promises to yield a number of new drugs in the foreseeable future. The second pharmacologic approach, used in diseases such as peptic ulcer disease (Chapter 46, Integrative Inflammation Pharmacology: Peptic Ulcer Disease), asthma (Chapter 47, Integrative Inflammation Pharmacology: Asthma), and gout (Chapter 48, Integrative Inflammation Pharmacology: Gout), involves modification of the underlying pathophysiologic stimulus, thus removing the impetus for inflammation. The difference between these two approaches is, at times, indistinct and will continue to blur as the pathophysiology of chronic inflammatory disease is better understood at a molecular level. This chapter provides sufficient background in the physiology of inflammation and the immune system to understand the subsequent chapters in this section of the textbook. The treatment is necessarily brief, with an emphasis on pharmacologically relevant targets of the inflammatory response. The chapter is organized into four parts. First, a general overview of the immune system is presented. Second, the molecular signals mediating cellular communication and inflammation are introduced. Third, the immune and inflammatory cells and signaling molecules are discussed in

Inflammation and the immune system are closely intertwined. Inflammation is a complex web of responses to tissue injury and infection, characterized by the classic signs of rubor (redness), calor (heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). The immune system comprises the cells and soluble factors, such as antibodies and complement proteins, that mediate the inflammatory response; these cells and factors both eliminate the inciting inflammatory stimulus and initiate the process of immunologic memory. A normal inflammatory response is an acute process that resolves after removal of the inciting stimulus. Diseases of inflammation and immunity can occur due to inappropriate inflammation or when the normal inflammatory response progresses to chronic inflammation, either because of a long-term inappropriate response to a stimulus (for example, allergies) or because the offending agent is not removed (for example, chronic infection, transplantation, and autoimmunity). Two pharmacologic strategies are used to target the pathophysiology of immune diseases. The first involves modification of the signaling mediators of the inflammatory process or suppression of components of the immune system. This is the rationale for drugs that affect eicosanoid pathways (Chapter 42, Pharmacology of Eicosanoids), histamine (Chapter 43, Histamine Pharmacology), and

729

CHAPTER 41 / Principles of Inflammation and the Immune System 731

Bone marrow

Pluripotent hematopoietic stem cell

Trilineage myeloid stem cell

Lymphoid stem cell

Megakaryocyte

Blood and tissues Granulocytes

Lymphocytes y

B cell

T cell

Neutrophil

Eosinophil

Basophil

Effector cells

Plasma cell

Activated T cell

Erythroblast

Blood Mast cell

Monocyte/ macrophage

Precursor

Monocyte

Platelets

Erythrocyte

Tissue cells

Mast cell

Macrophage

FIGURE 41-1.

Development of cells of the immune system. All hematopoietic cells develop from the pluripotent hematopoietic stem cell. This cell gives rise to the lymphoid stem cell and the trilineage myeloid stem cell. The lymphoid stem cell and its progenitor cells (not shown) give rise to mature lymphocytes (B cells and T cells), the cells that mediate adaptive immune responses. When exposed to specific antigens, B cells differentiate into antibody-producing plasma cells, and T cells adopt an activated phenotype. The myeloid stem cell and its progenitor cells, including megakaryocytes, erythroblasts, and myeloid precursors (not shown), proliferate and differentiate into mature neutrophils, eosinophils, basophils, mast cells, monocytes, platelets, and erythrocytes. In the tissues, monocytes differentiate into macrophages, and mast cell precursors differentiate into mast cells. (See Fig. 44-1 for more details about the differentiation of cell lineages in the bone marrow.)

innate immune roles; the biology of these cell types is beyond the scope of this text. “Granulocyte” is a descriptive term based on the appearance of the cytoplasmic granules within these cells. Neutrophils, the most abundant cell type of the innate immune system, are phagocytic cells primarily responsible for defense against bacterial infection. These cells envelop invading bacteria in phagocytic vesicles and destroy the bacteria within these vesicles using enzymes such as myeloperoxidase. Eosinophils are circulating granulocytes primarily involved in defense against parasitic infections. Because parasites are often too large to engulf, eosinophils attach to a parasite’s exterior and secrete cytotoxic substances directly on the parasite. Both basophils (circulating) and mast cells (tissue-resident) bind IgE antibody, display this IgE on the cell surface, and maintain histamine-containing granules that are released when exogenous antigen binds to and crosslinks the IgE. Basophils and mast cells are important in

allergic responses. Eosinophils and basophils are so named because they exhibit eosinophilic and basophilic patterns, respectively, when stained with Wright-Giemsa stain. Antigen-Presenting Cells Antigen-presenting cells (APCs) process the macromolecules (especially proteins) of an invading agent to display the processed fragments on the surface of the APC. In this form, the fragments serve as molecular fingerprints used by cells of the adaptive immune system to recognize the invading agent. APCs are important initiators of immune responses because, in addition to displaying nonself antigens to T cells (see below), they provide the costimulatory signals that are necessary for T-cell activation. The concept of costimulation, in which two separate signals are required to initiate an immune response to a stimulus, is discussed below. Monocytes that exit the bloodstream and take up residence in the tissues can differentiate into macrophages.

CHAPTER 41 / Principles of Inflammation and the Immune System 733

A MHC class I

Protein fragment

CD8 binding site

MHC class I protein β2 microglobulin

Protein fragments Cytoplasmic protein

Secretory protein Endoplasmic reticulum Nucleated cell

B MHC class II

CD4 binding site

Protein fragment MHC class II protein

Protein Endocytosis

Degradation

Protein fragments

Antigen-presenting cell

FIGURE 41-2.

Class I and class II major histocompatibility complex proteins. A. A representative fraction of cytoplasmic proteins are proteolytically degraded in the cytosol, and the protein fragments are transported to the endoplasmic reticulum (ER). A fraction of secretory proteins are degraded directly in the ER. MHC class I protein, in association with ␤2 microglobulin, binds a fragment of the degraded cytoplasmic or secretory protein in the ER. The MHC class I:protein fragment complex is transported to the cell surface, where it serves as a fingerprint for the diversity of proteins expressed by that cell. The CD8 binding site on MHC class I ensures that the class I protein:antigen complex interacts only with cytotoxic T cells, which express CD8. All nucleated human cells express MHC class I proteins. B. Antigen-presenting cells phagocytose and degrade bacteria and other foreign agents, generating protein fragments that bind to MHC class II protein in the ER. The MHC class II:protein fragment complex is transported to the cell surface, where it serves to display all the potentially nonself antigens that have been ingested by that cell. The CD4 binding site on MHC class II ensures that the class II protein:antigen complex interacts only with helper T cells, which express CD4. Professional antigen-presenting cells (B cells, macrophages, and dendritic cells) are usually the only cell types that express MHC class II proteins, but other cells can be induced to express class II proteins and present antigens under some circumstances.

MHC class I proteins at the surface of the cell, and the immune system will recognize that cell as virally infected. Antigens presented by MHC class I proteins are recognized by T cells bearing the cell surface protein CD8. (The designation “CD” stands for “cluster of differentiation” or “cluster designation” and is a system for naming an ever-growing list of cell-associated antigens—now numbering in the hundreds— that are present on leukocytes and other cell types. Each antigen must be defined by at least two different monoclonal antibodies in order to earn the “CD” designation.) MHC class II proteins display protein fragments derived from endocytic vesicles. In contrast to class I proteins, which are expressed on all nucleated cells, MHC class II proteins are expressed mostly on antigen-presenting cells (e.g., macrophages and dendritic cells), although some other cell types can be induced to express MHC class II proteins. Endocytic vesicles contain antigenic protein fragments derived from infectious agents after phagocytosis and proteolytic processing of those agents. Therefore, the protein fragments expressed on MHC class II proteins generally identify extracellular foreign agents (e.g., bacteria). As discussed below, T cells bearing the cell surface protein CD4 recognize antigens presented by MHC class II proteins. In the process, these T cells stimulate the antigen-presenting cells to produce soluble factors called cytokines and chemokines, which, in turn, aid the T cells in responding to the antigen. In general, then, the protein fragments bound to MHC class I identify infected cells, whereas the fragments bound to MHC class II identify infectious agents. However, because of the phenomenon of cross-presentation, some proteins generated in the cytosol can be presented by MHC class II to CD4⫹ T cells, and some phagocytosed antigens can be presented by MHC class I to CD8⫹ T cells. Immune Diversity While MHC proteins provide a mechanism for distinguishing infected cells and infectious agents from uninfected cells, somatic gene recombination and other processes for generating diversity provide a mechanism for generating a specific response to an infection. By recombination, immunoglobulin and T-cell receptor genes semirandomly create millions of modular three-dimensional protein structures, referred to as variable regions. Recombined variable regions may undergo somatic hypermutation to create additional diversity that, in the aggregate, can recognize almost any structure. This is the primary mechanism by which the immune system generates an astounding diversity of immune responses. Humoral and Cellular Immunity Adaptive immunity is generally divided into humoral immunity and cellular immunity. In the basic (simplified) model of the immune system, the primary cells mediating these branches of the immune system are referred to as B cells and T cells, respectively (Table 41-1). The humoral response involves the production of antibodies specific for an antigen. These antibodies are secreted by plasma cells (differentiated B cells) and are therefore effective primarily against extracellular infectious agents (such as many bacteria). In contrast, the cellular response involves activation and clonal expansion of T cells that recognize a specific antigen. Some T cells recognize infected cells and then lyse those cells using cytotoxic proteins called perforins and granzymes. Cellular immune

734 Principles of Inflammation and Immune Pharmacology

responses are therefore effective against many intracellular infectious agents (such as viruses). In addition to their role in cellular immunity, T cells control the extent of immune responses. Each T cell evolves so that it is activated by only one specific MHC:antigen complex. All T cells express an MHC:antigen-specific T-cell receptor (TCR). T cells are divided into cytotoxic T cells (TC) and helper T cells (TH) based on the type of coreceptor expressed and the function imparted by that coreceptor (Fig. 41-3). TC cells are the mediators of cellular adaptive immunity. These cells express the CD8 coreceptor, which recognizes a constant (i.e., antigen-independent) domain on MHC class I proteins. This coreceptor function allows the antigen-specific TCR on TC cells to bind a specific class I MHC:antigen complex with sufficiently high affinity that the TC cell is activated by the cell expressing the class I MHC:antigen complex.

A Cytotoxic T cell T cell receptor

MHC class I protein

β2 microglobulin

Antigen

CD8

Cytotoxic T cell

Virus-infected cell

B Helper T cell MHC class II protein T cell receptor Antigen

IL-2R IL-2

Helper T cell

FIGURE 41-3.

CD28

B7

CD4

Antigen-presenting cell

Activation of cytotoxic and helper T cells. T cells mediate and regulate the cellular immune response. A. Cytotoxic T cells (TC) are the primary mediators of cellular immunity. These cells express T-cell receptors (TCR) and CD8. The TCR identifies nonself antigens bound to MHC proteins, and CD8 ensures that TC cells interact only with cells expressing MHC class I proteins. In the example shown, the interaction of a TC cell with the MHC class I protein of a virus-infected cell leads to activation of the TC cell and subsequent killing of the virus-infected cell. B. Helper T cells (TH) are the primary regulators of cellular immunity. These cells express TCR and CD4. CD4 binds to MHC class II proteins on antigen-presenting cells (APC); this interaction ensures that TH cells interact only with cells expressing MHC class II proteins. An additional degree of specificity is provided by the interaction of CD28 on TH cells with proteins of the B7 family on APC; this “costimulatory signal” is required for TH activation. In the example shown, the interaction of a TH cell with the MHC class II and B7 proteins of an antigenpresenting cell leads to activation of the TH cell. The activated TH cell secretes IL-2 and expresses the IL-2 receptor (IL-2R); this autocrine pathway stimulates further TH-cell proliferation and activation. IL-2 and other cytokines secreted by the TH cell activate not only TH cells, but also TC cells and B cells.

Specific activation of the TC cell initiates a chain of events, including the secretion of membrane-penetrating perforins and apoptosis-inducing granzymes, that results in the death of the cell displaying the foreign antigen. TH cells are primarily the regulators of adaptive immunity. TH cells are identified by their expression of the CD4 coreceptor, which recognizes an antigen-independent domain on MHC class II proteins. This coreceptor function allows the antigen-specific TCR on TH cells to bind a specific class II MHC:antigen complex with sufficiently high affinity that the TH cell is activated by the antigen-presenting cell. In addition to initiating and strengthening the immune response, TH cells control the type of immune response by producing one or another set of cytokines. TH cells can be generally divided into TH1 and TH2 subtypes based on the cytokines produced by the cells. TH1 cells characteristically produce IFN-␥ and IL-2, and these cytokines influence the development of cellmediated immune responses of both CD8⫹ TC cells and other CD4⫹ TH cells. In contrast, TH2 cells characteristically produce IL-4, IL-5, and IL-10, and these cytokines enhance antibody production by B cells. The TH2 cell subtype is more often associated with autoimmunity (see Chapter 45). In addition to regulating adaptive immunity, TH cells can mediate immunity by secreting cytokines that activate phagocytic cells to kill infecting microbes more efficiently. Subtypes of TH cells other than TH1 and TH2 have been discovered, and some of these subtypes are important in human disease. For example, the cytokine IL-23 stimulates naive CD4⫹ cells to differentiate into TH17 cells, and these cells produce IL-17 isoforms that recruit neutrophils and amplify the immune response. Drugs that block the maturation or growth of TH17 cells are becoming available for clinical use. Tolerance and Costimulation Diversity in the variable regions of immunoglobulins and T-cell receptors creates the potential for some of these molecules to recognize and attack native proteins, a circumstance termed “autoimmunity.” There are two primary mechanisms for avoiding autoimmunity. The first is clonal deletion, in which T cells die during development when they express high-affinity receptors that recognize self-antigen. In a second process referred to as tolerance or anergy, cells of the immune system undergo a carefully regulated series of steps during development to ensure that mature immune cells do not recognize native proteins. Costimulation—the requirement for multiple simultaneous signals to initiate an immune response—ensures that stimulation of a single immune receptor does not activate a damaging immune reaction. Signal 1 provides specificity, while signal 2 is permissive, ensuring that an inflammatory response is appropriate. Regulation of costimulatory molecules is a mechanism whereby the innate immune system regulates the extent of an immune response. If antigen is presented without a coincident costimulatory signal (i.e., without innate immune activation), then anergy results, whereby a cell becomes unreactive and will not respond to further antigenic stimuli. Drugs that induce anergy could be therapeutically attractive, because such agents could allow long-term acceptance of an organ graft or limit the extent of an autoimmune disease. For T cells, signal 1 is mediated by the MHC:antigen:TCR interaction. Signal 2 is mediated predominantly by the interaction of CD28 on T cells with B7-1 (also called CD80) or B7-2 (CD86) on activated antigen-presenting cells (Fig. 41-4).

CHAPTER 41 / Principles of Inflammation and the Immune System 735

Antigen recognition

T cell response Cytokine receptor

A No costimulation

MHC

TCR

CD4

Naive T cell

Resting APC

B With costimulation

MHC

CD4

Activated APC

No response

TCR

B7

CD28

IL-2R

Activated T cell

Cytokines

IL-2

T cell proliferation and differentiation

FIGURE 41-4.

Costimulation in the T-cell activation pathway. Two signals are required for activation of a T-cell response to antigen. A. If an antigen-presenting cell (APC) presents an antigen to a T cell in the absence of an appropriate costimulatory signal, the T cell does not respond and may become anergic. B. If an APC presents both the antigen and a costimulatory molecule such as B7, the T cell proliferates and differentiates in response to the antigenic stimulus. Cytokines secreted by the activated APC augment T-cell activation.

Resting T cells present CD28, which can bind either B7-1 or B7-2. B7-1 and B7-2 are not normally present on antigenpresenting cells, but their expression is increased by the innate immune system during an immune response to a pathogen. The lack of expression of B7 molecules in the absence of an innate immune response may help to limit inappropriate adaptive immune responses. When a T cell receives both signal 1 and signal 2, expression of IL-2, T-cell activation, and clonal expansion of TH cells specific for that foreign epitope occur. Activated T cells eventually down-regulate CD28 expression

A

B

T cells recognize antigen Signal leading to expression of CD40L

and up-regulate CTLA-4 expression. CTLA-4, like CD28, binds B7-1 and B7-2 but with much higher affinity than CD28. In contrast to the activating CD28 signal, interaction of CTLA-4 with B7-1 or B7-2 inhibits T-cell proliferation. This may be a physiologic mechanism for self-limitation of the immune response. CD40 ligand (CD40L) is another mediator of costimulation. Activated T cells express CD40L (CD154). CD40 is expressed on antigen-presenting cells, including B cells and macrophages (Fig. 41-5). Interaction of TH-cell CD40L with

Activated T cells express CD40L

C

APCs express B7 and secrete T cell-activating cytokines

Signal leading to expression of B7 CD40

CD40

Cytokine receptor

CD40

CD40L

CD40L

Antigen

CD4

APC

CD28

CD28

T cell

APC

B7

Activated T cell

APC

CD28

Cytokines

Activated T cell

Enhanced T cell proliferation and differentiation Costimulation and the CD40–CD40L interaction. A. An antigen-presenting cell (APC) presents MHC class II-bound antigen to a CD4⫹ T cell. T-cell recognition of antigen initiates an intracellular signaling cascade that leads to expression of CD40 ligand (CD40L) at the T-cell surface. B. CD40L on the activated T cell binds to CD40 on the surface of the APC. Activation of CD40 generates an intracellular signaling cascade that leads to expression of B7 on the APC surface. C. Enhanced T-cell proliferation and differentiation are promoted by costimulation of the T cell by MHC class II antigen (which binds to the T-cell receptor), CD40 (which binds to T-cell CD40L), and B7 (which binds to T-cell CD28). Cytokines secreted by the activated APC augment T-cell proliferation and differentiation.

FIGURE 41-5.

738 Principles of Inflammation and Immune Pharmacology A Normal

B Inflammation

sialyl-Lewisx (s-Lex) Neutrophil

Rolling adhesion

Blood flow Blood vessel lumen

Tight binding

Diapedesis

Migration

IL-8R LFA-1

IL-8 ICAM-1

E-selectin

Subendothelial space

Endothelial cells

CD31

Basement membrane Chemokine (IL-8)

FIGURE 41-6.

Overview of the inflammatory response. A. Leukocytes circulating in the blood interact with selectins expressed on the surface of vascular endothelial cells. In the absence of inflammation, the interaction between leukocytes and endothelial cells is weak, and leukocytes either flow past or roll along the endothelium. Neutrophil rolling is mediated by the interaction between endothelial cell E-selectin and neutrophil sialyl-Lewisx (s-Lex). B. During the inflammatory response, endothelial cells up-regulate their expression of intercellular adhesion molecules (ICAMs). ICAM expression increases the potential for strong binding interactions between leukocytes and the activated endothelial cells. For example, ICAM-1 on endothelial cells binds tightly to LFA-1 on neutrophils. The enhanced cell–cell interaction leads to margination of leukocytes onto endothelial cell surfaces and initiates the process of leukocyte diapedesis and transmigration from the vascular space into extravascular tissues. Leukocytes migrate through injured tissue in response to chemokines such as IL-8, which are inflammatory mediators released by injured cells and by other immune cells that have already reached the site of injury.

one further stimulus to activate their killing machinery. Foreign substances must be coated by an opsonin before they can be ingested (phagocytosed) by leukocytes. Opsonins are molecular adaptors that coat foreign surfaces and signal leukocytes that a particle should be attacked. The major opsonins consist of complement, immunoglobulins (antibodies), and collectins (plasma proteins that bind to certain microbial carbohydrates). The interaction of a phagocytic cell with an opsonized particle initiates engulfment and destruction of the offending agent. This step is also a crucial point of interaction between innate and adaptive immunity. Antigen-presenting cells process engulfed particles and present their antigens to B cells and T cells, which then react to the antigens. In the introductory case, Mark’s cut presumably allowed bacteria to penetrate his skin barrier, leading to infection. The presence of these bacteria initiated an inflammatory response that included phagocytosis of bacteria by APCs, presentation of bacterial antigens to TH cells, activation and expansion of TH cells, TH-cell activation of further APC-mediated phagocytosis, and synthesis and secretion of antibodies specific for the bacteria.

Resolution Tissue repair and re-establishment of homeostasis are the final events in the acute inflammatory response. The same mediators that activate inflammation also initiate a cascade of tissue repair; this process is mediated by the release of growth factors and cytokines, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor-2 (bFGF-2), transforming growth factor-␤1 (TGF-␤1), IL-1, and TNF-␣. These factors act as mitogens for endothelial cells and fibroblasts and ultimately stimulate healing and scar formation through angiogenesis (formation of new blood vessels) and the formation of granulation tissue. In the introductory case, the granulation tissue and eventual scar will be the only record of Mark’s acute inflammatory event. Of note, angiogenesis

can be a pathologic state when it is associated with abnormal blood vessel growth or tumor growth, and pharmacologic inhibitors of angiogenesis are currently being used to treat age-related macular degeneration (where abnormal blood vessels obscure vision) and as antineoplastic agents (see Chapter 39, Pharmacology of Cancer: Signal Transduction).

 CHRONIC INFLAMMATION Chronic inflammation is a pathologic state characterized by the continued and inappropriate response of the immune system to an inflammatory stimulus. Chronic inflammation accounts for the symptoms of many autoimmune diseases and may be an important cause of organ transplant rejection. In contrast to the acute inflammatory response, which is dominated by neutrophils, one of the hallmarks of chronic inflammation is the predominance of macrophages. Activated macrophages secrete collagenases and growth factors in addition to inflammatory mediators such as proteases and eicosanoids. These secreted products initiate and maintain a cycle of tissue injury and repair, leading to tissue remodeling. Over time, chronic inflammation can cause relentless tissue destruction. Promising treatments for chronic inflammation could include cytokine inhibitors that neutralize mediators of the signaling cascades that perpetuate chronic inflammation. These agents are discussed in Chapter 45.

 CONCLUSION AND FUTURE DIRECTIONS The immune system intricately regulates the response to tissue injury and infection. A complete review of immunology is beyond the scope of this text; instead, the discussion in this chapter presents a broad overview and highlights elements of immunity that may be addressed pharmacologically. Innate immune mechanisms respond to patterned elements

CHAPTER 41 / Principles of Inflammation and the Immune System 739

shared among a class of infectious agents, such as bacterial lipopolysaccharide or viral RNA. The innate immune system also processes these agents and presents them to lymphocytes, thereby activating the adaptive immune system. The adaptive immune system develops a response specific to an infectious agent or inflammatory stimulus. As part of the inflammatory response, the adaptive immune response also has mechanisms that mediate tolerance to distinguish self from nonself; dysregulation of these mechanisms may lead to chronic inflammation and autoimmune disease. Many anti-inflammatory drugs work in whole or in part by depleting populations of innate or adaptive immune cells; this concept is discussed in more detail in Chapter 45, Pharmacology of Immunosuppression. The chemical mediators of the inflammatory response— including histamine, complement, eicosanoids, and cytokines —are also major targets of current pharmacologic therapies. Macromolecules are playing an increasingly important role in modulation of these chemical mediators; for example, a number of anticytokine antibodies, including inhibitors of tumor necrosis factor-␣, have been developed for the treatment of rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel disease. A second approach to modulation of inflammatory responses has been to target the intracellular signaling cascades responsible for initiation of immune responses. An example of such a drug is cyclosporine, discussed in Chapter 45. As the number of agents available for treatment of immune disorders grows, it will become increasingly important to determine whether macromolecular agents

and small-molecule signaling inhibitors can be used in combination to target multiple steps in inflammatory pathways.

Disclosure Lloyd B. Klickstein is an employee and stockholder of Novartis, Inc., which manufactures or distributes drugs that act by mechanisms discussed in this chapter (for example, ranibizumab).

Suggested Reading Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. (Advances in understanding the innate immune system.) Dinarello CA. Anti-inflammatory agents: present and future. Cell 2010; 140:935–950. (A signaling-oriented overview of targets for development of new anti-inflammatory agents.) Ibelgaufts H. COPE: Cytokines & Cells Online Pathfinder Encyclopedia. Available at: http://www.copewithcytokines.de/cope.cgi. (Website that describes all known actions of cytokines.) Littman DR, Rudensky AY. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 2010;140:845–858. (Discusses advances in T-cell subsets and regulatory T-cell biology.) Murphy KM, Travers P, Walport M. Janeway’s immunobiology. 7th ed. New York: Garland Publishing; 2007. (A general immunology textbook.) Pier GB, Lyczak JB, Wetzler L. Immunology, infection and immunity. Washington, DC: ASM Press; 2004. (A detailed text with a focus on immunologic mechanisms.) Zola H, Swart B, Banham A, et al. CD molecules 2006: human cell differentiation molecules. J Immunol Meth 2007;319:1–5. (Summarizes classification of molecules with the “CD” designation.)

42 Pharmacology of Eicosanoids David M. Dudzinski and Charles N. Serhan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 740-741 PHYSIOLOGY OF EICOSANOID METABOLISM . . . . . . . . . . . 740 Generation of Arachidonic Acid and Omega-3 Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Cyclooxygenase Pathway . . . . . . . . . . . . . . . . . . . . . . . . . 742 Prostaglandins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744 Thromboxane and Prostacyclin . . . . . . . . . . . . . . . . . . 744 Lipoxygenase Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . 745 Leukotrienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745 Lipoxins, Resolvins, Protectins, and Maresins . . . . . . . 748 Epoxygenase Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 748 Isoprostanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748 Metabolic Inactivation of Local Eicosanoids . . . . . . . . . . . 748 Integrated Inflammation Schema . . . . . . . . . . . . . . . . . . . 752 PATHOPHYSIOLOGY OF EICOSANOIDS . . . . . . . . . . . . . . . . 752 Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753 Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . 753 Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753 Glomerulonephritis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754 Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 754

PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 754 Phospholipase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 754 Cyclooxygenase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 754 Traditional Nonselective Inhibitors: NSAIDs . . . . . . . . . 754 Acetaminophen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756 Selection of the Appropriate NSAID . . . . . . . . . . . . . . . 756 COX-2 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757 Prostanoid Receptor Mimetics . . . . . . . . . . . . . . . . . . . . . 758 Thromboxane Antagonists . . . . . . . . . . . . . . . . . . . . . . . . 758 Leukotriene Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . 758 Lipoxygenase Inhibition. . . . . . . . . . . . . . . . . . . . . . . . 758 5-Lipoxygenase Activating Protein (FLAP) Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759 Leukotriene Synthesis Inhibitors . . . . . . . . . . . . . . . . . 759 Leukotriene Receptor Antagonists . . . . . . . . . . . . . . . . 759 Lipoxins, Aspirin-Triggered Lipoxins, Resolvins/ Protectins/Maresins, and Lipoxin-Stable Analogues . . . . . 759 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 759 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

 INTRODUCTION

 PHYSIOLOGY OF EICOSANOID METABOLISM

Autacoids are substances that are rapidly synthesized in response to specific stimuli, act quickly in the immediate environment, and remain active for only a short time before degradation. Eicosanoids represent a chemically diverse family of autacoids that are mostly derived from arachidonic acid. Research on eicosanoids continues to elucidate their critical roles in inflammatory, neoplastic, and cardiovascular physiology and pathophysiology. Numerous pharmacologic interventions in eicosanoid pathways—including the nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, leukotriene inhibitors, and others—are useful in the clinical management of inflammation, pain, and fever. Given the diverse bioactivities of eicosanoids, future research in eicosanoid physiology and pharmacology may lead to the development of new therapeutics for the treatment of asthma, inflammatory diseases, autoimmune diseases, glomerulonephritis, cancer, cardiovascular diseases, and other clinical conditions.

740

Eicosanoids are centrally involved in a number of metabolic pathways that exhibit diverse roles in inflammation and cellular signaling. The vast majority of these pathways center on reactions involving the metabolism of arachidonic acid (Fig. 42-1). The following section considers the biochemical steps leading to arachidonic acid generation, and then discusses the cyclooxygenase, lipoxygenase, epoxygenase, and isoprostane pathways of arachidonic acid metabolism. The word eicosanoid arises from the Greek root for twenty, and the term classically refers to 20-carbon molecules derived from arachidonic acid oxygenation. The term eicosanoid also applies broadly to various other molecules—such as resolvins, protectins, and maresins—that are derived from docosahexaenoic acid, a 22-carbon precursor. The term docosanoids is sometimes also used to describe these 22carbon structures.

Cleavage site

O R

P O

R = choline or ethanolamine

O

O-

O

Arachidonate Acyl O

Phospholipids Phospholipase A2

COOH Cytochrome P450 epoxygenases

Non-enzymatic Isoprostanes

Arachidonic acid Lipoxygenase pathways

Lipoxins Leukotrienes

Cyclooxygenase pathways

Epoxyeicosatetraenoic acids (EETs)

Prostaglandins Prostacyclin Thromboxane

CHAPTER 42 / Pharmacology of Eicosanoids 743 COOH

Vasodilation Inhibits platelet aggregation

Smooth muscle contraction Inhibits platelet aggregation

Arachidonic acid

IP DP COOH

HO

COX-1 and COX-2: cyclooxygenase activity

O

NSAIDs, COX-2 inhibitors

COOH

O

OH

PGD2

HO

OH

O

PGI2

COOH

O OOH

Hydrolysis

O

Prostacyclin synthase (endothelium)

COOH

PGD2 isomerase (brain, mast cells)

PGG2

HO

COX-1 and COX-2: peroxidase activity HO

OH

Inactive

6-keto-PGF1α O

COOH

O

Vasoconstriction Platelet activation Thromboxane antagonists

OH

Thromboxane synthase (platelets)

TP

PGF2α reductase (uterus, lung)

PGH2

HO

COOH O O

PGE2 isomerase (macrophages, mast cells)

COOH

OH

HO

TxA2 O

Nonenzymatic hydrolysis

COOH

FP

OH

HO

O OH

PGE2

TxB2

FIGURE 42-2.

EP1 Inactive

Smooth muscle contraction Bronchoconstriction Abortion

OH

COOH HO

OH

PGF2α

EP4 EP2

EP3

Vasodilation Hyperalgesia Fever Diuresis Immunomodulation

Prostaglandin biosynthesis, function, and pharmacologic inhibition. The biosynthetic pathways from arachidonic acid to prostaglandins, prostacyclin, and thromboxane are depicted. Tissue-specific enzyme expression determines the tissues in which the various PGH2-derived products are biosynthesized. NSAIDs and COX-2 inhibitors are the most important classes of drugs that modulate prostaglandin production. Thromboxane antagonists and PGE2 synthase inhibitors are promising pharmacologic strategies that are currently in development. COX, cyclooxygenase; PG, prostaglandin; Tx, thromboxane; DP, PGD2 receptor; EP, PGE2 receptor; FP, PGF2␣ receptor; IP, PGI2 receptor; TP, TxA2 receptor; NSAID, nonsteroidal anti-inflammatory drug. Note that DP, EP, FP, IP, and TP, are all G protein-coupled receptors.

COOH

OH Prostanoid backbone

746 Principles of Inflammation and Immune Pharmacology Stimuli

Ca2+

PLA2

COOH

Arachidonic acid

5-Lipoxygenase

Zileuton, FLAP inhibitors OOH COOH

5-HPETE

5-Lipoxygenase

Zileuton, FLAP inhibitors O COOH

LTA4 LTA4 hydrolase

H2O

LTC4 synthase

Glutathione

OH COOH

OH

OH S

COOH H N

COOH

HN H2N

O O

LTB4 COOH

γ-glutamyl transpeptidase

LTC4

Carboxypeptidase A

BLT1 OH OH

COOH COOH

Major source: Neutrophils (BLT1) Actions: Activation of neutrophils - Margination - Migration - Degranulation - Superoxide anion generation - Eicosanoid synthesis

S S H N

OH HN

COOH

H2N

H2N

O O

O

LTD4

COOH

LTF4

Plasma exudation

γ-glutamyl transpeptidase

Dipeptidase OH

COOH S OH H2N O

LTE4 Major source: Mast cells, basophils, eosinophils Actions:

FIGURE 42-4.

Bronchoconstriction Vasoconstriction Decreased coronary blood flow Decreased cardiac contractility Plasma exudation

Zafirlukast Montelukast CysLT1

Leukotriene biosynthesis, function, and pharmacologic inhibition. The biosynthetic pathways from arachidonic acid to the leukotrienes are shown. Zileuton and 5-lipoxygenase activating protein (FLAP) inhibitors prevent the conversion of arachidonic acid to 5-HPETE and LTA4; zileuton has been used in the chronic management of asthma. Zafirlukast and montelukast are antagonists at CysLT1, the receptor for all cysteinyl leukotrienes (mainly LTC4 and LTD4); these drugs are used in the chronic management of asthma. The cysteinyl leukotrienes also interact with CysLT2 (not shown). BLT1 and BLT2 are LTB4-related G protein-coupled receptors; BLT1 is the major LTB4 receptor. BLT2 is the G protein-coupled receptor for HHT, a cyclooxygenase product (see text for details; not shown).

CHAPTER 42 / Pharmacology of Eicosanoids 747

COOH

Arachidonic acid 15-Lipoxygenase 5-Lipoxygenase Peroxidase

OOH COOH

COOH

5-HPETE

OH 15-HETE

5-Lipoxygenase

5-Lipoxygenase

OOH O

COOH

COOH

LTA4

OH 5-hydroperoxy, 15-hydroxyeicosatetraenoic acid

5-Lipoxygenase

15-Lipoxygenase

COOH O

OH Epoxytetraene Hydrolysis

Hydrolysis

OH

OH

COOH

COOH

OH

OH

OH

OH LXA4

FPR2/ALX

LXB4

FIGURE 42-5. Lipoxin biosynthesis. Two main routes lead to biosynthesis of the lipoxins. In each pathway, sequential lipoxygenase reactions are required, followed by hydrolysis. The immediate precursor of the lipoxins is epoxytetraene; hydrolysis of epoxytetraene yields the lipoxins. Left pathway: Arachidonic acid is converted to 15-HETE by sequential activity of 15-lipoxygenase and peroxidase. 15-HETE is converted by 5-lipoxygenase to the chemical intermediate 5-hydroperoxy, 15-hydroxyeicosatetraenoic acid, and 5-lipoxygenase acts on this intermediate to form an epoxytetraene. Right pathway: Arachidonic acid is converted to 5-HPETE by 5-lipoxygenase, and 5-HPETE is converted to LTA4 by further action of 5-lipoxygenase. LTA4 is converted to epoxytetraene by 15-lipoxygenase. Common pathway: Epoxytetraene is hydrolyzed to the active lipoxins LXA4 and LXB4. The lipoxins have both anti-inflammatory and pro-resolving roles, are counter-regulators of leukotriene action, and regulate many cytokines and growth factors. LXA4 is a highly selective agonist for the G protein-coupled receptor FPR2/ALX. In a transcellular reaction, platelet 12-lipoxygenase can also catalyze the formation of LXA4 from neutrophil-derived LTA4; the detailed mechanism remains to be elucidated (not shown; see also Fig. 42-7).

CHAPTER 42 / Pharmacology of Eicosanoids 749

A H

H COOH

H(O)O

Aspirin-acetylated COX-2 COOH

Eicosapentaenoic acid (EPA)

18-hydroperoxy-EPA 5-lipoxygenase HOOC O(O)H

5(6)-epoxyresolvin Esynthase reaction COOH O

OH

HO

5(6)-epoxy-18-hydroxy-EPA

5-hydroperoxy, 18-hydroxy-EPA

Enzymatic epoxide hydrolysis

Peroxidase

OH OH

COOH

HO OH COOH

Resolvin E1 (RvE1)

Regulates neutrophil infiltration Regulates dendritic cell function and IL-12 production Promotes resolution Reduces colitis Protects from osteoclast-mediated bone destruction

FIGURE 42-6.

HO

Resolvin E2 (RvE2)

Regulates neutrophil infiltration

Resolvins, protectins, and maresins: biosynthesis and actions of novel families of omega-3-derived mediators. A. EPA is the precursor to E-series resolvins. B and C. DHA is the precursor to D-series resolvins, protectins, and maresins. Some of the major endogenous anti-inflammatory and proresolving functions are listed below some of the mediators. In addition, resolvin D1 regulates neutrophil infiltration and resolvin D2 enhances microbial phagocytosis and clearance. (continued)

750 Principles of Inflammation and Immune Pharmacology B HOOC

HOOC

HO OH

HO

HO

OH

OH

Resolvin D1 (RvD1)

Resolvin D2 (RvD2)

7(8)-epoxide intermediate

5-lipoxygenase H

H HOOC

Lipoxygenase COOH

OOH

5-lipoxygenase

Docosahexaenoic acid (DHA)

17-hydroperoxy-DHA

4(5)-epoxide intermediate

OH HOOC

COOH OH OH OH

Resolvin D3 (RvD3)

FIGURE 42-6.

(continued)

HO OH

Resolvin D4 (RvD4)

CHAPTER 42 / Pharmacology of Eicosanoids 751

C

H

H

H

H

COOH

COOH

Docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA) 12/15-lipoxygenases

Lipoxygenase

O(O)H HOOC

COOH

OOH

17-hydroperoxy-DHA

14-hydroperoxy-DHA

Enzymatic epoxidation and hydrolysis

Enzymatic epoxidation and conversion

COOH

COOH

OH

OH OH

OH

Protectin D1 (PD1)

Regulates neutrophil and T cell infiltration Regulates TNF and interferon production Promotes resolution Reduces peritonitis and airway inflammation Protects brain from ischemia/reperfusion injury Mitigates kidney ischemia injury

FIGURE 42-6.

(continued)

Maresin 1 (MaR1)

Regulates neutrophil infiltration Promotes resolution

Platelet

LTC4

LTA4

LXA4 LXB4

AA A

Leukocyte

5-Lipoxygenase/FLAP

LTA4 LTA4 hydrolase

LTB4

Prostacyclin Prostacyclin synthase

COX

AA Endothelial cell

LTA4

LTC4 LTC4 synthase

Salicylate class

Propionic acid class

O OH

OH O

O

Ibuprofen

O

Aspirin Acetic acid class (Phenyl acetic acids)

Oxicam class

O

OH

HO

O

Cl

N H

H N

N

N S O

O

Cl

Piroxicam Diclofenac

Acetic acid class (Indole acetic acids)

Aminophenol class O

O OH

NH

O N O OH

Cl

O

Indomethacin

Acetaminophen

Fenamate class

Ketone class

OH

O H N

O

Nabumetone Mefenamate

756 Principles of Inflammation and Immune Pharmacology

inactivated by aspirin, the aspirin-modified COX-2 enzyme retains a distinct part of its catalytic activity and can form a new product, 15-(R)-HETE, from arachidonic acid. By analogy to lipoxin biosynthesis (Fig. 42-5), 5-LOX then converts 15-(R)-HETE to 15-epi-lipoxins, which are relatively stable stereoisomers (carbon 15-position epimers) of lipoxins that are collectively called aspirin-triggered lipoxins (ATLs). 15-Epi-lipoxins mimic the functions of lipoxins as antiinflammatory agents. 15-Epi-lipoxins may represent another endogenous mechanism of anti-inflammation, and their production mediates at least part of the anti-inflammatory effects of aspirin. Development of 15-epi-lipoxin analogues could lead to anti-inflammatory drugs that do not have the adverse effects associated with COX-1 inhibition. Aspirin is generally well tolerated. Its major toxicities are the gastropathy and nephropathy common to all NSAIDs. Long-term aspirin therapy can lead to gastrointestinal ulceration and hemorrhage, nephrotoxicity, and hepatic injury. NSAIDs should be used cautiously, if at all, in patients with renal insufficiency and heart failure. Two unique toxicities are aspirin-induced airway hyperreactivity in asthmatics (so-called aspirin-sensitive asthma) and Reye’s syndrome. The prevalence of aspirin sensitivity among patients with asthma is approximately 10%. Exposure to aspirin in these patients leads to ocular and nasal congestion along with severe airway obstruction. Aspirin-sensitive patients are also reactive to some other NSAIDs, including indomethacin, naproxen, ibuprofen, mefenamate, and phenylbutazone. One possible etiology of aspirin/NSAID sensitivity in asthmatics is that exposure to these drugs leads to increased levels of leukotrienes, which are implicated in the pathogenesis of asthma (see Fig. 42-1). Reye’s syndrome is a condition characterized by hepatic encephalopathy and liver steatosis in young children. Aspirin therapy during the course of a febrile viral infection has been implicated as a potential etiology of the liver damage. Although a causal link between aspirin and Reye’s syndrome has not been definitively established, aspirin is generally not administered to children because of the fear of Reye’s syndrome. Acetaminophen is widely used in children instead of aspirin. Propionic Acid Derivatives

Propionic acid NSAIDs include ibuprofen, naproxen, ketoprofen, and flurbiprofen. Ibuprofen is a relatively potent analgesic used in rheumatoid arthritis (as in the case of Ms. G, to relieve intermittent pain), osteoarthritis, ankylosing spondylitis, gout, and primary dysmenorrhea. Naproxen has a long plasma half-life, is 20 times more potent than aspirin, directly inhibits leukocyte function, and causes less severe gastrointestinal adverse effects than aspirin. Acetic Acid Derivatives

Acetic acid NSAIDs include the indole acetic acids— indomethacin, sulindac, and etodolac—and the phenylacetic acids diclofenac and ketorolac (a substituted phenylacetic acid derivative). Besides inhibiting cyclooxygenase, many of the acetic acid NSAIDs promote the incorporation of unesterified arachidonic acid into triglyceride, thus reducing the availability of the substrate for cyclooxygenase and lipoxygenase. Indomethacin is a direct inhibitor of neutrophil motility, but it is not tolerated by patients as well as ibuprofen. Diclofenac also reduces intracellular arachidonic

acid concentrations by altering cellular fatty acid transport. Diclofenac is a more potent anti-inflammatory than indomethacin and naproxen and is used widely in the treatment of pain associated with renal stones. Ketorolac is primarily employed for its strong analgesic properties, particularly in postsurgical patients; however, in part due to its potency and adverse effects, ketorolac is used for no more than 3–5 days. The acetic acid NSAIDs are mostly used to relieve symptoms in the long-term treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and other musculoskeletal disorders. Use of acetic acid NSAIDs causes gastrointestinal ulceration and, rarely, hepatitis and jaundice. Indomethacin also has a specific use in promoting the closure of a patent ductus arteriosus in newborns by inhibiting the vasodilatory eicosanoids PGE2 and PGI2. Oxicam Derivatives

Piroxicam is as efficacious as aspirin, naproxen, and ibuprofen in the treatment of rheumatoid arthritis and osteoarthritis, but may be better tolerated. Piroxicam has additional effects in the modulation of neutrophil function by inhibiting collagenase, proteoglycanase, and the oxidative burst. Because of its extremely long half-life, piroxicam can be administered once daily. As with other NSAIDs, piroxicam displays gastrointestinal adverse effects such as gastric ulceration, and it prolongs the bleeding time because of its antiplatelet effect. Fenamate Derivatives

The two fenamate NSAIDs are mefenamate and meclofenamate. Both inhibit cyclooxygenases but also antagonize prostanoid receptors to various degrees. Because fenamates have less anti-inflammatory activity and are more toxic than aspirin, there is little advantage to their use. Mefenamate is used only for primary dysmenorrhea, and meclofenamate is used in the treatment of rheumatoid arthritis and osteoarthritis. Ketone NSAIDs

Nabumetone is a ketone prodrug that is oxidized in vivo to the active acid form. Compared to other nonselective NSAIDs, nabumetone has preferential activity against COX-2. The incidence of gastrointestinal adverse effects is relatively low, although headache and dizziness are frequently reported. Acetaminophen Acetaminophen is sometimes classified with the NSAIDs, but it is technically not an NSAID: although acetaminophen has analgesic and antipyretic effects similar to aspirin, the anti-inflammatory effect of acetaminophen is insignificant because of its weak inhibition of cyclooxygenases. Nonetheless, acetaminophen therapy can be valuable in patients, such as children, who are at risk for the adverse effects of aspirin. The most important adverse effect of acetaminophen is hepatotoxicity. Modification of acetaminophen by hepatic cytochrome P450 enzymes produces a reactive metabolite, which is normally detoxified by conjugation with glutathione. An overdose of acetaminophen can overwhelm glutathione stores, leading to cellular and oxidative damage and, in severe cases, to acute hepatic necrosis (see Chapter 5, Drug Toxicity). Selection of the Appropriate NSAID The anti-inflammatory, analgesic, and antipyretic effects of the NSAIDs appear to vary among the many agents in

O

O

O

O S

S H2N N

N O

CF3

O

Celecoxib

Rofecoxib

O

O S

OH

O

H2N

S N H

O

N N

N S O

O

Meloxicam

Valdecoxib

CHAPTER 42 / Pharmacology of Eicosanoids 759

A

via its receptors on neutrophils, inhibits LTB4 biosynthesis by regulating arachidonic acid release and, possibly, by interfering with the influx of calcium. Furthermore, adenosine is thought to have a role in limiting cell and tissue injury during inflammation. High cell turnover at inflammatory sites generates high local concentrations of adenosine, which may decrease LTB4 biosynthesis and reduce leukocyte recruitment and activation. Selective adenosine receptor agonists could be considered for development as pharmacologic agents in the control of inflammation.

O HO N

NH2

S

Zileuton

B O

O

O S

O N H

N

HN

O O

Zafirlukast

COOH

S Cl

N

HO

Montelukast

FIGURE 42-10. Leukotriene pathway inhibitors. A. Zileuton is a 5lipoxygenase inhibitor that blocks the biosynthesis of leukotrienes from arachidonic acid. B. Zafirlukast and montelukast are CysLT1 receptor antagonists. All three drugs are approved for the prophylaxis and chronic treatment of asthma in both adults and children. None of these drugs is effective in the treatment of acute asthma attacks.

zileuton is not as widely used as the other antileukotriene asthma drugs (see below). 5-Lipoxygenase Activating Protein (FLAP) Inhibition Interfering with the role of FLAP could represent an alternative approach to the selective inhibition of 5-LOX activity and leukotriene function. Recall that 5-LOX is activated after the enzyme translocates to the nuclear membrane and docks with FLAP and that FLAP binds arachidonic acid released by phospholipase A2 and shuttles it to the 5-LOX active site. FLAP inhibitors have been developed that both prevent and reverse LOX binding to FLAP and block the arachidonic acid binding site, but no FLAP inhibitors are currently available for clinical use. Leukotriene Synthesis Inhibitors Other than zileuton, no specific inhibitors of the enzymes involved in leukotriene synthesis are available for clinical use. Specific LTA4 hydrolase inhibitors, which block LTB4 biosynthesis, are currently in development. Adenosine, acting

Leukotriene Receptor Antagonists Leukotriene receptor antagonism represents a receptor-based mechanism for inhibiting leukotriene-mediated bronchoconstriction and other effects (Fig. 42-4). Cysteinyl leukotriene receptor (CysLT1) antagonists are effective against asthma induced by antigen, exercise, cold, or aspirin. These agents significantly improve bronchial tone, pulmonary function tests, and asthma symptoms. Montelukast and zafirlukast (Fig. 42-10B) are the currently available cysteinyl leukotriene receptor antagonists; the main clinical application for these antagonists is in the treatment of asthma. More potent CysLT1 antagonists are in development, including pobilukast, tomelukast, and verlukast. Further research will likely elucidate cysteinyl leukotriene receptor subtypes and their respective tissue distributions, which could offer the possibility of tissue-targeted antagonism and the application of these tissue-selective antagonists to other conditions such as rheumatoid arthritis, inflammatory bowel disease, and various allergic disorders.

Lipoxins, Aspirin-Triggered Lipoxins, Resolvins/Protectins/Maresins, and Lipoxin-Stable Analogues Lipoxins, ATLs, and the omega-3-derived resolvins, protectins, and maresins all offer the potential to antagonize the inflammatory actions of leukotrienes and other inflammatory mediators and to promote resolution of inflammation. Stable, oral and parenteral analogues of these compounds could represent a new approach to treatment, since they are agonists of endogenous anti-inflammation and pro-resolution pathways rather than direct enzyme inhibitors or receptor antagonists. Because lipoxins are endogenous regulators, they would be expected to have selective actions with few adverse effects. Stable analogues of lipoxins and ATLs are currently being developed, and second-generation lipoxin-stable analogues have shown efficacy in enhancing the resolution of recurring bouts of acute inflammation in skin inflammation and gastrointestinal inflammation models. This new approach to the treatment of inflammation remains to be established in human trials.

 CONCLUSION AND FUTURE DIRECTIONS Eicosanoids are critical mediators of homeostasis and of many pathophysiologic processes, especially those involving host defense and inflammation. Arachidonic acid is the important substrate and is converted into prostaglandins, thromboxanes, prostacyclin, leukotrienes, lipoxins, isoprostanes, and epoxyeicosatetraenoic acids (EETs). Prostaglandins have diverse roles in vascular tone regulation, gastrointestinal regulation, uterine physiology, analgesia, and inflammation.

760 Principles of Inflammation and Immune Pharmacology

Prostacyclin and thromboxane coordinately control vascular tone, platelet activation, and thrombogenesis. Leukotrienes (LTC4, LTD4) are the chief mediators of bronchoconstriction and airway hyperactivity; LTB4 is a major activator of leukocyte chemotaxis and infiltration. Lipoxins antagonize the effects of leukotrienes, reduce the extent of inflammation, and activate resolution pathways. Pharmacologic interventions at many critical points in these pathways are useful in limiting inflammatory sequelae. Glucocorticoids inhibit several steps in eicosanoid generation, including the rate-determining step involving phospholipase A2. However, chronic glucocorticoid use is associated with many serious adverse effects, including osteoporosis, muscle wasting, and abnormal carbohydrate metabolism. Cyclooxygenase inhibitors block the first step of prostanoid synthesis and prevent the generation of prostanoid mediators of inflammation. Lipoxygenase inhibitors, FLAP inhibitors, leukotriene synthesis inhibitors, and leukotriene receptor antagonists prevent leukotriene signaling, thereby limiting inflammation and its deleterious effects. Future drug development efforts will allow selective targeting of eicosanoid pathways involved in many clinical conditions. Systems biology has revealed mechanisms underlying inflammatory disease and has created a new discipline of resolution pharmacology. Essential omega-3 fatty acids, in particular eicosapentaenoic acid and docosahexaenoic acid, are precursors to pro-resolving and anti-inflammatory SPM that serve a physiologic role leading to programmed resolution of inflammation (Fig. 42-6). These new bioactive

mediators are many times more potent than their respective omega-3 precursors and, hence, may mediate the essential and beneficial effects of omega-3 fatty acids. In the near future, resolvins and protectins may be developed as new therapeutic agents to promote resolution of inflammation.

Suggested Reading Brink C, Dahlen SE, Drazen J, et al. International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors. Pharmacol Rev 2003;55:195–227. (International consensus report on eicosanoid receptors and their antagonists.) Gilroy DW, Perretti M. Aspirin and steroids: new mechanistic findings and avenues for drug discovery. Curr Opin Pharmacol 2005;5:1–7. (Reviews the anti-inflammatory actions of aspirin-triggered lipoxins and the discovery of annexin and related compounds in the actions of glucocorticoids.) Patrono C, Baigent C. Low-dose aspirin, coxibs, and other NSAIDS: a clinical mosaic emerges. Mol Interv 2009;9:31–39. (Reviews the data regarding cardiovascular risks of NSAIDs and coxibs.) Psaty BM, Furberg CD. COX-2 inhibitors—lessons in drug safety. N Engl J Med 2005;352:1133–1135. (Reviews issues surrounding withdrawal of COX-2 selective inhibitors.) Serhan CN. Resolution phases of inflammation: novel endogenous antiinflammatory and pro-resolving lipid mediators and pathways. Annu Rev Immunol 2007;25:101–137. (Reviews the pathways mediating resolution of inflammation.) Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 2008;8:249–261. (Reviews advances in the role of eicosanoid pathways and novel lipid mediators in resolution programs of inflammation.) Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Ann Rev Pharmacol Toxicol 1998;38:97–120. (Historic overview of prostaglandin research, including discussion of the pharmacologic manipulation of these pathways.)

43 Histamine Pharmacology Cindy Chambers, Joseph C. Kvedar, and April W. Armstrong

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 765-766 PHYSIOLOGY OF HISTAMINE . . . . . . . . . . . . . . . . . . . . . . . . 765 Histamine Synthesis, Storage, and Release . . . . . . . . . . . 765 Actions of Histamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765 Histamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768 Clinical Manifestations of Histamine Pathophysiology. . . . 769 Histamine and Anaphylaxis . . . . . . . . . . . . . . . . . . . . . . . 769 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 769 H1-Antihistamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . 769

Classification of First- and Second-Generation H1-Antihistamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770 Pharmacologic Effects and Clinical Uses . . . . . . . . . . . 771 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 Other Antihistamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 773 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773

 INTRODUCTION

level of this metabolite is used to determine the amount of histamine that has been released systemically. Histamine synthesis and storage can be divided into two “pools”: a slowly turning over pool and a rapidly turning over pool. The slowly turning over pool is located in mast cells and basophils. Histamine is stored in large granules in these inflammatory cells, and the release of histamine involves complete degranulation of the cells. Degranulation can be triggered by allergic processes, anaphylaxis, or cellular destruction from trauma, cold, or other insults. This pool is termed slowly turning over because several weeks are required to replenish the stores of histamine after degranulation has occurred. The rapidly turning over pool is located in gastric ECL cells and in histaminergic CNS neurons. These cells synthesize and release histamine as required for gastric acid secretion and neurotransmission, respectively. Unlike mast cells and basophils, ECL cells and histaminergic neurons do not store histamine. Instead, the production and release of histamine in these cells depend on physiologic stimuli. In the gut, for example, histidine decarboxylase is activated after the ingestion of food.

Histamine is a biogenic amine found in many tissues, including mast cells, basophils, lymphocytes, neurons, and gastric enterochromaffin-like cells. It is an autacoid—that is, a molecule secreted locally to increase or decrease the activity of nearby cells. Histamine is a major mediator of allergic and inflammatory processes: it also has significant roles in the regulation of gastric acid secretion, neurotransmission, and immune modulation. Knowledge of the diverse actions of histamine has led to the development of a number of widely used pharmacologic agents that regulate the effects of histamine in pathologic states. This chapter focuses on the pharmacologic actions of H1-antihistamines; H2-antihistamines are discussed in Chapter 46, Integrative Inflammation Pharmacology: Peptic Ulcer Disease.

 PHYSIOLOGY OF HISTAMINE Histamine Synthesis, Storage, and Release Histamine is synthesized from the amino acid L-histidine. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to 2-(4-imidazolyl)ethylamine, commonly known as histamine (Fig. 43-1). The synthesis of histamine occurs in mast cells and basophils of the immune system, enterochromaffin-like (ECL) cells in the gastric mucosa, and certain neurons in the central nervous system (CNS) that use histamine as a neurotransmitter. Oxidative pathways in the liver rapidly degrade circulating histamine to inert metabolites. One major metabolite of histamine, imidazole acetic acid, can be measured in the urine, and the

Actions of Histamine Histamine has a wide spectrum of actions involving many organs and organ systems. To understand the roles of histamine, it is useful to consider the physiologic effects of histamine in each tissue (Table 43-1). These effects include actions on smooth muscle, vascular endothelium, afferent nerve terminals, heart, gastrointestinal tract, and CNS. On smooth muscle, histamine causes some muscle fibers to contract and others to relax. Histamine dilates all terminal 765

NH2

N HN

HO

O

Histidine Decarboxylation (L-histidine decarboxylase)

NH2

N HN

Histamine

Ring methylation (Imidazole-Nmethyltransferase)

NH2

N

Oxidative deamination (mainly Diamine oxidase)

OH

N O

HN

N

Methyl histamine

ImAA

Oxidation (Monoamine oxidase)

OH

N N

Conjugation with ribose

O

Methyl ImAA

ImAA riboside

768 Principles of Inflammation and Immune Pharmacology

expressed on cardiac muscle cells, on some immune cells, and on certain postsynaptic neurons in the central nervous system. H2 receptors on parietal cells activate a G proteindependent cyclic AMP cascade, leading to enhanced proton pump-mediated delivery of protons into the gastric fluid. Whereas H1 and H2 receptor subtypes have been well characterized, H3 and H4 receptor subtypes and their downstream actions are areas of active investigation. H3 receptors are predominantly located on presynaptic neurons in distinct regions of the CNS, including the cerebral cortex, basal ganglia, and tuberomammillary nucleus of the hypothalamus. H3 receptors appear to function as both autoreceptors and heteroreceptors, thereby limiting the synthesis and release of histamine as well as other neurotransmitters, including dopamine, acetylcholine, norepinephrine, GABA, and serotonin. This complex interaction between histamine and various neurotransmitter systems contributes to histamine’s widespread effects on CNS functions, including wakefulness, appetite, and memory. H3 receptors have also been localized in the peripheral nervous system and appear to limit histaminergic actions in gastric mucosa and bronchial smooth muscle. The downstream effects of H3 receptor activation are mediated via a decrease in Ca2⫹ influx. H4 receptors are localized to cells of hematopoietic origin, primarily mast cells, eosinophils, and basophils. H4 receptors share 40% homology with H3 receptors and bind many H3 receptor agonists, although with lower affinity. Coupling

of the H4 receptor to Gi/o leads to decreased cAMP and activation of phospholipase C␤, and downstream events result in increased intracellular Ca2⫹. H4 receptors are of particular interest because they are thought to play an important role in inflammation; activation of H4 receptors mediates histamine-induced leukotriene B4 production, adhesion molecule up-regulation, and chemotaxis of mast cells, eosinophils, and dendritic cells.

 PATHOPHYSIOLOGY Histamine is an essential mediator of immune and inflammatory responses. Histamine plays a prominent role in the IgEmediated type I hypersensitivity reaction, also known as the allergic reaction. In a localized allergic reaction, an allergen (antigen) first penetrates an epithelial surface (e.g., skin, nasal mucosa). The allergen can also be delivered systemically, as in the case of an allergic response to penicillin. With the aid of T-helper (TH) cells, the allergen stimulates B lymphocytes to produce IgE antibodies that are specific for that allergen. The IgE then binds to Fc receptors on mast cells and basophils, in a process known as sensitization. Once these immune cells are “sensitized” with IgE antibodies, they are able to detect and respond rapidly to a subsequent exposure to the allergen. Upon such an exposure, the allergen binds to and cross-links the IgE/Fc receptor complexes, triggering cell degranulation (Fig. 43-2).

A Initial exposure Allergen

B cell

IgE Mast cell

Mast cell Capillary

Granules IgE

B Subsequent exposure Allergen

Edema fluid

Crosslinked IgE

Histamine

Degranulated mast cell

Mast cell degranulation

FIGURE 43-2.

Pathophysiology of the IgE-mediated hypersensitivity reaction. Allergen-induced mast cell degranulation requires two separate exposures to the allergen. A. On initial exposure, the allergen must penetrate mucosal surfaces so that it can encounter cells of the immune system. Activation of the immune response causes B lymphocytes to secrete allergen-specific IgE antibodies. These IgE molecules bind to Fc receptors on mast cells, leading to sensitization of the mast cells. B. On subsequent exposure, the multivalent allergen cross-links two IgE/Fc receptor complexes on the mast cell surface. Receptor cross-linking causes the mast cell to degranulate. Local histamine release results in an inflammatory response, shown here as edema.

A

αq/11 β

αq/11

γ

GTP

GDP

Inactive state

Active state X

Agonist (histamine) B

N

N

Histamine

General structure (X = C, O, or omitted)

αq/11 β

γ

αq/11 GTP

GDP

Inactive state

Ethers or ethanolamines

Cl O

N

Active state

N

N

Inverse agonist (H1-antihistamines)

H1-antihistamine

Alkylamines

C

Diphenhydramine

Chlorpheniramine

Ethylenediamines

Phenothiazines N

αq/11 β GDP

γ

N

αq/11 GTP

N N

N

S

Inactive state

Active state Tripelennamine

Promethazine Piperazines

Piperidines

N N

N Cyclizine

Cyproheptadine

H N

N

H N

S N

HN

C N

Cimetidine

H N

O N

H N

S NO2 Ranitidine

CHAPTER 43 / Histamine Pharmacology 773

Similar to H3 receptors, H4 receptors couple with Gi/o to decrease intracellular cAMP concentrations. Because H4 receptors are selectively expressed on cells of hematopoietic origin, especially mast cells, basophils, and eosinophils, there is considerable interest in elucidating the role of the H4 receptor in the inflammatory process. H4 receptor antagonists represent a promising area of drug development to treat inflammatory conditions that involve mast cells and eosinophils.

 CONCLUSION AND FUTURE DIRECTIONS Histamine plays a key role in diverse physiologic processes including allergy, inflammation, neurotransmission, and gastric acid secretion. Drugs targeting H1 and H2 receptors have substantially increased the pharmacologic options for treatment of allergy and peptic ulcer disease. While most H1antihistamines demonstrate similar efficacy in the treatment of allergic rhinitis and urticaria, significant differences exist in the adverse effect profiles of first- and second-generation H1-antihistamines. The more recent elucidation of the H3 and H4 receptor subtypes has renewed interest in the role of histamine in CNS-related disorders. H3-specific receptor targeting may provide new therapies for a number of cognitive, neuroendocrine, and neuropsychiatric conditions. Clinical and preclinical research is currently under way evaluating prototypic H3 antagonists in pathological processes such as sleep-wake disorders (narcolepsy and insomnia),

neuropsychiatric diseases (Alzheimer’s disease, ADHD, dementia, depression, and schizophrenia), neurologic disorders (epilepsy), nociceptive processes (neuropathic pain), and feeding and energy homeostasis (obesity and diabetes). The H4 receptor is also an exciting molecular target for drug development, as it is thought to play an important role in inflammatory conditions involving mast cells and eosinophils. Agents directed against H4 receptors might one day be employed to treat a variety of inflammatory conditions, such as asthma, allergic rhinitis, inflammatory bowel disease, and rheumatoid arthritis.

Suggested Reading Leurs R, Church MK, Taglialatea M. H1-antihistamines: inverse agonism, anti-inflammatory actions and cardiac effects. Clin Exp Allergy 2002;32:489–498. (Mechanism-based discussion of H1-antihistamines as inverse agonists.) Nicolas JM. The metabolic profile of second-generation antihistamine. Allergy 2000;55:46–52. (Discussion of differences among second-generation drugs.) Sander K, Kottke T, Stark H. Histamine H3 receptor antagonists go to clinics. Biol Pharm Bull 2008;31:2163–2181. (Comprehensively reviews the current state of H3 receptor antagonist research.) Simons FE. Advances in H1-antihistamines. N Engl J Med 2004;351:2203– 2217. (Comprehensively summarizes the mechanism of action and clinical uses of H1-antihistamines.) Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov 2008;7:41–53. (Reviews the role of histamine in inflammation and immune modulation, with emphasis on the role of the H4 receptor.)

44 Pharmacology of Hematopoiesis and Immunomodulation Andrew J. Wagner, Ramy A. Arnaout, and George D. Demetri

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 776-777 PHYSIOLOGY OF HEMATOPOIESIS . . . . . . . . . . . . . . . . . . . . 776 Central Role of Hematopoietic Growth Factors . . . . . . . . . 778 Multilineage Growth Factors . . . . . . . . . . . . . . . . . . . . 779 Lineage-Specific Growth Factors . . . . . . . . . . . . . . . . 779 Erythrocyte Production (Erythropoiesis) . . . . . . . . . . . . . . 779 Erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779 Leukocyte Production (Myelopoiesis and Lymphopoiesis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Granulocyte-Stimulating Factors . . . . . . . . . . . . . . . . . 781 Lymphocyte-Stimulating Factors . . . . . . . . . . . . . . . . . 782 Platelet Production (Thrombopoiesis) . . . . . . . . . . . . . . . . 782 Thrombopoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 782 Agents That Stimulate Erythrocyte Production . . . . . . . . . 783 Recombinant Human Erythropoietin (rhEPO) and Darbepoetin (NESP). . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Agents That Induce Fetal Hemoglobin (HbF) . . . . . . . . . . . 783 5-Azacytidine and Decitabine . . . . . . . . . . . . . . . . . . . 784 Hydroxyurea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784 Butyrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784 Agents That Stimulate Leukocyte Production . . . . . . . . . . 784 Recombinant Human G-CSFs (Filgrastim and PEG-Filgrastim) and GM-CSF (Sargramostim) . . . . . . . 784 Agents That Stimulate Platelet Production . . . . . . . . . . . . 785 Thrombopoietin and Pharmacologic Analogues . . . . . . 785 Interleukin-11 (rhIL-11 [Oprelvekin]) . . . . . . . . . . . . . . 785 Immunomodulatory Agents with Antineoplastic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785 Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785 Levamisole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785 Interleukin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785 Tretinoin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 786 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

 INTRODUCTION

that stimulate their production, and the pharmacologic agents used to increase blood cell production. An outline of the immunomodulatory agents used in anticancer therapy is also presented.

A number of clinical situations are characterized by deficiencies of red blood cells, white blood cells, or platelets— cells of the hematopoietic system. This chapter describes the pharmacologic agents that can be used to stimulate production of hematopoietic cells; it is also important to note the nonpharmacologic alternatives, which could include transfusion and bone marrow transplantation. Blood cell production is controlled physiologically by hematopoietic growth factors, a diverse but functionally overlapping group of glycoproteins produced by the body in response to certain signals. For example, hypoxia stimulates production of the erythroid lineage growth factor erythropoietin, which in turn stimulates the production of erythrocytes in an attempt to relieve the hypoxia. The main pharmacologic strategy used to stimulate the production of blood cells is to administer exogenous growth factors or synthetic growth factor analogues. This chapter provides an introduction to the cells of the hematopoietic system, the growth factors

776

 PHYSIOLOGY OF HEMATOPOIESIS The cells of the hematopoietic system are functionally diverse (Table 44-1). Red blood cells, or erythrocytes, carry oxygen; many types of white blood cells, from granulocytes and macrophages to lymphocytes, fight infection and help protect against cancer; and platelets help control bleeding. Nonetheless, these cells all have one feature in common: they all develop from a common cell in the bone marrow called the pluripotent hematopoietic stem cell (Fig. 44-1). Hematopoietic stem cells are induced to differentiate along committed lineages into red blood cells, white blood cells, or platelets through interactions with glycoproteins called hematopoietic growth factors.

778 Principles of Inflammation and Immune Pharmacology

Bone marrow

Multipotent stem cells

Pluripotent hematopoietic stem cell

SCF, IL-6, Flt3L

Lymphoid stem cell

Trilineage myeloid stem cell (CFU-S) IL-3, GM-CSF, IL-6

Committed progenitor cells

IL-5

SCF, Flt3L, IL-7

GM-CSF

CFU-Eo IL-5

TPO, IL-11

CFU-Mix

CFU-G/M

M-CSF

G-CSF

CFU-M

Pro-B

Flt3L CFUMega

CFU-G

TPO

Pro-NK

IL-15

Pro-T

IL-7

BFU-E

EPO Thymus CFU-E

Morphologically recognizable precursor cells

EPO

Eosinophiloblast

Monoblast

Myeloblast

Megakaryoblast Proerythroblast

Mature cells

Blood and tissues

Eosinophil

Monocyte/ macrophage

Neutrophil

Platelets

Erythrocyte

B cell

NK cell

T cell

FIGURE 44-1.

Development of cells of the hematopoietic system. Mature cells of the hematopoietic system all develop from pluripotent stem cells that reside in the bone marrow. The type of mature cell that develops is dependent on the extracellular milieu and the exposure of stem cells and progenitor cells to specific growth factors. The pluripotent stem cell differentiates into a trilineage myeloid stem cell (CFU-S) or a lymphoid stem cell. Depending on the growth factors that are present, CFU-S cells differentiate into granulocytes (eosinophils, neutrophils), monocyte/macrophages, platelets, or erythrocytes. Lymphoid stem cells differentiate into B cells, natural killer (NK) cells, or T cells. Except for the terminal differentiation of pro-T cells to mature T cells, which takes place in the thymus, the differentiation of all hematopoietic stem cells, progenitor cells, and precursor cells occurs in the bone marrow. Of the growth factors illustrated here, G-CSF, GM-CSF, erythropoietin (EPO), and IL-11 are currently used as therapeutic agents. BFU, burst-forming unit; CFU, colony-forming unit; CSF, colony-stimulating factor; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin.

Central Role of Hematopoietic Growth Factors Hematopoietic growth factors and cytokines constitute a heterogeneous group of molecules that regulate blood cell production, maturation, and function. Nearly 36 such factors have been identified, ranging in size from 9 to 90 kDa. The membrane-associated receptors for these factors belong to at least six receptor superfamilies, and genes encoding

the factors are found on 11 different chromosomes. Conceptually, growth factors can be divided into two groups: multilineage (also called general or early-acting or pleiotropic) growth factors, which stimulate multiple lineages, and lineage-specific (also called lineage-dominant or late-acting) growth factors, which stimulate differentiation and survival of a single lineage. Many growth factors and

Normal or high O2

CoCl2 Iron chelation Antioxidants

Low O2

HIF-1α

HIF-1α PHD

PHD O2 or CO

HIF-1α

OH

VHL complex

Ub Ub Ub Ub Ub

HIF-1α

OH

Nucleus

26S proteasome HIF-1α HIF-1β Ub Ub

HIF-1α fragments

Transcription of VEGF, PDGF-β, TGF-α, EPO genes

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IL-5 is produced by a subset of helper T cells. This growth factor selectively promotes the differentiation, adhesion, degranulation, and survival of eosinophils. As such, IL-5 is believed to have an important role in the pathophysiology of allergic reactions and asthma. Lymphocyte-Stimulating Factors Regulatory proteins called interleukins control lymphocyte development and activation. To date, more than 30 members of this family have been defined. Family members are numbered IL-1, IL-2, and so forth. Interleukins regulate not only lymphocyte differentiation but also multiple and overlapping aspects of the innate and adaptive immune responses, including stimulation of T cells and macrophages. Several interleukins are described above as granulocyte-stimulating factors; others are discussed below in the context of platelet production. IL-2 and IL-7 are two interleukins critical to white blood cell differentiation. IL-2 is a 45-kDa protein produced by T cells. Because it drives proliferation of T cells and B cells, IL-2 once received much attention as a potential immunostimulant. Investigations of this hypothesis showed, however, that mice lacking IL-2 exhibit lymphoproliferative rather than lymphopenic diseases. This unexpected finding underscores the principle that growth factors and immune cells have diverse functions in vivo, including, as in this case, regulatory or suppressive (tolerogenic) effects as well as stimulatory effects. This finding also points out that uncontrolled proliferation can ensue if differentiation is not regulated normally, a process that may underlie some types of cancer. IL-7, produced by cells in the spleen, thymus, and bone marrow stroma, is a multilineage lymphostimulatory growth factor that enhances the growth and differentiation of B cells and T cells. The interferons constitute a second family of regulatory proteins that modulate lymphocyte growth and activity. Like the interleukins, these proteins can stimulate the activity of T cells and macrophages. Interferons have prominent antiviral actions and are used in the treatment of infections such as hepatitis B and C (see Chapter 37, Pharmacology of Viral Infections). Other effects of interferons include promoting the terminal differentiation of lymphocytes, suppressing cell division (in some situations), and exerting direct cytotoxic effects on cells under stress. The three types of interferons— called IFN-␣, IFN-␤, and IFN-␥—have different biological actions. The cellular effects of interferons, like those of growth factors, are mediated by specific cell surface receptors and JAK-STAT signal transduction cascades.

Platelet Production (Thrombopoiesis) Platelets—sometimes called thrombocytes—are essential for clot formation. These small cells, which lack a nucleus and do not synthesize new proteins, have a half-life of about 9 or 10 days in the circulation. The production of platelets, like that of all formed elements of the hematopoietic system, is controlled by both multilineage and lineage-specific growth factors (Fig. 44-3). The most important multilineage growth factors that stimulate platelet production are IL-11, IL-3, GM-CSF, stem cell factor, and IL-6. Not surprisingly, these factors also stimulate the production of erythrocytes because platelets and erythrocytes share a common progenitor, the CFU-Mix cell. Whether CFU-Mix cells become erythrocytes or platelets depends on their subsequent exposure to

Megakaryoblast

Myeloid stem cell

IL-11 TPO

IL-11

Platelets

TPO

IL-3 GM-CSF SCF Early

IL-6

Late Stage of megakaryocytopoiesis

FIGURE 44-3.

Growth factors involved in platelet production. A number of growth factors are involved in platelet production (megakaryocytopoiesis). IL-11 acts primarily in the early stages; this growth factor stimulates production of GMCSF and acts synergistically with IL-3 and stem cell factor (SCF) to increase the proliferation and differentiation of megakaryocyte progenitors. IL-6 and thrombopoietin (TPO) act primarily in the late stages of megakaryocytopoiesis. Both recombinant human IL-11 (oprelvekin) and TPO receptor agonists (eltrombopag and romiplostim) can be used therapeutically to increase platelet production.

lineage-specific growth factors. Differentiation into BFU-E and other cells of the erythroid lineage is promoted by erythropoietin. In contrast, differentiation into CFU-Mega cells and then into megakaryocytes (which then form platelets) is promoted by the lineage-specific growth factor thrombopoietin (Fig. 44-1). Thrombopoietin Thrombopoietin (TPO) is produced in the liver and, to a lesser extent, in the proximal convoluted tubule of the kidney. Like erythropoietin, thrombopoietin is a heavily glycosylated protein (35 kDa) that has its major effect on a single cell lineage; also like erythropoietin, thrombopoietin signals through a JAK-STAT transduction cascade. However, unlike erythropoietin, thrombopoietin is not regulated in its activity at the level of gene expression, because thrombopoietin is expressed constitutively. Instead, by an interesting functional mechanism, circulating levels of thrombopoietin are regulated by the thrombopoietin receptor (also known as Mpl), which is the protein product of the gene c-mpl. Structurally and functionally, the thrombopoietin receptor resembles the receptors for IL-3, erythropoietin, and GMCSF. It is found both on platelet progenitors—CFU-S, CFUMix, CFU-Mega, and megakaryocytes—and on platelets themselves. Thrombopoietin has different effects on these cell types, however. On platelet progenitors, the binding of thrombopoietin to its receptor promotes cell growth and differentiation. In contrast, thrombopoietin receptors on platelets act as molecular sponges to bind excess thrombopoietin and thereby prevent platelet overproduction if platelets are in adequate supply. Thrombopoietin also enhances platelet function by sensitizing these cells to the proaggregatory effects of thrombin and collagen (see Chapter 22, Pharmacology of Hemostasis and Thrombosis).

 PHARMACOLOGIC CLASSES AND AGENTS The hematopoietic growth factors used clinically can be divided into two groups. First, recombinant or synthetic growth factor analogues are used to treat deficiencies of

CHAPTER 44 / Pharmacology of Hematopoiesis and Immunomodulation 785

G-CSF. Of note, however, these patients also received a higher dose of cyclophosphamide than patients who did not develop AML/MDS. GM-CSF is associated with fever, arthralgia, edema, and pleural and pericardial effusion. G-CSF and GMCSF are proteins and must be administered parenterally, typically by daily injection over the course of several weeks.

Agents That Stimulate Platelet Production A low platelet count, or thrombocytopenia, is an important adverse effect of many cancer chemotherapeutic agents, occasionally limiting the doses that can be delivered with acceptable safety and tolerability. The complications of thrombocytopenia include increased bleeding risk and platelet transfusion requirement; in turn, platelet transfusion is associated with an increased risk of infection, febrile reaction, and, rarely, graft-versus-host disease. Research into the pharmacologic management of chemotherapy-induced thrombocytopenia has focused on the thrombopoietin (TPO) analogues recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (see below). To date, however, only recombinant human IL-11 (rhIL-11 or oprelvekin) has been approved by the FDA for this indication. These agents all have the potential to increase megakaryocytopoiesis (platelet production) in a dose-dependent fashion; although these drugs stimulate some multipotent as well as committed precursor cells, they do not significantly increase the hematocrit or white blood cell count. Importantly, these agents must all be administered prophylactically because there is a 1–2 week delay from drug administration to a clinically significant increase in platelet count. Thrombopoietin and Pharmacologic Analogues Cloning of the thrombopoietin gene in 1994 led to the development of two thrombopoietin analogues. The first, rhTPO, is a full-length, glycosylated analogue; the second, PEG-rHuMGDF, consists of the N-terminal 163 amino acids of thrombopoietin conjugated to polyethylene glycol (PEG). Like natural thrombopoietin, both rhTPO and PEGrHuMGDF bind to Mpl (the endogenous receptor for thrombopoietin, named for its role in murine myeloproliferative leukemia), and activation of Mpl is the basis for the effect of these drugs. Both rhTPO and PEG-rHuMGDF have been tested as prophylactic agents to minimize chemotherapyinduced thrombocytopenia, and both can cause a 2- to 10-fold increase in the platelet count. One caution is that stimulation of platelet production could lead to thrombosis if the platelets that are produced are also activated. A small trial of PEG-rHuMGDF suggests that this drug is safe to use in treating the thrombocytopenia associated with AML, even though AML cells may also express the TPO receptor. The heavily bioengineered variants of natural TPO (e.g., PEG-rHuMGDF) have recently been dropped from clinical development because of an excess risk of developing anti-TPO autoantibodies, which could suppress natural platelet production. The testing of full-length rhTPO continues; there are no reports to date of neutralizing antibodies in patients who receive this lightly bioengineered agent, which differs from native human TPO only in its glycosylation pattern. Two new TPO receptor agonists have recently been approved by the FDA for treatment of thrombocytopenia due

to refractory immune thrombocytopenic purpura (ITP), an autoimmune disease caused by autoantibodies directed against the patient’s own platelets. These drugs include eltrombopag, a small-molecule TPO receptor agonist, and romiplostim, a recombinant IgG1 Fc-peptide fusion protein that also binds and activates the TPO receptor. By activating the TPO receptor, both molecules induce a transient increase in the platelet count. However, worsening thrombocytopenia may develop after cessation of treatment with these agents, and bone marrow toxicity manifesting as bone marrow fibrosis and other conditions has also been reported. Interleukin-11 (rhIL-11 [Oprelvekin]) Recombinant human IL-11 (rhIL-11), also called oprelvekin, is the only drug currently approved for the prevention of severe thrombocytopenia in patients receiving myelosuppressive chemotherapy. Oprelvekin is produced in Escherichia coli and differs from natural IL-11 only in its lack of the Nterminal proline residue. rhIL-11 causes a dose-dependent increase in the platelet count and in the number of megakaryocytes in the bone marrow. The practical goal of treatment with oprelvekin is to maintain the platelet count above 20,000/␮L (normal range, 150,000–450,000/␮L) in order to minimize the risk of life-threatening bleeding. However, the use of rhIL-11 is associated with significant adverse effects, especially fatigue and fluid retention. Atrial fibrillation has also been observed, and rhIL-11 should be used with caution in any patient with underlying heart disease. The undesirable actions of rhIL-11 likely result from pleiotropic effects of this factor on receptors distributed outside the hematopoietic system. It is unclear whether the therapeutic benefit of this agent outweighs the risk of systemic adverse effects.

Immunomodulatory Agents with Antineoplastic Applications Interferons Clinical investigation has led to the use of interferons as therapeutic agents against a number of different malignancies, with moderate success. However, the multiple and overlapping effects of these proteins have made it difficult to determine the drugs’ mechanism of action in any given clinical situation. Induction of antitumor-directed immunity, terminal differentiation of cycling tumor cells, and direct cytotoxic effects have all been hypothesized to have important roles in the treatment of different malignancies. Interferons are also used to treat certain viral infections and are discussed in greater detail in Chapter 37. Levamisole Levamisole was known as an antihelminthic agent for decades before its anticancer effects were discovered. In combination with the antimetabolite 5-fluorouracil (see Chapter 38), this drug is now approved for use in the treatment of colon cancer. Although its mechanism of action remains uncertain, levamisole is thought to cause macrophages and T cells to secrete cytokines (such as IL-1) and other factors that suppress tumor growth. Interleukin-2 Interleukin-2 (IL-2) is approved by the FDA for the treatment of melanoma. At therapeutic doses, however, this cytokine has relatively low efficacy and relatively high toxicity.

786 Principles of Inflammation and Immune Pharmacology

See Chapter 45, Pharmacology of Immunosuppression, for more information about IL-2. Tretinoin Tretinoin, or all-trans retinoic acid (ATRA), is a ligand of the retinoic acid receptor (RAR). ATRA is used in the treatment of acute promyelocytic leukemia. This disease is characterized by a translocation t(15;17) in which part of the RAR␣ gene is fused to the PML gene, creating a fusion protein that induces a block to differentiation and thereby allows development of the leukemia. Treatment with ATRA stimulates differentiation of these cells into more normal granulocytes. In some patients, the induction of differentiation can lead to a life-threatening overproduction of white blood cells. ATRA can also induce a rapidly progressive syndrome of fever, acute respiratory distress with pulmonary infiltrates, edema and weight gain, and multisystem organ failure. Therapy with high doses of glucocorticoids is often an effective treatment for this ATRA syndrome.

 CONCLUSION AND FUTURE DIRECTIONS The production of cells of the hematopoietic system— red blood cells (erythrocytes), white blood cells (neutrophils, monocytes, lymphocytes, and other cell types), and platelets—is controlled by a variety of proteins called growth factors and cytokines. Cancer chemotherapy, malignant infiltration of the bone marrow, and other conditions can cause deficiencies in these cell populations (anemia, neutropenia, and/or thrombocytopenia). The agents currently used to treat these deficiencies are mainly recombinant analogues of the natural growth factors or agonists of the growth factor receptors. Thus, the erythropoietin analogues rhEPO and darbepoetin treat anemia; the G-CSF and GM-CSF analogues filgrastim, PEG-filgrastim, and sargramostim treat neutropenia; and rhIL-11 and the thrombopoietin receptor agonists rhTPO, eltrombopag, and romiplostim treat thrombocytopenia. Several agents affecting the hematopoietic system are also used to treat sickle cell disease, a common autosomal recessive disease caused by a point mutation in the ␤ globin gene. These agents (hydroxyurea, 5-azacytidine, and decitabine) increase expression of fetal hemoglobin (HbF) and thereby restore normal erythrocyte structure and function. Several other drugs, including recombinant forms of the immunostimulatory

interferon proteins, levamisole, and retinoic acid, are used to treat certain cancers, although their precise mechanisms of action remain unknown. Other agents that activate hematopoiesis continue to be identified. Preclinical evidence suggests that daily injections of a parathyroid hormone analogue (PTH 1-34) promote blood cell development, perhaps by activating stimulatory receptors on osteoblasts that neighbor hematopoietic stem cells. These observations have led to clinical trials of PTH in enhancing stem cell production for transplantation and in protecting hematopoietic stem cells from the cytotoxic effects of chemotherapy. Studies designed to tease apart the complex overlapping functionalities of hematopoiesis-regulating proteins are likely to provide a source of more selective pharmacologic interventions in the future.

Suggested Reading Demetri GD. Anaemia and its functional consequences in cancer patients: current challenges in management and prospects for improving therapy. Br J Cancer 2001;84:31–37. (Reviews the use and effectiveness of recombinant human erythropoietin.) Hankins J, Aygun B. Pharmacotherapy in sickle cell disease—state of the art and future prospects. Br J Haematol 2009;145:296–308. (Reviews use of hydroxyurea and decitabine.) Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet 2003;362(9392):1255–1260. (Describes unfavorable outcome in head and neck cancer patients receiving epoetin beta.) Kaushansky K. Lineage-specific hematopoietic growth factors. N Engl J Med 2006;354:2034–2045. (Reviews hematopoietic growth factors.) Kuter DJ. Thrombopoietin and thrombopoietin mimetics in the treatment of thrombocytopenia. Annu Rev Med 2009;60:193–206. (Reviews recent advances in treatment of thrombocytopenia, including use of romiplostim and eltrombopag.) Singh AK, Szczech L, Tang KL, et al. Correction of anemia with epoetin alfa in chronic kidney disease. N Engl J Med 2006;355:2085–2098. Pfeffer MA, Burdmann EA, Chen CY, et al. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease. N Engl J Med 2009;361:2019–2032. (Clinical trials of erythropoiesis-stimulating agents in patients with anemia and chronic kidney disease.) Smith TJ, Khatcheressian J, Lyman GH, et al. Update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline. J Clin Oncol 2006;24:3187–3205. (American Society of Clinical Oncology guidelines for the use of myeloid growth factors.) Vansteenkiste J, Pirker R, Massuti B, et al. Double-blind, placebo-controlled, randomized phase III trial of darbepoetin alfa in lung cancer patients receiving chemotherapy. J Natl Cancer Inst 2002;94:1211–1220. (Evidence for clinical effectiveness of darbepoetin.)

45 Pharmacology of Immunosuppression April W. Armstrong, Ehrin J. Armstrong, and Lloyd B. Klickstein

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 790-791 PATHOPHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790 Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790 Solid Organ Rejection . . . . . . . . . . . . . . . . . . . . . . . . . 790 Graft-Versus-Host Disease (GVHD) . . . . . . . . . . . . . . . 792 Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 792 Inhibitors of Gene Expression . . . . . . . . . . . . . . . . . . . . . . 793 Glucocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793 Cytotoxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794 Antimetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794 Alkylating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796 Specific Lymphocyte-Signaling Inhibitors . . . . . . . . . . . . . 796 Cyclosporine and Tacrolimus . . . . . . . . . . . . . . . . . . . . 796 mTOR Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

Cytokine Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798 TNF-␣ Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798 IL-12/IL-23p40 Inhibitors . . . . . . . . . . . . . . . . . . . . . . 799 IL-1 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 Cytokine Receptor Antagonists . . . . . . . . . . . . . . . . . . 800 Depletion of Specific Immune Cells . . . . . . . . . . . . . . . . . 800 Polyclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 800 Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 800 LFA-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801 Inhibition of Costimulation . . . . . . . . . . . . . . . . . . . . . . . . 801 Abatacept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801 Blockade of Cell Adhesion . . . . . . . . . . . . . . . . . . . . . . . . 802 Natalizumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802 Inhibition of Complement Activation . . . . . . . . . . . . . . . . . 802 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 802 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802

 INTRODUCTION

most organ transplantation occurs between unrelated individuals. Donor and recipient tissues express different MHC class I molecules, and recipient immune cells therefore recognize the transplanted tissues as foreign. This is termed alloimmunity, and it occurs when the recipient’s immune system attacks a transplanted organ. In the case of a bone marrow or stem cell transplant, graft-versus-host disease (GVHD) can result when donor lymphocytes mount an assault on recipient tissues.

Patients with autoimmune disease and patients who have received transplanted tissues or organs typically require therapy with immunosuppressive drugs. Immunosuppressive agents have been in use for more than 50 years, beginning with corticosteroids, antimetabolites, and alkylating agents. These early agents assisted in the treatment of previously incurable conditions, but their lack of specificity led to many serious adverse effects. Over the past 20 years, the field of immunosuppression has shifted to specific inhibitors of immunity that affect distinct immune pathways. This shift is important both because of the greater efficacy and reduced toxicity of these agents, and because, as the mechanisms of these agents are discovered, insights are gained into the operation of the immune system.

 PATHOPHYSIOLOGY Transplantation The first transplant performed successfully in humans was a kidney transplant between identical twins. No immunosuppression was used, and the individuals did well. Currently, 790

Solid Organ Rejection Transplant rejection of solid organs can be divided into three major phases according to the time to onset. These phases, hyperacute, acute, and chronic rejection, are caused by different mechanisms and are therefore treated differently. The following three sections examine each of these processes, and Table 45-1 summarizes their differences. Hyperacute Rejection

Hyperacute rejection is mediated by preformed recipient antibodies against donor antigen. Because these antibodies are present at the time of organ implantation, hyperacute rejection occurs almost immediately after reperfusion of the transplanted organ. In fact, the surgeon can observe the

792 Principles of Inflammation and Immune Pharmacology

directed against endothelial cells and is thus also known as acute vascular rejection. Like acute cellular rejection, acute humoral rejection can usually be prevented by immunosuppression of the recipient after transplantation. Even with immunosuppression, however, episodes of acute rejection can occur months or even years after transplantation. Chronic Rejection

Chronic rejection is believed to be both humoral and cellular in nature and does not occur until months or years after transplantation. Because hyperacute and acute rejection are generally well controlled by donor/recipient matching and immunosuppressive therapy, chronic rejection is now the most common life-threatening pathology associated with organ transplantation. Chronic rejection is thought to result from chronic inflammation caused by the response of activated T cells to donor antigen. Activated T cells release cytokines that recruit macrophages into the graft. The macrophages induce chronic inflammation that leads to intimal proliferation of the vasculature and scarring of the graft tissue. The chronic changes eventually lead to irreversible organ failure. Other contributing nonimmune factors can include ischemia-reperfusion injury and infection. No effective treatment regimens are currently available to eliminate chronic rejection. It is believed, however, that several experimental therapies have a reasonable chance of reducing chronic rejection. Especially promising is the possibility of developing tolerance through elimination of costimulation (see below). Graft-Versus-Host Disease (GVHD) Leukemia, primary immunodeficiency, and other conditions can be treated with bone marrow or peripheral stem cell transplantation. In this procedure, hematopoietic and immune function is restored after the patient’s bone marrow has been eradicated by aggressive chemotherapy and/or radiation therapy. GVHD is a major complication of allogeneic bone marrow or stem cell transplantation. GVHD is an alloimmune inflammatory reaction that occurs when transplanted immune cells attack the cells of the recipient. The severity of GVHD ranges from mild to life-threatening and typically involves the skin (rash), gastrointestinal tract (diarrhea), lungs (pneumonitis), and liver (veno-occlusive disease). GVHD can often be ameliorated by removing T cells from the donor bone marrow before transplantation. Mild-to-moderate GVHD can also be beneficial when donor immune cells attack recipient tumor cells that have survived the aggressive chemotherapy and radiation therapy. (In the case of leukemia, this is called the graft-versus-leukemia effect, or GVL.) Therefore, although removing donor T cells from the “graft” reduces the risk of GVHD, this may not be the best approach for marrow transplants used in antineoplastic therapy.

in the thymus (T cells) and bone marrow (B cells). Central tolerance ensures that the majority of immature autoreactive T and B cells do not develop into self-reactive clones. The thymus and bone marrow do not express every antigen in the body, however; a number of proteins are expressed only in specific tissues. For this reason, peripheral tolerance is also important. Peripheral tolerance results from deletion of autoreactive T cells by Fas-Fas ligand-mediated apoptosis, activation of T suppressor cells, or induction of T-cell anergy due to antigen presentation in the absence of costimulation. Although breakdown in tolerance lies at the center of virtually all autoimmune diseases, the inciting stimulus leading to loss of tolerance is often unknown. Genetic factors may play a role, in that the presence of certain MHC subtypes may predispose T cells to the loss of self-tolerance. For example, human leukocyte antigen (HLA)-B27 is causally related to many forms of autoimmune spondylitis. Several other autoimmune diseases are linked to specific HLA loci, supporting an association, if not a causal role, for genetic predisposition to autoimmunity. Molecular mimicry, whereby epitopes from infectious agents are similar to self-antigens, can also lead to a breakdown of tolerance and may be the mechanism underlying poststreptococcal glomerulonephritis. A number of other processes, including failure of T-cell apoptosis, polyclonal lymphocyte activation, and exposure of cryptic self-antigens, have also been hypothesized to lead to autoimmunity. The details of these mechanisms are beyond the scope of this book; however, the result of each is a loss of tolerance. Once self-tolerance has been compromised, the specific expression of autoimmunity can take one of three general forms (Table 45-2). In some diseases, production of autoantibodies against a specific antigen causes antibody-dependent opsonization of cells in the target organ, with subsequent cytotoxicity. One example is Goodpasture’s syndrome, which results from autoantibodies against collagen type IV in the renal glomerular basement membrane. In some autoimmune vasculitis syndromes, circulating antibody–antigen complexes deposit in blood vessels, causing inflammation and injury to the vessels. Two examples of immune-complex disease are mixed essential cryoglobulinemia and systemic lupus erythematosus. Finally, T-cell-mediated diseases are caused by cytotoxic T cells that react with a specific self-antigen, resulting in destruction of the tissue(s) expressing that antigen. One example is type 1 diabetes mellitus, in which the cytotoxic T cells react against self-antigens in pancreatic ␤-cells. The pharmacologic therapy for autoimmune diseases does not yet match the exquisite specificity of the offending biological process. Most currently available pharmacologic agents cause generalized immunosuppression and do not target the specific pathophysiology. Better understanding of the molecular pathways leading to autoimmune diseases should reveal new pharmacologic targets that can be used to suppress the specific autoimmune response before disease arises.

Autoimmunity Autoimmune diseases occur when the host immune system attacks its own tissues, mistaking self-antigen for foreign. The typical result is chronic inflammation in the tissue(s) expressing the antigen. Autoimmune diseases are most commonly due to a breakdown of self-tolerance, both central and peripheral. Central tolerance refers to the specific clonal deletion of autoreactive T and B cells during their development from precursor cells

 PHARMACOLOGIC CLASSES AND AGENTS Pharmacologic suppression of the immune system utilizes eight mechanistic approaches (Fig. 45-1): 1. Inhibition of gene expression to modulate inflammatory responses 2. Depletion of expanding lymphocyte populations with cytotoxic agents

4

Cytokines

Cytokine receptor T cell

MHC class II TCR 3

1

CD4

B7

CD28 5

6

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Cell surface receptor

Cl

on

al

ex pa n si o n of

7

Tissues

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CHAPTER 45 / Pharmacology of Immunosuppression 795

N O2

N N

S N

N N

N H

Azathioprine

+

Glutathione

S N

HN N

N H

Mercaptopurine

FIGURE 45-2. Formation of mercaptopurine from azathioprine. Azathioprine is a prodrug form of the antimetabolite 6-mercaptopurine. Mercaptopurine is formed by the cleavage of azathioprine in a nonenzymatic reaction with glutathione. Although mercaptopurine can also be used directly as a cytotoxic agent, azathioprine has greater oral bioavailability and a longer duration of action and is more immunosuppressive than mercaptopurine.

5-fluorouracil, 6-mercaptopurine, and mycophenolic acid, also promote apoptosis of activated T cells. MTX may be such a versatile drug because of its combined antineutrophil, anti-T-cell, and antihumoral effects. Mycophenolic Acid and Mycophenolate Mofetil

Mycophenolic acid (MPA) is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the formation of guanosine (see Fig. 38-3). Because MPA has low oral bioavailability, it is usually administered as a sodium salt or in its prodrug form, mycophenolate mofetil (MMF), both of which have greater oral bioavailability (Fig. 45-3). MMF is increasingly used in the treatment of immune-mediated disease because of its high selectivity and profound effect on lymphocytes. MPA and MMF both act primarily on lymphocytes. Two main factors contribute to this selectivity. First, as discussed in Chapter 38, lymphocytes are dependent on the de novo pathway of purine synthesis, whereas most other tissues rely heavily on the salvage pathway. Because IMPDH is required for de novo synthesis of guanosine nucleotides but not for the salvage pathway, MPA affects only cells such as lymphocytes that rely on de novo purine synthesis. Second, IMPDH is expressed in two isoforms, type I and type II. MPA preferentially inhibits type II IMPDH, the isoform expressed mainly in lymphocytes. Together, these factors confer on MPA and MMF selectivity against T and B cells, with relatively low toxicity to other cells. Inhibition of IMPDH by MPA reduces intracellular guanosine levels and elevates intracellular adenosine levels, with many downstream effects on lymphocyte activation and activity. MPA has a cytostatic effect on lymphocytes, but can also induce apoptosis of activated T cells leading to the elimination of reactive clones of proliferative cells. Because guanosine is required for some glycosylation reactions, the reduction in guanosine nucleotides leads to decreased expression of

adhesion molecules that are required for recruitment of several immune cell types to sites of inflammation. Furthermore, because guanosine is a precursor of tetrahydrobiopterin (BH4), which regulates inducible nitric oxide synthase (iNOS), the reduction in guanosine levels leads to decreased NO production by neutrophils. Endothelial NOS (eNOS), which controls vascular tone and is regulated by Ca2⫹ and calmodulin, is not affected by changes in guanosine levels, again demonstrating the considerable selectivity of MPA. As noted above, clinical studies comparing MMF and AZA have shown MMF to be more efficacious in preventing acute rejection of kidney transplants. Animal models show that chronic rejection is also reduced more effectively in recipients treated with MMF than in those treated with AZA or cyclosporine. The efficacy of MMF in treating chronic rejection may be related to its inhibition of both the lymphocyte and the smooth muscle cell proliferation characteristic of chronic rejection. MMF is also efficacious in the treatment of autoimmune disease. In rheumatoid arthritis, levels of rheumatoid factor, immunoglobulin, and T cells are reduced by treatment with MMF. MMF is frequently used in the initial therapy of lupus nephritis. There have also been isolated reports of successful treatment of myasthenia gravis, psoriasis, autoimmune hemolytic anemia, and inflammatory bowel disease with MMF. The most common adverse effect of MMF is gastrointestinal discomfort, which is dose-dependent and can include nausea, diarrhea, soft stools, anorexia, and vomiting. Leflunomide

Activated lymphocytes both proliferate and synthesize large quantities of cytokines and other effector molecules, and these processes require increased DNA and RNA synthesis. Therefore, agents that reduce intracellular nucleotide pools have effects on these activated cells. Leflunomide is an

O

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O

OH OH

O O O

Mycophenolic acid

FIGURE 45-3. Mycophenolic acid and mycophenolate mofetil. Mycophenolate mofetil (MMF) has greater oral bioavailability than mycophenolic acid (MPA). Orally administered mycophenolate mofetil is absorbed into the circulation, where plasma esterases rapidly cleave the ester bond to yield mycophenolic acid. Both agents inhibit inosine monophosphate dehydrogenase type II (IMPDH II), an enzyme crucial for de novo synthesis of guanosine. Because of its greater oral bioavailability, MMF (or the sodium salt of MPA) is typically used.

796 Principles of Inflammation and Immune Pharmacology

FIGURE 45-4. Inhibition of pyrimidine synthesis by leflunomide. De novo pyrimidine synthesis depends on the oxidation of dihydroorotate to orotate, a reaction that is catalyzed by dihydroorotate dehydrogenase. Leflunomide inhibits dihydroorotate dehydrogenase and thereby inhibits pyrimidine synthesis. Because lymphocytes are dependent on de novo pyrimidine synthesis for cell proliferation and clonal expansion after immune cell activation, depletion of the pyrimidine pool inhibits lymphocyte expansion. Experimentally, leflunomide appears to inhibit preferentially the proliferation of B cells; the reason for this preferential action is unknown.

O-

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N-Carbamoylaspartate H+ Dihydroorotase H2O

inhibitor of pyrimidine synthesis, specifically blocking the synthesis of uridylate (UMP) by inhibiting dihydroorotate dehydrogenase (DHOD). DHOD is a key enzyme in the synthesis of UMP (Fig. 45-4), which is essential for the synthesis of all pyrimidines. (See Chapter 38 for a review of pyrimidine synthesis.) Experimentally, leflunomide has been shown to be most effective in reducing B-cell populations, but a significant effect on T cells has also been observed. Leflunomide is currently approved for use in rheumatoid arthritis, but the drug has also shown significant efficacy in the treatment of other immune diseases, including systemic lupus erythematosus and myasthenia gravis. Leflunomide prolongs transplant graft survival and limits GVHD in animal models. The most significant adverse effects of leflunomide are diarrhea and reversible alopecia. Leflunomide undergoes significant enterohepatic circulation, resulting in a prolonged pharmacologic effect. If leflunomide must be removed quickly from a patient’s system, cholestyramine may be administered. By binding to bile acids, cholestyramine interrupts the enterohepatic circulation and causes a “washout” of leflunomide.

O HN ON H

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Dihydroorotate NAD+ Dihydroorotate dehydrogenase

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Alkylating Agents

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Cyclophosphamide (Cy) is a highly toxic drug that alkylates DNA. The mechanism of action and uses of Cy are discussed extensively in Chapter 38; therefore, the discussion here is limited to Cy’s utility in treating diseases of the immune system. Because Cy has a major suppressive effect on B-cell proliferation but can enhance T-cell responses, the use of Cy in immune diseases is limited to disorders of humoral immunity, particularly systemic lupus erythematosus. Another use under consideration for Cy is the suppression of antibody formation against xenotransplant grafts. Adverse effects of Cy are severe and widespread, including leukopenia, cardiotoxicity, alopecia, and an increased risk of cancer because of mutagenicity. The risk of bladder cancer is especially notable because Cy produces a carcinogenic metabolite, acrolein, which is concentrated in the urine. When high-dose Cy is administered by intravenous infusion, acrolein can be detoxified by co-administration of mesna (a sulfhydryl-containing compound that neutralizes the reactive moiety of acrolein).

Specific Lymphocyte-Signaling Inhibitors Cyclosporine and Tacrolimus The discovery in 1976 that cyclosporine (CsA; also referred to as cyclosporin A) is a specific inhibitor of T-cell-mediated immunity enabled widespread whole-organ transplantation. In fact, CsA made heart transplantation a legitimate alternative in the treatment of end-stage heart failure. CsA is a cyclic decapeptide isolated from a soil fungus, Tolypocladium inflatum.

Cyclosporine

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IL-2 mRNA

798 Principles of Inflammation and Immune Pharmacology

IL-2

Sirolimus

IL-2 receptor

mTOR FKBP p70 S6 kinase

PHAS-1

Translation of selected mRNAs needed for cell cycle progression

FIGURE 45-6.

Mechanism of action of sirolimus. IL-2 receptor signal transduction involves a complex set of protein–protein interactions that lead to increased translation of selected mRNAs encoding proteins required for T-cell proliferation. Specifically, IL-2 receptor activation initiates an intracellular signaling cascade that leads to phosphorylation of the molecular target of rapamycin (mTOR). mTOR is a kinase that phosphorylates and thereby regulates the activity of PHAS-1 and p70 S6 kinase. PHAS-1 inhibits the activity of a factor (eIF4E) required for translation, and p70 S6 kinase phosphorylates proteins involved in protein synthesis (not shown). The net effect of mTOR activation is to increase protein synthesis, thereby promoting the transition from G1 to S phase of the cell cycle. Sirolimus (also known as rapamycin) crosses the plasma membrane and binds to intracellular FK-binding protein (FKBP). The sirolimus–FKBP complex inhibits mTOR, thereby inhibiting translation and causing T cells to arrest in G1. Everolimus and zotarolimus are sirolimus analogues that act by the same mechanism.

TNF-␣. TNF-␣ stimulates macrophages to produce cytotoxic metabolites, thereby increasing phagocytic killing activity. TNF-␣ also stimulates production of acute-phase proteins, has pyrogenic effects, and fosters local containment of the inflammatory response. Some of these effects are indirect and are mediated by other cytokines induced by TNF-␣. TNF-␣ has been implicated in numerous autoimmune diseases. Rheumatoid arthritis, psoriasis, and Crohn’s disease are three disorders in which inhibition of TNF-␣ has demonstrated therapeutic efficacy. Rheumatoid arthritis illustrates the central role of TNF-␣ in the pathophysiology of autoimmune diseases (Fig. 45-7). Although the initial stimulus for joint inflammation is still debated, it is thought that macrophages in a diseased joint secrete TNF-␣, which activates endothelial cells, other monocytes, and synovial fibroblasts. Activated endothelial cells up-regulate adhesion molecule expression, resulting in recruitment of inflammatory cells to the joint. Monocyte activation has a positive feedback effect on T-cell and synovial fibroblast activation. Activated synovial fibroblasts secrete interleukins, which recruit additional inflammatory cells. With time, the synovium hypertrophies and forms a pannus that leads to destruction of bone and cartilage in the joint, causing the characteristic deformity and pain of rheumatoid arthritis. Five therapies interfering with TNF-␣ activity have been approved. Etanercept is a soluble TNF receptor dimer that

Macrophage

TNF Endothelial cells

artery smooth muscle cells and thereby reducing the rate of in-stent restenosis that results from neointimal proliferation of vascular smooth muscle cells.

Monocyte/macrophage

IL-1

Cytokine Inhibition Cytokines are critical signaling mediators in immune function. Cytokines are also pleiotropic; that is, they exert different effects depending on the target cell and overall cytokine milieu. For this reason, pharmacologic uses of cytokines or cytokine inhibitors may have unpredictable effects. Anticytokine therapy has been in clinical use for immunologically mediated diseases for more than a decade. The first anticytokine agent approved for clinical use was etanercept, an anti-TNF-␣ drug developed for rheumatoid arthritis. During the initial clinical studies, some patients with severe, drugrefractory rheumatoid arthritis literally got up from their wheelchairs and walked after receiving etanercept. This dramatic efficacy ushered in a new age of biological therapies for autoimmune disease, and the number of new drugs that inhibit proinflammatory cytokines continues to grow rapidly. TNF-␣ Inhibitors Tumor necrosis factor-␣ (TNF-␣) is a cytokine central to many aspects of the inflammatory response. Macrophages, mast cells, and activated TH cells (especially TH1 cells) secrete

Activated endothelial cells

IL-8

Synovial fibroblast

T cell

Matrix metalloproteases PGE2 IL-6 Cartilage degradation

Leukocyte adhesion and diapedesis

FIGURE 45-7. Proposed roles for tumor necrosis factor in rheumatoid arthritis. Tumor necrosis factor (TNF) is secreted by activated macrophages in an affected joint, where this cytokine has multiple proinflammatory effects. First, TNF activates endothelial cells to up-regulate their expression of cell surface adhesion molecules (shown as projections on endothelial cells) and undergo other phenotypic changes that promote leukocyte adhesion and diapedesis. Second, TNF has a positive feedback effect on nearby monocytes and macrophages, promoting their secretion of cytokines such as IL-1. In turn, IL-1 activates T cells (among other functions), and the combination of IL-1 and TNF stimulates synovial fibroblasts to increase their expression of matrix metalloproteases, prostaglandins (especially PGE2), and cytokines (such as IL-6) that degrade the joint cartilage. Synovial fibroblasts also secrete IL-8, which promotes neutrophil diapedesis.

CHAPTER 45 / Pharmacology of Immunosuppression 799

links the extracellular, ligand-binding domain of human TNF receptor type II to the Fc domain of human immunoglobulin G1 (IgG1); infliximab is a partially humanized mouse monoclonal antibody against human TNF-␣; adalimumab is a fully human IgG1 monoclonal antibody against TNF-␣ (Fig. 45-8). Certolizumab pegol is a pegylated anti-TNF-␣ monoclonal antibody fragment that lacks the Fc portion of the antibody; as a result, unlike infliximab and adalimumab, certolizumab does not cause antibody-dependent cell-mediated cytotoxicity or fix complement in vitro. Golimumab is a fully human IgG1 monoclonal antibody against TNF-␣ that has a longer half-life than the other anti-TNF-␣ agents.

Extracellular domain of human p75 TNF receptor

s s s s

Fc domain of human IgG1

CH2

CH2

CH3

CH3

Etanercept

VH

VH CH1

VL

CH1

VL

CL

CL s s s s CH2

CH2

CH3

CH3

Infliximab

Human

FIGURE 45-8.

Mouse

Anti-TNF agents. Shown is the molecular domain organization of etanercept and infliximab. Etanercept consists of the extracellular domain of the human TNF receptor fused to the Fc domain of human IgG1. This “decoy” receptor binds TNF-␣ and TNF-␤ in the circulation, preventing the access of these cytokines to target tissues. Infliximab is a partially humanized monoclonal antibody against TNF-␣. The variable heavy chain (VH) and variable light chain (VL) regions are derived from mouse antihuman sequences, while the remainder of the antibody (the constant regions, denoted by CH and CL) is composed of human antibody sequences. This modification of the original mouse anti-TNF-␣ monoclonal antibody reduces the development of neutralizing antibodies against infliximab. Additional TNF-␣ inhibitors include adalimumab, a fully human monoclonal antibody; certolizumab pegol, a pegylated monoclonal antibody fragment; and golimumab, a fully human monoclonal antibody (not shown).

Although all of these agents target TNF-␣, etanercept is somewhat less specific because it binds to both TNF-␣ and TNF-␤. Infliximab, adalimumab, certolizumab, and golimumab are specific for TNF-␣ and do not bind TNF-␤. The Fc portions of infliximab, adalimumab, and golimumab may also have specific activity with respect to complement fixation and binding to Fc receptors on effector cells. The immune effector actions of these agents may be relevant to their mechanisms of action because TNF-␣ is expressed on the surface of cells, especially macrophages, and the cell-surface form is cleaved to yield the soluble cytokine. Anti-TNF-␣ agents with effector functions may have different biological effects than agents that do not bind Fc receptors or fix complement. Etanercept is approved for use in rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis; infliximab is approved for use in rheumatoid arthritis, Crohn’s disease, ulcerative colitis, plaque psoriasis, and ankylosing spondylitis; adalimumab is approved for use in rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, and Crohn’s disease. Certolizumab is approved for the treatment of Crohn’s disease. Golimumab is approved for use in adults with rheumatoid arthritis (in combination with methotrexate), psoriatic arthritis, and ankylosing spondylitis. It is important to note that high levels of TNF-␣ are likely mediators of underlying pathophysiologic processes. However, although treatment with an anti-TNF-␣ agent often improves disease symptoms, it may not reverse the underlying pathophysiology. Therefore, upon drug discontinuation, maintenance of clinical response is uncertain. Etanercept, infliximab, adalimumab, certolizumab, and golimumab are proteins and must be administered parenterally. Orally active inhibitors of TNF-␣ and inhibitors of TNF-␣ converting enzyme (TACE) are under investigation. A number of important adverse effects must be considered when administering TNF inhibitors. All patients should undergo screening for tuberculosis before initiating therapy because of increased risk of reactivating latent tuberculosis. Any patient developing an infection while taking a TNF-␣ inhibitor should undergo evaluation and aggressive antibiotic treatment. Epidemiologic surveillance has also suggested that there may be an increased risk of demyelinating disease with anti-TNF therapy, although it has not yet been determined whether the relationship is causal. IL-12/IL-23p40 Inhibitors New biological therapies for the treatment of T-cell-mediated diseases include antibodies to IL-12 and IL-23. IL-12 and IL-23 are cytokines involved in natural killer cell activation and CD4⫹ T-cell differentiation and activation. IL-12, a heterodimer composed of p40 and p35 subunits, directs the differentiation of naïve T cells into TH1 cells, which secrete IL-2, IFN-␥, and TNF-␣. IL-23 is also a heterodimer that has the same p40 subunit covalently linked to a p19 subunit. IL-23 directs the differentiation of naïve T cells into TH17 cells, which secrete IL-17 and IL-22. Ustekinumab is a high-affinity IgG1 human monoclonal antibody that binds to the p40 subunit shared by IL-12 and IL-23. Ustekinumab is approved for use in psoriasis and is in late-stage clinical trials for the treatment of multiple sclerosis and psoriatic arthritis. Adverse effects include an increased risk of infections.

CHAPTER 45 / Pharmacology of Immunosuppression 801

antibody not involved in binding to the antigen are changed to the corresponding human sequences. Antibodies can be partially or fully humanized, depending on the extent of these changes. Humanization limits the likelihood of production of human antibodies against the therapeutic antibody, increasing the clinical effectiveness of the antibody and allowing its long-term use (see Chapter 53, Protein Therapeutics). A more recent approach to the preparation of therapeutic antibodies is to prepare the antibody in an experimental animal bearing a human immune system or to use an in vitro human antibody system. This strategy generates fully human antibodies that do not require further manipulation to render them nonimmunogenic. Anti-CD20 mAb

Rituximab is a chimeric, partially humanized anti-CD20 monoclonal antibody. CD20 is expressed on the surface of all mature B cells, and administration of rituximab causes profound depletion of circulating B cells. Originally approved for the treatment of CD20⫹ non-Hodgkin’s lymphoma (see Chapter 39), rituximab has also been approved for use in rheumatoid arthritis refractory to TNF-␣ inhibitors. Several additional anti-CD20 antibodies are in clinical development; ofatumumab is a fully human anti-CD20 monoclonal antibody that recognizes an epitope distinct from that of rituximab. Ofatumumab is approved for use in chronic lymphocytic leukemia. Anti-CD25 mAb

Daclizumab and basiliximab are monoclonal antibodies against CD25, the high-affinity IL-2 receptor. IL-2 mediates early steps in T-cell activation. Because CD25 is expressed only on activated T cells, anti-CD25 antibody therapy selectively targets T cells that have been activated by an MHCantigen stimulus. Daclizumab is administered prophylactically in renal transplantation to inhibit acute organ rejection. It is also used as a component of general immunosuppressive regimens after organ transplantation. Daclizumab is typically administered in a five-dose regimen, with the first administration immediately after transplantation and then four additional doses at 2-week intervals. This type of dosing regimen, in which drug is administered for a limited period immediately after transplantation, is referred to as induction therapy. Anti-CD52 mAb

Campath-1 (CD52) is an antigen expressed on most mature lymphocytes and on some lymphocyte precursors. An antibody against this antigen was originally tested in rheumatoid arthritis and found to cause prolonged and sustained depletion of all T cells, often lasting for years. The reason for the sustained lymphocyte depletion is unknown. Anti-CD52 mAb therapy did lead to some improvement in the symptoms of arthritis; however, the sustained depletion of lymphocytes and concern about infections precluded further study of this antibody in autoimmune conditions. Under the generic name alemtuzumab, anti-CD52 mAb has been approved as an adjunctive therapy in the treatment of B-cell chronic lymphocytic leukemia—a condition in which sustained suppression of the leukemic cells is desirable. LFA-3 LFA-3 (also called CD58) is the counter-receptor for CD2, an antigen expressed at high levels on the surface

of memory effector T cells. Interaction of CD2 on T cells with LFA-3 on antigen-presenting cells promotes increased T-cell proliferation and enhanced T-cell-dependent cytotoxicity. Because the memory effector T-cell population is elevated in patients with psoriasis, a pharmacologic agent that disrupts the CD2–LFA-3 interaction was tested for use in psoriasis. Alefacept is an LFA-3/Fc fusion protein that interrupts CD2–LFA-3 signaling by binding to T-cell CD2, and thereby inhibits T-cell activation. Additionally, the Fc portion of alefacept may activate NK cells to deplete the immune system of memory effector T cells. Clinically, alefacept significantly decreases the severity of chronic plaque psoriasis. Because CD2 is expressed on other adaptive immune cells, administration of alefacept also causes a dose-dependent reduction in CD4 and CD8 T-cell populations. Its use is therefore contraindicated in patients with HIV, and patients taking alefacept may have an increased risk of serious infection. Alefacept therapy may also be associated with an increased risk of malignancy, primarily skin cancer.

Inhibition of Costimulation Costimulation refers to the paradigm that cells of the immune system typically require two signals for activation (see Chapter 41, Principles of Inflammation and the Immune System). If a first signal is provided in the absence of a second signal, the target immune cell may become anergic rather than activated. Because induction of anergy could lead to long-term acceptance of an organ graft or limit the extent of an autoimmune disease, inhibition of costimulation represents a viable strategy for immunosuppression. Several therapeutic agents inhibit costimulation by blocking the second signal required for cell activation, and more such agents are under development. Abatacept Abatacept consists of CTLA-4 fused to an IgG1 constant region. Abatacept complexes with costimulatory B7 molecules on the surface of antigen-presenting cells. When the antigenpresenting cell interacts with a T cell, MHC:antigen–TCR interaction (“signal 1”) occurs, but the complex of B7 with abatacept prevents delivery of a costimulatory signal (“signal 2”), and the T cell develops anergy or undergoes apoptosis. By this mechanism, abatacept therapy appears to be effective in down-regulating specific T-cell populations. Abatacept is approved for the treatment of rheumatoid arthritis that is refractory to methotrexate or TNF-␣ inhibitors. Clinically, abatacept significantly improves symptoms of rheumatoid arthritis in patients who fail to respond to methotrexate or TNF-␣ inhibitors. The major adverse effects of abatacept are exacerbations of bronchitis in patients with preexisting obstructive lung disease and increased susceptibility to infection. Abatacept should not be administered concurrently with TNF-␣ inhibitors or anakinra because the combination carries an unacceptably high risk of infection. Belatacept is a close structural congener of abatacept that has increased affinity for B7-1 and B7-2. In a large clinical trial, belatacept was as effective as cyclosporine at inhibiting acute rejection in renal transplant recipients. Belatacept is currently under further investigation as an immunosuppressant for organ transplantation.

802 Principles of Inflammation and Immune Pharmacology

Blockade of Cell Adhesion The recruitment and accumulation of inflammatory cells at sites of inflammation is an essential element of most autoimmune diseases; the only exceptions to this rule are autoimmune diseases that are purely humoral, such as myasthenia gravis. Drugs that inhibit cell migration to sites of inflammation may also inhibit antigen presentation and cytotoxicity, thus providing multiple potential mechanisms of beneficial action. Natalizumab Alpha-4 integrins are critical to immune-cell adhesion and homing. The ␣4␤1 integrin mediates immune-cell interactions with cells expressing vascular cell adhesion molecule 1 (VCAM-1), while the ␣4␤7 integrin mediates immune-cell binding to cells expressing mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Natalizumab is a monoclonal antibody against ␣4 integrin that inhibits immune-cell interactions with cells expressing VCAM-1 or MAdCAM-1. Natalizumab was approved for the treatment of relapsing multiple sclerosis. During postmarketing surveillance of the drug, however, several patients treated with natalizumab developed progressive multifocal leukoencephalopathy (PML), a rare demyelinating disorder caused by infection with JC virus. This finding resulted in voluntary withdrawal of the drug. After further FDA investigation, it was decided to resume testing of natalizumab and to add a warning to the product label regarding the possible association. Natalizumab was subsequently reapproved for use in the treatment of multiple sclerosis and Crohn’s disease.

Inhibition of Complement Activation The complement system mediates a number of innate immune responses (see Chapter 41). Recognition of foreign proteins or carbohydrates leads to sequential activation of complement proteins and eventual assembly of the membrane attack complex, a multiprotein structure that can cause cell lysis. Patients with paroxysmal nocturnal hemoglobinuria (PNH) have acquired defects in complement regulatory proteins, leading to inappropriate activation of complement and complement-mediated lysis of erythrocytes. Eculizumab is a humanized monoclonal antibody against C5, a complement protein that mediates late steps in complement activation and triggers assembly of the membrane attack complex. Eculizumab is approved for the treatment of PNH; it significantly decreases hemoglobinuria and the need for erythrocyte transfusions in patients with this disorder. Genetic evidence indicates that complement activation may play an etiologic role in age-dependent macular degeneration, suggesting that inhibitors of the complement cascade could be useful local therapies for this disease.

 CONCLUSION AND FUTURE DIRECTIONS Several approaches are available for the pharmacologic suppression of adaptive immunity, ranging from the relatively low-specificity approaches represented by glucocorticoids and cytotoxic agents to the more specific approaches represented by cell-signaling inhibitors and antibody therapies. Glucocorticoids induce profound suppression of the inflammatory response and immune system, but cause many

adverse effects, most of which are due to drug effects on cells outside the immune system. Glucocorticoid receptor modulators are being sought that retain the anti-inflammatory effects of glucocorticoids but have less severe adverse effects on metabolism and bone mineral homeostasis. Cytotoxic agents target DNA replication; although immune cells are highly susceptible to these drugs, so too are other normal cells such as those in the gastrointestinal epithelium. The cytotoxic agent mycophenolate mofetil is highly selective, both because lymphocytes depend on de novo purine synthesis and because mycophenolic acid preferentially targets the inosine monophosphate dehydrogenase isoenzyme expressed in lymphocytes. Lymphocyte-signaling inhibitors—such as cyclosporine, tacrolimus, sirolimus, and everolimus, which target intracellular signal transduction pathways necessary for T-cell activation—are also reasonably selective. Many new inhibitors of intracellular signaling in lymphocytes are under investigation; inhibition of the Janus kinase family appears particularly promising. Cytokine inhibitors interrupt soluble signals mediating immune-cell activation. TNF-␣ inhibitors—such as etanercept, infliximab, and adalimumab—represent an expanding class of drugs. Promising new targets include cytokines associated with TH17 cells, among others. The concept of preventing immune-cell activation has also been extended to the blockade of costimulation represented by the antirheumatic agent abatacept. Specific depletion of B cells is a well-established therapy for lymphomas and rheumatoid arthritis. Specific depletion of T cells may be beneficial in organ transplantation: antithymocyte globulin, OKT3, and daclizumab are antibodies against T-cell-specific epitopes. Several antibody therapeutics and small molecules are available that block immune-cell adhesion and homing, and more such agents are under development. New research is yielding novel ideas for the manipulation of the immune system. For example, microRNAs (miRNAs) have been shown to have important regulatory roles in immunity, and experimental manipulations of animal models of disease have suggested that selective modulation of miRNA may enable a greater degree of control of immunosuppression.

Disclosure Lloyd Klickstein is an employee and stockholder of Novartis, Inc., which manufactures or distributes drugs discussed in this chapter, including cyclosporine, mycophenolate sodium, everolimus, canakinumab, and basiliximab.

Suggested Reading Allison AC. Mechanisms of action of mycophenolate mofetil. Lupus 2005;14(Suppl 1):s2–8. (Review of mycophenolate mofetil.) Murphy K, Travers P, Walport M. Janeway’s immunobiology: the immune system in health and disease. 7th ed. New York: Garland Publishing; 2007. (Discussion of autoimmunity and transplantation immunity.) Lindsay MA. microRNAs and the immune response. Trends Immunol 2008;29:343–351. (Discusses role that microRNAs may have in regulating inflammation.) Nucleotide biosynthesis. In: Berg JM, Tymoczko JL, Stryer L, eds. Biochemistry. 6th ed. New York: W. H. Freeman and Company; 2007. (Review of nucleotide biosynthesis.) Vincenti F, Larsen C, Durrbach A, et al. Costimulation with belatacept in renal transplantation. N Engl J Med 2005;353:770–781. (Clinical trial demonstrating noninferiority of belatacept relative to cyclosporine.)

46 Integrative Inflammation Pharmacology: Peptic Ulcer Disease Dalia S. Nagel and Helen M. Shields

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 807-808 PHYSIOLOGY OF GASTRIC ACID SECRETION . . . . . . . . . . . 807 Neurohormonal Control of Gastric Acid Secretion . . . . . . . 807 Phases of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . 809 Protective Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809 PATHOPHYSIOLOGY OF PEPTIC ULCER DISEASE . . . . . . . . 810 Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810 NSAIDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811 Acid Hypersecretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811 Other Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 812 Agents That Decrease Acid Secretion . . . . . . . . . . . . . . . . 812 H2 Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . 812

Proton Pump Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 813 Anticholinergic Agents . . . . . . . . . . . . . . . . . . . . . . . . 816 Agents That Neutralize Acid . . . . . . . . . . . . . . . . . . . . . . . 816 Agents That Promote Mucosal Defense . . . . . . . . . . . . . . 816 Coating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816 Prostaglandins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816 Agents That Modify Risk Factors . . . . . . . . . . . . . . . . . . . 816 Diet, Tobacco, and Alcohol. . . . . . . . . . . . . . . . . . . . . . 816 Treatment of H. pylori Infection . . . . . . . . . . . . . . . . . . 816 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 817 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817

 INTRODUCTION

 PHYSIOLOGY OF GASTRIC ACID SECRETION

A peptic ulcer is a break in the mucosa of the stomach (gastric ulcer) or duodenum (duodenal ulcer). Four-and-a-half million people in the United States suffer from active peptic ulcer disease, and 500,000 new cases of peptic ulcer disease are diagnosed each year. The lifetime prevalence of peptic ulcer disease is approximately 10%, and the estimated annual cost for treatment exceeds one billion dollars. There are several pathophysiologic mechanisms for peptic ulcer disease, and clinical management therefore requires multiple pharmacologic strategies. This chapter describes the physiology of gastric acid secretion and the pathophysiology underlying the formation of peptic ulcers. The pharmacologic agents used in the treatment of peptic ulcer disease are then discussed in relation to the pathophysiology that is interrupted by these drugs.

Neurohormonal Control of Gastric Acid Secretion Hydrochloric acid is secreted into the stomach by parietal cells, which are located in oxyntic glands in the fundus and body of the stomach. The parietal cell actively transports H⫹ across its apical canalicular membranes via H⫹/K⫹ ATPases (proton pumps) that exchange intracellular H⫹ for extracellular K⫹. Three neurohormonal secretagogues regulate this process: histamine, gastrin, and acetylcholine (ACh). Each of these secretagogues binds to and activates specific receptors on the basolateral membrane of the parietal cell, thereby initiating the biochemical changes necessary for active transport of H⫹ out of the cell.

807

CHAPTER 46 / Integrative Inflammation Pharmacology: Peptic Ulcer Disease 809 Lumen

Cl-

K+

Apical membrane

Canaliculus K+

H+/K+ ATPase Parietal cell

Tubulovesicle containing inactive H+/K+ ATPase

Translocation/ fusion

Endoplasmic reticulum

ATP H+

ADP

Ca2+

Protein kinases ATP cAMP

IP3 Basolateral membrane

αs GTP

β

γ

AC

β

αq

γ

H2 M3

β

γ

GTP

DAG

CCKB PLC

Histamine Gastrin

ACh

ECL cell

Nerve

Blood vessel

FIGURE 46-1.

Control of parietal cell acid secretion. Stimulation of parietal cell acid secretion is modulated by paracrine (histamine), neuroendocrine (acetylcholine [ACh]), and endocrine (gastrin) pathways, which activate their respective receptors (H2, M3, and CCKB). H2 receptor activation increases cAMP, which activates protein kinase A. M3 and CCKB receptor activation stimulates release of Ca2⫹ by the Gq-mediated IP3/DAG pathway; these signals may also stimulate protein kinase C activity. Protein kinase activation results in translocation of cytoplasmic tubulovesicles containing inactive H⫹/K⫹ ATPase to the apical membrane. Fusion of tubulovesicles with the apical membrane activates H⫹/K⫹ ATPase, which pumps H⫹ ions into the stomach lumen. An apical membrane Cl⫺ channel couples Cl⫺ efflux to H⫹ efflux, and an apical membrane K⫹ channel recycles K⫹ out of the cell. The net result of this process is the rapid extrusion of HCl into the stomach lumen. In addition to its direct effect on CCKB receptors on parietal cells, gastrin also stimulates CCKB receptors on ECL cells to promote histamine release (not shown).

Phases of Gastric Acid Secretion

Protective Factors

Gastric secretions increase considerably during a meal. There are three phases of gastric acid secretion. The cephalic phase includes responses to sight, taste, smell, and thought of food. “Sham feedings,” experiments in which food is chewed but not swallowed, trigger an increase in acid secretion mediated by vagal stimulation and increased gastrin secretion. Mechanical distension of the stomach and ingestion of amino acids and peptides stimulate the gastric phase. Distension activates stretch receptors in the wall of the stomach that are linked to short intramural nerves and vagal fibers. Luminal nutrients, such as amino acids, are strong stimulants for gastrin release. Gastrin travels via the blood to the oxyntic mucosa and stimulates ECL cells to release histamine. An important negative feedback on acid secretion in this phase is acid (pH ⬍3)-mediated inhibition of gastrin release from antral G cells. Acid secretion is also inhibited by release of somatostatin from antral D cells. The intestinal phase involves stimulation of gastric acid secretion by digested protein in the intestine. Gastrin plays a major role in mediating this phase as well.

Factors that protect the gastric mucosa include gastric mucus, prostaglandins (discussed above and in Chapter 42, Pharmacology of Eicosanoids), gastric and duodenal bicarbonate, restitution (repair), and blood flow. The epithelial cells of the stomach secrete mucus, which acts as a lubricant that protects the mucosal cells from abrasions. Composed of hydrophilic glycoproteins that are viscous and have gelforming properties, the mucus layer enables formation of an uninterrupted layer of water at the luminal surface of the epithelium. Together, the mucus and water layers attenuate potential damage due to the acidic environment of the gastric lumen. Prostaglandins stimulate mucus secretion, whereas NSAIDs and anticholinergic medications inhibit mucus production. In addition, H. pylori disrupts the mucus layer (see below). Bicarbonate protects the gastric epithelium by neutralizing gastric acid. Bicarbonate is secreted by epithelial cells at the luminal surface of the gastric mucosa, in gastric pits, and at the luminal surface of the duodenal mucosa. Bicarbonate secretion in the duodenum serves to neutralize acid entering the intestine from the stomach.

Urease activity

G cell

pH

Gastrin Cell proliferation Somatostatin Acid secretion

Helicobacter pylori

D cell Inflammatory mediators

Parietal cell

Duodenal ulcer disease

A Systemic effects Inhibition of cyclooxygenase

Gastric acid secretion Bicarbonate/mucus production Blood flow

Prostaglandins

NSAID Expression of intercellular adhesion molecules in gastric vascular endothelium

Neutrophil adherence to vascular endothelial cells

Mucosal damage due to neutrophilderived free radicals and proteases

B Topical injury

Stomach lumen

Gastric epithelial cell

(pH ~ 2)

(pH ~ 7) O

O OH

O-

H+ + O

O O

NSAID (aspirin) weak acid

O

Cell damage

812 Principles of Inflammation and Immune Pharmacology Bismuth

Antacids Lumen

H. pylori

Antibiotics

Coating agents

Cl-

Proton pump inhibitors Canaliculus

Mucus cell

Parietal cell

Mucus cell K+

H+/K+ ATPase

Tubulovesicle containing inactive H+/K+ ATPase

K+

Translocation/ fusion

Endoplasmic reticulum

ATP H+

ADP

Ca2+

Protein kinases ATP cAMP

IP3

αs GTP

β

γ

AC

β

DAG

M3

CCKB PLC

Muscarinic antagonists ACh

ECL cell

γ

GTP

H2 H2 blockers Histamine

β

αq

γ

Gastrin Nerve

Blood vessel

FIGURE 46-4. Sites of action of drugs used to treat peptic ulcer disease. H2 receptor antagonists (H2 blockers) inhibit activation of the histamine H2 receptor by endogenous histamine. Muscarinic antagonists inhibit signaling through the M3 muscarinic acetylcholine (ACh) receptor. Proton pump inhibitors decrease the activity of the H⫹/K⫹ ATPase on the canalicular membrane of the parietal cell. Antacids neutralize acid in the stomach lumen. Coating agents provide a protective layer on the epithelial surface of the gastric mucosa. Bismuth and antibiotics act to eradicate H. pylori from the mucus layer coating the gastric mucosa. H. pylori infection is an important contributing factor in the pathogenesis of peptic ulcer disease.

 PHARMACOLOGIC CLASSES AND AGENTS Several pathophysiologic mechanisms can lead to peptic ulcer disease, and clinical management requires consideration of multiple pharmacologic options. The available agents can be divided into drugs that: (1) decrease acid secretion; (2) neutralize acid; (3) promote mucosal defense; and (4) modify risk factors (Fig. 46-4).

Agents That Decrease Acid Secretion H2 Receptor Antagonists The discovery of H2 receptor antagonists by Black and colleagues in the 1970s significantly changed the treatment of peptic ulcer disease. These investigators identified a second histamine receptor (H1 was the first; see Chapter 43, Histamine Pharmacology) and elucidated its role in gastric acid secretion. H2 receptor antagonists (also called H2 blockers) reversibly and competitively inhibit the binding of histamine to H2 receptors, resulting in suppression of gastric acid secretion. H2 receptor antagonists also indirectly decrease gastrinand acetylcholine-induced gastric acid secretion. Four H2 receptor antagonists are available: cimetidine, ranitidine, famotidine, and nizatidine (Fig. 46-5). H2 receptor

antagonists are absorbed rapidly from the small intestine. Peak plasma concentrations are achieved within 1–3 hours. Elimination of H2 receptor antagonists involves both renal excretion and hepatic metabolism. It is therefore important to decrease the dose of these drugs for patients with liver or kidney failure. An exception is nizatidine, which is eliminated primarily by the kidney. All four drugs are well tolerated in general. Occasional minor adverse effects include diarrhea, headache, muscle pain, constipation, and fatigue. H2 receptor antagonists may induce confusion and hallucinations in some patients. These adverse effects in the central nervous system are uncommon, however, and are typically associated with intravenous administration of the H2 receptor antagonist. Additional adverse effects specific to cimetidine, the first H2 receptor antagonist to be developed, are discussed below. Several clinically significant drug–drug interactions can occur with H2 receptor antagonists. For example, ketoconazole, a drug that requires an acidic medium for gastric absorption, has reduced uptake in the alkaline environment created by H2 receptor antagonists. As a second example, H2 receptor antagonists compete for renal tubular secretion of procainamide and certain other drugs. Cimetidine inhibits many cytochrome P450 enzymes and thus can interfere with the hepatic metabolism of numerous

CHAPTER 46 / Integrative Inflammation Pharmacology: Peptic Ulcer Disease 813

NH2

N

proton pump inhibitors have also been developed, including esomeprazole (the [S]-enantiomer of omeprazole), rabeprazole, lansoprazole, dexlansoprazole (the [R]-enantiomer of lansoprazole), and pantoprazole (Fig. 46-6). All of the proton pump inhibitors are prodrugs that require activation in the acidic environment of the parietal cell canaliculus. Oral formulations of these drugs are enteric-coated to prevent premature activation. The prodrug is converted to its active sulfenamide form in the acidic canalicular environment, and the sulfenamide reacts with a cysteine residue on the H⫹/K⫹ ATPase to form a covalent

HN

Histamine (imidazole ring) H N

N

H N

S N

HN

C N

Cimetidine (imidazole ring) O

NH H N

O

H N

N

O

S

N

NO2

N N H2N

O

NH

O

N

N

S

S S

O

Omeprazole

Ranitidine (furan ring) NH2 O

N

S

NH2

N

O

S

O

NH2

Famotidine (thiazole ring)

Esomeprazole NO2

N N

O

H N

N

S HN

S

HN S N

O

Nizatidine (thiazole ring)

O

Rabeprazole

FIGURE 46-5.

Histamine H2 receptor antagonists. H2 receptor antagonists share moieties related to histamine, providing a structural rationale for inhibition of the H2 receptor. For a more detailed description of the structure of these agents, see the legend to Figure 43-5.

O

NH

N

S N

drugs. For example, cimetidine can decrease the metabolism of lidocaine, phenytoin, quinidine, theophylline, and warfarin, facilitating the accumulation of these drugs to toxic levels. Cimetidine appears to inhibit P450 enzymes to a greater extent than the other H2 receptor antagonists, and an H2 receptor antagonist other than cimetidine may be preferred when the patient is receiving other medications. Cimetidine crosses the placenta and is secreted into breast milk, and is therefore not recommended for use during pregnancy or when nursing. Cimetidine can have antiandrogenic effects because of its action as an antagonist at the androgen receptor, resulting in gynecomastia (enlarged breasts) in men and, rarely, galactorrhea (discharge of milk) in women. Proton Pump Inhibitors Proton pump inhibitors block the parietal cell H⫹/K⫹ ATPase (proton pump). Compared to H2 receptor antagonists, proton pump inhibitors are superior at suppressing acid secretion and promoting peptic ulcer healing. Omeprazole is the prototype proton pump inhibitor. Several other

O

F F

Lansoprazole

F

O O O O

F F

N

S

N

NH

Pantoprazole

FIGURE 46-6.

Proton pump inhibitors. The proton pump inhibitors are a family of structurally related prodrugs that are all activated by the mechanism shown in Figure 46-7. Note that esomeprazole is the (S )-enantiomer of omeprazole, which is formulated as a racemic mixture of (R )- and (S )-enantiomers. Dexlansoprazole (not shown ) is the (R )-enantiomer of lansoprazole.

Canaliculus

Parietal cell

pH < 2.0 Omeprazole K+

ATP H+

ADP

Omeprazole pH 7.1 (cytoplasm) Freely crosses cell membrane pH 7.4 (blood) Omeprazole

O

O

H+ N S

N NH

Exposed to acidic O environment of parietal cell canaliculus

O

N+ S N

N

Reacts rapidly to form a covalent disulfide

S

N+ N

S

H+/K+ ATPase

NH

O

O

Omeprazole (prodrug)

Active sulfenamide

O

Sulfenamide-H+/K+ ATPase complex (inactive enzyme)

816 Principles of Inflammation and Immune Pharmacology

of this interaction remains uncertain, however, as observational studies have revealed conflicting results, and at least one large clinical trial has found no significant difference in adverse clinical outcomes (cardiovascular death, myocardial infarction, or stroke) between individuals treated with clopidogrel alone and individuals treated concomitantly with clopidogrel and a proton pump inhibitor. Some studies also suggest an increased risk of hip fracture in patients who take proton pump inhibitors for an extended period of time. Research on this topic has yielded conflicting evidence to date: some studies suggest that proton pump inhibitor therapy may decrease gastric absorption of insoluble calcium by raising gastric pH, but other studies suggest that omeprazole may decrease bone resorption by inhibiting osteoclastic vacuolar H⫹/K⫹ ATPase. Use of proton pump inhibitors during hospital admission has been shown to increase the risk for hospital-acquired pneumonia, C. difficile infection, and enteric infections with Salmonella and Escherichia coli. This increased risk may be related to compromise of a normal defense mechanism (i.e., gastric acid) by the proton pump inhibitor, allowing ingested organisms to escape acid-mediated destruction. Anticholinergic Agents Anticholinergic agents such as dicyclomine antagonize muscarinic ACh receptors on parietal cells and thereby decrease gastric acid secretion. However, anticholinergic agents are seldom used in the treatment of peptic ulcer disease because they are not as effective as H2 receptor antagonists or proton pump inhibitors. These agents also have many adverse effects, including dry mouth, blurred vision, cardiac arrhythmia, and urinary retention.

Agents That Neutralize Acid Antacids are used on an as-needed basis for symptomatic relief of dyspepsia. These agents neutralize hydrochloric acid by reacting with the acid to form water and salts. The most widely used antacids are mixtures of aluminum hydroxide and magnesium hydroxide. The hydroxide ion reacts with hydrogen ions in the stomach to form water, while the magnesium and aluminum react with bicarbonate in pancreatic secretions and with phosphates in the diet to form salts. Common adverse effects associated with these antacids include diarrhea (magnesium) and constipation (aluminum). When antacids containing aluminum and magnesium are taken together, constipation and diarrhea may be avoided. Antacids containing aluminum can bind phosphate; the resulting hypophosphatemia can cause weakness, malaise, and anorexia. Patients with chronic kidney disease should avoid magnesium-containing antacids because they can lead to hypermagnesemia. Sodium bicarbonate reacts rapidly with HCl to form water, carbon dioxide, and salt. Antacids containing sodium bicarbonate have high amounts of sodium; in patients with hypertension or fluid overload, sodium-containing antacids can result in significant sodium retention. Calcium carbonate is less soluble than sodium bicarbonate; it reacts with gastric acid to produce calcium chloride and carbon dioxide. Calcium carbonate is not only useful as an antacid, but can also serve as a calcium supplement for prevention of osteoporosis. The high calcium content of this antacid formulation may cause constipation.

Agents That Promote Mucosal Defense Agents that promote mucosal defense are used in the symptomatic relief of peptic ulcer disease. These drugs include coating agents and prostaglandins. Coating Agents Sucralfate, a complex salt of sucrose sulfate and aluminum hydroxide, is a coating agent used to alleviate the symptoms of peptic ulcer disease. Sucralfate has little ability to alter gastric pH. Instead, in the acidic environment of the stomach, this complex forms a viscous gel that binds to positively charged proteins and thereby adheres to gastric epithelial cells (including areas of ulceration). The gel protects the luminal surface of the stomach from degradation by acid and pepsin. Because sucralfate is poorly soluble, there is little systemic absorption and no systemic toxicity. Constipation is one of the few adverse effects. In addition, sucralfate may bind to drugs such as quinolone antibiotics, phenytoin, and warfarin and limit their absorption. Colloidal bismuth is a second coating agent used in peptic ulcer disease. Bismuth salts combine with mucus glycoproteins to form a barrier that protects an ulcer from further damage by acid and pepsin. Bismuth agents may stimulate mucosal bicarbonate and prostaglandin E2 secretion and thereby also protect the mucosa from acid and pepsin degradation. Colloidal bismuth has been found to impede the growth of H. pylori and is frequently used as part of a multidrug regimen for the eradication of H. pylori-associated peptic ulcers (see below). Prostaglandins Prostaglandins can be used in the treatment of peptic ulcer disease (see Chapter 42), specifically in the treatment of NSAID-induced ulcers. NSAIDs are ulcerogenic because they inhibit prostaglandin synthesis and thereby interrupt the “gastroprotective” functions of PGE2, which include reduced gastric acid secretion and enhanced bicarbonate secretion, mucus production, and blood flow. Misoprostol is a prostaglandin analogue used to prevent NSAID-induced peptic ulcers. Its most frequent adverse effects are abdominal discomfort and diarrhea. In clinical practice, these adverse effects often interfere with patient adherence. Misoprostol is contraindicated in women who are (or may be) pregnant because of the possibility of generating uterine contractions that could result in abortion (see Chapter 29, Pharmacology of Reproduction).

Agents That Modify Risk Factors Diet, Tobacco, and Alcohol As in the introductory case, diet therapy typically involves recommendations to avoid caffeine-containing products because of their ability to increase acid secretion. Avoidance of alcohol and cigarette smoking is also advised. Excessive alcohol intake is directly toxic to the mucosa and is associated with erosive gastritis and an increased incidence of peptic ulcers. Cigarette smoking is thought to decrease the production of duodenal bicarbonate and diminish mucosal blood flow, leading to a delay in ulcer healing. Treatment of H. pylori Infection Elimination of H. pylori can lead to cure of H. pyloriassociated peptic ulcers. Treatment for H. pylori infection

47 Integrative Inflammation Pharmacology: Asthma Joshua M. Galanter and Stephen Lazarus

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 820-821 PHYSIOLOGY OF AIRWAY SMOOTH MUSCLE TONE AND IMMUNE FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 820 Physiology of Airway Smooth Muscle Contraction. . . . . . . 820 Immune Function in the Airway . . . . . . . . . . . . . . . . . . . . 821 PATHOPHYSIOLOGY OF ASTHMA. . . . . . . . . . . . . . . . . . . . . 822 Asthma as a Bronchoconstrictive Disease . . . . . . . . . . . . 822 Asthma as an Inflammatory Disease . . . . . . . . . . . . . . . . 824 TH2 Cells and the Origin of Asthma . . . . . . . . . . . . . . . 824 Plasma Cells, IgE, Mast Cells, and Leukotrienes . . . . . 825 Eosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 827 Bronchodilators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827 Anticholinergics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827

␤-Adrenergic Agonists . . . . . . . . . . . . . . . . . . . . . . . . 827 Methylxanthines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829 Magnesium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829 Anti-Inflammatory Agents . . . . . . . . . . . . . . . . . . . . . . . . 829 Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829 Cromolyns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830 Leukotriene Pathway-Modifying Agents . . . . . . . . . . . 830 Anti-IgE Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 831 Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831 Clinical Management of Asthma . . . . . . . . . . . . . . . . . . . . 832 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 832 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833

 INTRODUCTION

 PHYSIOLOGY OF AIRWAY SMOOTH MUSCLE TONE AND IMMUNE FUNCTION

Asthma is a chronic disease of the airways that is characterized by marked airway inflammation and exaggerated variability in airway caliber due to bronchial smooth muscle hyperresponsiveness. The symptoms of asthma include dyspnea and wheezing as well as mucus production and cough, particularly at night. Asthma is both an obstructive lung disease and an inflammatory disease; the obstructive component is characterized by bronchoconstriction, whereas the inflammatory component is marked by airway edema, goblet cell hyperplasia, mucus secretion, and infiltration and cytokine release by immune and inflammatory cells. Although the airway obstruction is generally reversible, asthma may, over time, cause airway remodeling and permanent deterioration in pulmonary function. Medications used to treat asthma act in one of two ways: by relaxing bronchial smooth muscle or by preventing and treating inflammation. This chapter approaches asthma as both a bronchoconstrictive and an inflammatory disease. After discussing the physiologic control of bronchial tone and the function of immune pathways in the airways, the chapter turns to the pathophysiology of asthma. Current therapies are then discussed, including the pharmacology of both bronchodilators and airway anti-inflammatory agents. 820

Asthma involves dysfunction in the pathways that regulate both smooth muscle tone and immune function in the airways. It is therefore important to review the normal physiology of these systems before discussing the pathophysiology of asthma.

Physiology of Airway Smooth Muscle Contraction As discussed in Chapter 8, Principles of Nervous System Physiology and Pharmacology, involuntary responses of smooth muscle are regulated by the autonomic nervous system. In the airways, sympathetic (adrenergic) tone causes bronchodilation and parasympathetic (cholinergic) tone causes bronchoconstriction. Bronchial smooth muscle tone is also regulated by nonadrenergic, noncholinergic (NANC) fibers that innervate the respiratory tree. Adrenergic innervation of the lung is concentrated primarily on pulmonary blood vessels and the submucosal glands. There is little direct adrenergic innervation of the bronchial smooth muscle. However, airway smooth muscle cells express ␤2-adrenergic receptors (and, to a lesser extent, ␤1-adrenergic receptors) that are responsive to circulating

CHAPTER 47 / Integrative Inflammation Pharmacology: Asthma 823

Allergen

Airway Airway epithelium

Goblet cell

Naive T-cell Antigen-presenting cell

CD4 IL-12

IL-4 Activated TH2 lymphocyte

CD4

Activated TH1 lymphocyte

CD4 IL-10

TH2 cytokines (IL-4, IL-5, IL-6, IL-9, IL-10, IL-13)

E i hil Eosinophil (MBP, ECP, leukotrienes, cytokines)

IFN-γ

Mast cell (histamine, leukotrienes, cytokines)

Plasma cellll Pl (IgE)

TH1 cytokines (IFN-γ, IL-2, TNF-α)

Inflammatory cell independent mechanisms (IL-13)

Low-level IgG response (physiologic response)

M h Macrophage Low level TH1 response (physiologic response)

Blood vessels

Goblet cell hyperplasia Airway edema

Submucosal gland Epithelium

Subepithelial fibrosis Cartilage

Smooth muscle hyperplasia and/or hypertrophy

FIGURE 47-1.

Smooth muscle Asthma

Normal airway (no asthma)

Origins of the asthmatic immune response. In nonatopic individuals, antigens derived from allergens are presented by antigen-presenting dendritic cells to engender a low-level, physiologic TH1 response. This response does not cause airway inflammation or bronchoconstriction (right side). Interferon-␥, produced by activated TH1 lymphocytes, inhibits a TH2 response. In individuals susceptible to asthma, allergen-derived antigens that are presented to immature CD4⫹ T cells cause these cells to differentiate into activated TH2 lymphocytes. The TH2 lymphocytes release cytokines that recruit other inflammatory cells, including eosinophils, mast cells, and IgE-producing B cells. Together, these cells produce an inflammatory response in the airway. Activated TH2 cells also induce an asthmatic response directly, in part through release of IL-13. The net result—airway hyperresponsiveness, mucus production by goblet cells, airway edema, subepithelial fibrosis, and bronchoconstriction—constitutes the asthmatic response (left side).

Airway smooth muscle contraction

Asthmatic response (hyperresponsiveness)

Hyperreactivity

Hypersensitivity Normal response

Stimulus (e.g., methacholine)

CHAPTER 47 / Integrative Inflammation Pharmacology: Asthma 825

Plasma Cells, IgE, Mast Cells, and Leukotrienes As noted above, an IgE-mediated type I hypersensitivity response is one mechanism by which allergens cause the pathologic and clinical manifestations of asthma (Fig. 47-3). The allergic response is initiated when a dendritic cell phagocytoses

an inhaled allergen. The dendritic cell presents the processed allergen to TH2 cells and activates them. The activated TH2 cells bind to and activate B lymphocytes via CD40 on the B-cell surface. Activated TH2 cells also generate IL-4 and IL-13, which induce B-cell transformation into IgE-producing plasma cells.

Airway Airway epithelium

Allergen MHC class II molecule IgE crosslinked by allergen

Antigenpresenting cell

TH2

FcεRI-bound IgE

T-cell receptor Neurotrophins

Omalizumab

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Histamine-releasing factor, neuropeptides, IL-9

IL-4 IL-4, IL-13 IL-5 IL-5

IL-4

Neuron

Pla Plasma cell

ECP

Mastt cellll

Histamine, leukotrienes, platelet-activating factor

Eosinophil i hil IL-5

Neuropeptides

Histamine, leukotrienes, cytokines

Chronic asthmatic reaction Bronchoconstriction Vasogenic edema Mucus hypersecretion Chronic inflammation Airway remodeling

MBP, ECP, leukotrienes, cytokines

Acute asthmatic reaction Bronchoconstriction Airway edema Mucus production

FIGURE 47-3. The allergic response in asthma. Asthma produces acute and chronic inflammatory responses in the airways. Antigen-presenting cells phagocytose and process allergens, presenting the antigens to CD4⫹ T cells. These cells differentiate into cytokine-producing TH2 lymphocytes. The activated TH2 cells release IL-4, IL-13, and IL-5, which recruit B cells and eosinophils. The B cells differentiate into IgE-producing plasma cells. The IgE binds to Fc␧RI receptors on mast cells and antigen-presenting cells. Upon re-exposure to the allergen, the IgE-bound Fc␧RI is cross-linked, inducing the mast cell to degranulate and release preformed and newly generated inflammatory mediators including histamine, cysteinyl leukotrienes, platelet-activating factor, and other cytokines. These cytokines cause acute airway inflammation and produce acute asthmatic symptoms (an asthma “attack” or exacerbation). Chronically, activated TH2 cells and mast cells produce circulating IL-5 that recruits eosinophils, and TH2 cells release products that stimulate local mast cells and neurons. Together, the inflammatory mediators and catabolic enzymes produced by eosinophils, mast cells, and neurons cause chronic airway inflammation and lead to airway remodeling. Omalizumab is a humanized monoclonal antibody against the Fc␧RI-binding domain of IgE. By preventing IgE from binding to the IgE receptor (Fc␧RI) on mast cells, omalizumab inhibits mast cell degranulation upon re-exposure to allergen and thereby modulates the acute asthmatic reaction. Omalizumab also down-regulates Fc␧RI on antigen-presenting cells, thereby diminishing antigen processing and presentation to CD4⫹ lymphocytes. Because fewer immature T cells are induced by allergen to differentiate into TH2 lymphocytes, the chronic asthmatic reaction is also blunted.

826 Principles of Inflammation and Immune Pharmacology

IgE circulates briefly in the bloodstream before binding to high-affinity IgE receptors (Fc␧RI) on mast cells. Upon re-exposure, the allergen binds to mast cell-bound IgE and cross-links the Fc␧RI receptors, thereby activating the mast cell. The activated mast cell degranulates, releasing its preformed inflammatory mediators. These molecules include histamine, proteolytic enzymes, and certain cytokines (such as platelet-activating factor). The activated mast cell also releases arachidonic acid from its plasma membrane and produces leukotrienes and prostaglandin D2 (Fig. 47-4). Acutely, mast-cell degranulation produces bronchoconstriction and airway inflammation. Histamine released by the mast cells promotes capillary leakage, leading to airway edema. Mast cells also release leukotriene C4 (LTC4), which is subsequently converted into LTD4 and LTE4 (see Chapter 42, Pharmacology of Eicosanoids). These three leukotrienes, called cysteinyl leukotrienes, are central to the pathophysiology of asthma because they induce marked bronchoconstriction. Leukotriene D4 is 1,000 times more potent than histamine in producing bronchoconstriction. Leukotrienes also cause mucus hypersecretion, capillary leakage, and vasogenic edema, and recruit additional inflammatory cells. The effect of the leukotrienes, though slower in onset, is more powerful and sustained than that of the preformed mediators. Because of their delayed yet potent inflammatory effect, leukotrienes were once called slow-reacting substance of anaphylaxis (SRS-A) before their actual structures were identified. Mast cells recruit other inflammatory cells via the release of cytokines. This produces a delayed reaction that develops 4 to 6 hours after exposure to allergen (Fig. 47-3). Mast cells also release tryptase, a protease that activates receptors on epithelial and endothelial cells, inducing the expression of adhesion molecules that attract eosinophils and basophils. Tryptase is also a smooth muscle mitogen, causing hyperplasia of airway smooth muscle cells and contributing to airway hyperresponsiveness. The production of IL-1, IL-2, IL-3, IL-4, IL-5, GM-CSF, interferon-␥, and TNF-␣ by mast cells contributes to chronic inflammation and the chronic asthmatic reaction. Finally, mast cells release proteases and proteoglycans that act on supporting airway structures to produce chronic changes in the airway (also called airway remodeling). Unlike the reversible component of bronchoconstriction that characterizes the acute asthmatic reaction, airway remodeling induced by chronic inflammation may cause irreversible impairment in pulmonary function. Eosinophils The major physiologic role of eosinophils is to defend against parasitic infections. Eosinophils originate in the bone marrow and are stimulated by IL-4, IL-5, and GM-CSF produced by TH2 lymphocytes and mast cells. Eosinophils migrate from the bloodstream to the airway by binding to specific adhesion molecules, particularly VCAM-1, and by traveling along chemokine gradients to sites of inflammation. Once recruited to the airway, eosinophils have a complex, multifunctional role in asthma. Activated eosinophils secrete cytotoxic granules that cause local tissue damage and induce airway remodeling, lipid mediators and neuromodulators that affect airway tone, and cytokines and chemokines that recruit other inflammatory cells. The toxic granules of eosinophils contain a number of cationic proteins—including major basic protein (MBP),

Nucleus

Cytosol Prostaglandins

Aspirin

Cyclooxygenase PLA2

Arachidonic acid 5-Lipoxygenase activating protein (FLAP) 5-Lipoxygenase Zileuton Glutathione Leukotriene A4 Leukotriene C4 Epoxide hydrolase (neutrophils, monocytes)

Leukotriene C4 synthase (mast cells, eosinophils)

Transporter

Extracellular space

Leukotriene B4 Leukotriene C4 Leukotriene D4

Leukotriene E4 BLT1

CysLT1 Montelukast Zafirlukast

Leukocyte Chemotaxis

Airway Smooth muscle contraction Eosinophil migration Airway edema

FIGURE 47-4. The leukotriene pathway in asthma. Leukotrienes are some of the most potent bronchoconstrictors known and are important mediators of inflammation in the airway. Drugs that inhibit leukotriene production or leukotriene receptor binding have a role in asthma therapy. Leukotrienes are formed when arachidonic acid is released from the inner leaflet of the plasma membrane by the action of phospholipase A2 (PLA2). Arachidonic acid is converted to leukotriene A4 by the action of 5-lipoxygenase. 5-Lipoxygenase is activated by the membranebound enzyme 5-lipoxygenase activating protein (FLAP). Leukotriene A4 is converted to leukotriene C4 by the action of leukotriene C4 synthase in mast cells and eosinophils, and leukotriene C4 is transported out of the cell. Leukotriene C4 is converted to leukotriene D4 and then to leukotriene E4; all three of these cysteinyl leukotrienes bind to CysLT1 receptors expressed on airway smooth muscle cells, leading to bronchoconstriction and airway edema. Leukotriene A4 is converted to leukotriene B4 by epoxide hydrolase in neutrophils and monocytes. Leukotriene B4 is transported out of the cell and binds to BLT1 receptors expressed on leukocytes, leading to leukocyte chemotaxis and recruitment. The leukotriene pathway can be inhibited by the 5-lipoxygenase inhibitor zileuton or by the CysLT1 receptor antagonists montelukast and zafirlukast. eosinophilic cationic protein (ECP), eosinophil peroxidase, and eosinophil-derived neurotoxin—that are directly damaging to the bronchial epithelium. For example, ECP can breach the integrity of target cell membranes by forming ion-selective, voltage-insensitive pores, and eosinophil peroxidase catalyzes the production of highly reactive oxygen species that oxidize target cell proteins and induce apoptosis.

828 Principles of Inflammation and Immune Pharmacology

but because it does not stimulate ␣-receptors, it does not cause peripheral vasoconstriction. Isoproterenol is not used frequently in current practice because of the availability of agents that are more selective for ␤2-receptors. The first agents to offer relative ␤2 selectivity were isoetharine and metaproterenol, although both drugs had moderate ␤1 effects. The newer drugs terbutaline, albuterol (also referred to as salbutamol), pirbuterol, and bitolterol bind to ␤2-adrenergic receptors 200–400 times more strongly than to ␤1-receptors and cause significantly fewer cardiac effects than the less selective adrenergic agonists. Albuterol was the first of the strongly ␤2-selective agents to be available in inhaled form, further reducing systemic effects. Modern inhaled ␤2-selective agonists were the first drugs to allow regular treatment of asthma with an acceptable adverse-effect profile. Nonetheless, at high doses, especially if taken orally, even these drugs can cause cardiac stimulation. In addition, since ␤2-adrenergic receptors are expressed in peripheral skeletal muscle, activation of these receptors by ␤2-selective agents can result in a tremor. Albuterol is a racemic mixture of two stereoisomers, Ralbuterol (or levalbuterol) and S-albuterol. Levalbuterol, which is now available as a pure enantiomer, has tighter binding to ␤2-receptors and is more ␤2-selective. In contrast, the S isomer induces airway hyperresponsiveness in animal models, although in clinical practice this effect has not been significant. Although racemic albuterol and levalbuterol produce similar response and adverse-effect profiles for most patients, a subset of patients may be more sensitive to the ␤1 effects of S-albuterol and may experience decreased tachycardia and palpitations when taking levalbuterol. ␤-Adrenergic receptors are coupled to the stimulatory G protein Gs (see Chapter 10). The ␣ subunit of Gs activates adenylyl cyclase, which catalyzes the production of cyclic adenosine monophosphate (cAMP). In the lung, cAMP causes a decrease in the intracellular calcium concentration and, via activation of protein kinase A, inactivates myosin light chain kinase and activates myosin light chain phosphorylase (Fig. 47-5). In addition, the ␤2-agonists open largeconductance calcium-activated potassium channels (KCa) and thereby hyperpolarize airway smooth muscle cells. The combination of decreased intracellular calcium, increased membrane potassium conductance, and decreased myosin light chain phosphorylation leads to smooth muscle relaxation and bronchodilation.

β2 agonist β2-adrenergic receptor

β

γ

Adenylyl cyclase

αs

Phosphodiesterase

GTP ATP

cAMP

AMP Theophylline

PKA Bronchodilation

There appears to be variability in clinical response among patients using ␤2-agonists. Some of this variability may be mediated through variants in the gene for the ␤2-adrenergic receptor. Researchers studying the effect of single nucleotide polymorphisms (SNPs) in the gene have found a common genetic variant that is associated with increased susceptibility to nocturnal asthma. Subjects homozygous for this genetic variant who receive regularly scheduled albuterol doses develop a decline in their peak expiratory flow rate (a measure of bronchoconstriction), while subjects without the polymorphism develop increased peak flow rates with scheduled albuterol use. Although the pharmacogenetics of the ␤2-adrenergic receptor are complicated and have yielded inconsistent associations, it is likely that some of the variability in drug response results from genetic influences. Most ␤2-adrenergic agonists have a rapid onset of action (15 to 30 minutes), a peak effect at 30 to 60 minutes, and a duration of action of approximately 4 to 6 hours. This time course of drug action makes the ␤2-agonists good candidates for use as asthma relievers (or rescue inhalers) during acute attacks. However, this profile also makes the ␤2-agonists poor candidates for control of nocturnal asthma and for prevention of attacks, although they can be used prophylactically before exposure to a known trigger such as exercise. Several newer agents, formoterol (and its enantiomerically pure form arformoterol, approved only for COPD) and salmeterol, are known as long-acting beta-agonists (LABAs). The LABAs were engineered with lipophilic side chains that resist degradation. As such, these agents have a 12- to 24-hour duration of action, making them good candidates for prevention of bronchoconstriction. Although formoterol and salmeterol are reasonable asthma controllers, these agents do not treat the underlying inflammation. In fact, regular use of formoterol or salmeterol may be associated with an increase in asthma deaths. Although the exact mechanism for this observation is unknown, it may occur because long-acting ␤-agonists can improve the chronic symptoms of asthma without affecting the underlying risk of a severe asthma exacerbation. Because patients may feel better on long-acting ␤-agonists, they may receive lower doses of inhaled corticosteroids or no inhaled corticosteroids at all. Since inhaled corticosteroids reduce the risk of an asthma exacerbation (see below), the reduction or withdrawal of inhaled corticosteroids may place patients at increased risk of asthma hospitalization and fatal asthma attack. For this reason, an FDA advisory committee has

FIGURE 47-5. Mechanism of the ␤2-agonists and theophylline. In airway smooth muscle cells, activation of protein kinase A by cAMP leads to phosphorylation of a number of intracellular proteins and thus to smooth muscle relaxation and bronchodilation. Any therapy that increases the level of intracellular cAMP can be expected to lead to bronchodilation. In practice, this can be accomplished in one of two ways: by increasing the production of cAMP or by inhibiting the breakdown of cAMP. cAMP production is stimulated by ␤2-agonist-mediated activation of ␤2-adrenergic receptors, which are G protein-coupled receptors. cAMP breakdown is inhibited by theophylline-mediated inhibition of phosphodiesterase.

CHAPTER 47 / Integrative Inflammation Pharmacology: Asthma 829

recommended that formoterol and salmeterol should be used only in combination with an inhaled corticosteroid. Because salmeterol has a slower onset of action than albuterol, it should not be used for acute asthma flares. Formoterol does have a rapid onset of action and can be used as a rescue inhaler, although it is not yet approved for this indication in the United States. One strategy has been to combine formoterol with an inhaled corticosteroid (budesonide) for use as needed in patients with mild asthma. Every time the patient uses this combination, the formoterol is available to provide acute relief of symptoms, but the patient also receives a dose of the inhaled corticosteroid to help quell the underlying inflammation. Methylxanthines Two methylxanthines, theophylline and aminophylline, are occasionally used in asthma treatment. The mechanism of action of these drugs is complex, but their primary bronchodilatory effect appears to be due to nonspecific inhibition of phosphodiesterase isoenzymes. Inhibition of phosphodiesterase types III and IV prevents cAMP degradation in airway smooth muscle cells, leading to smooth muscle relaxation by the cellular and molecular mechanisms detailed above (i.e., decreased intracellular calcium, increased membrane potassium conductance, and decreased myosin light chain phosphorylation). As shown in Figure 47-5, the bronchodilatory effect of methylxanthines results from perturbation of the same pathway that is initiated by ␤2-agonists, although methylxanthines act downstream of ␤2-adrenergic receptor stimulation. Methylxanthines also inhibit phosphodiesterase isoenzymes in inflammatory cells. Inhibition of phosphodiesterase type IV in T lymphocytes and eosinophils has an immunomodulatory and anti-inflammatory effect. By this mechanism, theophylline can control chronic asthma more effectively than would be expected on the basis of its bronchodilatory effect alone. Some of the adverse effects of methylxanthines, including cardiac arrhythmias, nausea, and vomiting, are also mediated by phosphodiesterase inhibition, although the responsible isoenzymes remain to be elucidated. Theophylline is a structural relative of caffeine, differing only by a single methyl group, and both caffeine and theophylline are adenosine receptor antagonists. Adenosine receptors are expressed on airway smooth muscle cells and mast cells, and antagonism of these receptors could play a role in preventing both bronchoconstriction and inflammation. In fact, coffee has been used to treat asthma. However, experiments with specific adenosine receptor antagonists that do not inhibit phosphodiesterase have shown little bronchodilation, suggesting that phosphodiesterase inhibition is the primary mechanism of action of methylxanthines. Nonetheless, adenosine receptor antagonism is responsible for many secondary effects of theophylline, including increased ventilation during hypoxia, improved endurance of diaphragmatic muscles, and decreased adenosine-stimulated mediator release from mast cells. In addition, some adverse effects of theophylline, such as tachycardia, psychomotor agitation, gastric acid secretion, and diuresis, are mediated through adenosine receptor antagonism. Because methylxanthines are nonselective and have multiple mechanisms of action, they cause multiple adverse effects and have a relatively narrow therapeutic index.

Moreover, there is significant interindividual variation in the metabolism of theophylline by the P450 isoenzyme CYP3A, and theophylline use is susceptible to drug–drug interactions with CYP3A inhibitors such as cimetidine and the azole antifungals. At supratherapeutic levels, theophylline produces nausea, diarrhea, vomiting, headache, irritability, and insomnia. At even higher doses, seizures, toxic encephalopathy, hyperthermia, brain damage, hyperglycemia, hypokalemia, hypotension, cardiac arrhythmias, and death can occur. For this reason, the role of theophylline in the treatment of chronic asthma has diminished. Theophylline is still used occasionally with routine monitoring of plasma drug levels when ␤-adrenergic agonists and corticosteroids are ineffective or contraindicated. Magnesium Magnesium ions inhibit calcium transport into smooth muscle cells and can interfere with intracellular phosphorylation reactions that induce smooth muscle contraction. For this reason, magnesium sulfate is commonly used as a tocolytic to cause uterine relaxation and to delay preterm labor. Magnesium has similar effects on airway smooth muscle, and it has been used experimentally in acute asthma exacerbations. Although the results of clinical studies have been variable, two meta-analyses have suggested a benefit to using magnesium sulfate in patients with severe asthma exacerbations presenting to the emergency department. Magnesium was not used in the introductory case, but it would have been a reasonable therapeutic option at the time of Mr. Y’s visit to the emergency department.

Anti-Inflammatory Agents As detailed above, allergic inflammation of the airways forms the pathophysiologic basis for asthma. To control persistent asthma and prevent exacerbations of acute asthma, treatment of all but the mildest forms of the disease should generally include anti-inflammatory agents. Corticosteroids have long been mainstays of asthma treatment, although the profound adverse effects of systemically administered corticosteroids remained problematic until the development of inhaled formulations. Three additional classes of drugs with anti-inflammatory mechanisms of action are used for the treatment of asthma: cromolyns, leukotriene pathway modifiers, and a humanized monoclonal anti-IgE antibody. Corticosteroids Inhaled corticosteroids are the chief preventive treatment for the vast majority of patients with asthma. Because inhaled corticosteroids produce higher local drug concentrations in the airway than an equivalent dose of systemically administered corticosteroids, a lower overall dose can be administered, reducing the likelihood of significant systemic effects. Corticosteroids alter the transcription of many genes. In general, corticosteroids increase the transcription of genes coding for the ␤2-adrenergic receptor and a number of anti-inflammatory proteins such as IL-10, IL-12, and IL-1 receptor antagonist (IL-1Ra). Corticosteroids decrease the transcription of genes coding for many pro-inflammatory (and other) proteins; examples include IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF-␣, GM-CSF, SCF, endothelial adhesion molecules, chemokines, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX), phospholipase A2, endothelin-1, and NK1-2 receptor. As described

CHAPTER 47 / Integrative Inflammation Pharmacology: Asthma 833

cure for asthma, but a therapeutic approach that treats both aspects of asthma by using anti-inflammatory medications and bronchodilators, along with the avoidance of known triggers, can be successful in achieving long-term clinical control and enabling successful management of the disease in most patients. As our understanding of the pathophysiology of asthma has improved, new targets for therapeutic intervention have become available. In general, research has focused on three areas: improving existing therapies by altering the ratio of benefit to adverse effect, devising new targeted therapies, and attempting to prevent or reverse permanent airway remodeling in long-standing asthma. One example of the first approach is the development of novel inhaled corticosteroids with reduced systemic effects. For example, research is continuing on selective glucocorticoid receptor modulators that retain anti-inflammatory activity while minimizing adverse effects. A number of inflammatory cytokine inhibitors are under development as potential new targeted therapeutics for asthma. However, the complex nature of asthma means that inhibition of a single pathway may not significantly affect the disease. For example, the anti-IL-5 monoclonal antibody mepolizumab has been explored as a potential treatment for asthma. Unfortunately, this drug showed no efficacy in multiple clinical trials, despite successfully reducing the number of circulating and airway eosinophils. A recent trial did find that mepolizumab could reduce the frequency of asthma exacerbations in a rare subgroup of patients with prednisonedependent asthma and sputum eosinophilia. Other studies are ongoing with inhibitors of IL-13 and IL-4 and with the inhibitory cytokine IL-10. For example, pitrakinra, a variant of IL-4 that blocks binding of IL-4 and IL-13 to the IL-4 receptor alpha, has shown some promise in early clinical studies. TNF-␣ (see Chapter 45) is a cytokine that is up-regulated in asthma and that recruits neutrophils and eosinophils into the airways. Etanercept (a recombinant fusion protein that inhibits TNF-␣) and infliximab (an anti-TNF-␣ monoclonal antibody) have also shown promising results in early clinical studies. Inhibition of phosphodiesterase type IV (PDE IV) is a new pharmacologic approach to reducing inflammation

in asthma. PDE IV hydrolyzes cAMP in a number of the inflammatory cell types involved in asthma pathophysiology, and studies have shown that increased intracellular cAMP inhibits the release of TNF-␣ and other cytokines from these cell types. Two PDE IV inhibitors, roflumilast and cilomilast, have been evaluated in advanced clinical trials for asthma and COPD. Unfortunately, the utility of both compounds has been limited by the development of dose-limiting nausea and vomiting that are thought to be due to inhibition of PDE IV in the brain. Thus, research is now focused on the discovery of nonemetogenic PDE IV inhibitors and on the development of an inhaled formulation.

Suggested Reading Barnes PJ. The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 2008;118:3546–3556. (Reviews the role of cytokines in the chronic asthmatic reaction and suggests targets for new drug development.) Chu EK, Drazen JM. Asthma: one hundred years of treatment and onward. Am J Respir Crit Care Med 2005;171:1203–1208. (Historic view of the evolution of asthma therapy over the last 100 years.) Fanta CH. Asthma. N Engl J Med 2009;360:1002–1014. (Discusses the clinical management of asthma, focusing on commonly prescribed therapeutics.) Guidelines for the Diagnosis and Management of Asthma (EPR-3). Available at: http://www.nhlbi.nih.gov/guidelines/asthma/ (This is most recent set of practice guidelines for the diagnosis and treatment of asthma, from the expert panel convened by the National Heart Lung and Blood Institute of the National Institutes of Health.) Hanania NA. Targeting airway inflammation in asthma: current and future therapies. Chest 2008;133:989–998. (A review of anti-inflammatory therapies for asthma, including inhaled corticosteroids, anti-IgE therapy, and novel treatments focused on immunomodulation.) Lemanske RG. Asthma therapies revisited: what have we learned. Proc Am Thorac Soc 2009;6:312–315. (Discusses the treatment of asthma, focusing on who and when to treat, and identifying the appropriate treatment.) Locksley RM. Asthma and allergic inflammation. Cell 2010;140:777–783. (Reviews the dysregulated interactions between airway epithelia and innate immune cells that initiate and maintain asthma.) Rhen T, Cidlowski JA. Anti-inflammatory action of glucocorticoids—new mechanisms for old drugs. N Engl J Med 2005;353:1711–1723. (Discusses the molecular mechanisms by which glucocorticoids act and efforts to develop novel glucocorticoids with improved adverse-effect profiles.)

48 Integrative Inflammation Pharmacology: Gout Ehrin J. Armstrong and Lloyd B. Klickstein

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 837-838 PHYSIOLOGY OF PURINE METABOLISM . . . . . . . . . . . . . . . 837 PATHOPHYSIOLOGY OF GOUT . . . . . . . . . . . . . . . . . . . . . . . 839 PHARMACOLOGIC CLASSES AND AGENTS . . . . . . . . . . . . . 840 Management of Acute Gout: Suppressors of Leukocyte Recruitment and Activation . . . . . . . . . . . . . . . . . . . . . . . 840 Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) . . . . . 840 Colchicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840 Glucocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841

Management of Chronic Gout: Agents That Lower Plasma Urate Concentration. . . . . . . . . . . . . . . . . . . . . . . 841 Agents That Decrease Uric Acid Synthesis. . . . . . . . . . 841 Agents That Increase Uric Acid Excretion. . . . . . . . . . . 842 Agents That Enhance Uric Acid Metabolism . . . . . . . . . 842 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 843 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843

 INTRODUCTION

the nucleotides guanine and adenine). The intermediates of purine metabolism are toxic to some cells, necessitating tight regulation of purine synthesis and degradation. Furthermore, the final breakdown product of purine metabolism is uric acid, which is barely soluble in blood or urine. Increased plasma levels of uric acid are the strongest risk factor for gout, although, for poorly understood reasons, not everyone with high plasma uric acid levels develops gout. Purines are synthesized via two general pathways: de novo synthesis and the salvage pathway (Fig. 48-1). The first step in the de novo pathway is the reaction of phosphoribosyl pyrophosphate (PRPP, a ribose sugar with two pyrophosphates attached) with glutamine. PRPP provides the ribose sugar as one precursor for the nascent nucleotide. Hydrolysis of the pyrophosphate in a later step makes the de novo pathway irreversible. Glutamine is the precursor for inosine monophosphate (IMP), a precursor that is common to adenine and guanine biosynthesis. The reaction of PRPP with glutamine is catalyzed by the enzyme amidophosphoribosyltransferase (amidoPRT). AmidoPRT is activated allosterically by high levels of PRPP; PRPP is thus both a substrate and an activator of amidoPRT. In general, the cellular level of PRPP is the most important determinant of de novo purine synthesis. High PRPP levels result in enhanced de novo purine synthesis, whereas low PRPP levels decrease the rate of synthesis. The salvage pathway is the second important mode of purine synthesis. The first step in the salvage pathway is catalyzed by the key regulatory enzyme hypoxanthineguanine phosphoribosyltransferase (HGPRT). HGPRT

Gout is a uniquely human disease. Most mammals possess uricase, an enzyme that metabolizes purine breakdown products into a freely water-soluble substance, allantoin. Humans, in contrast, excrete most purines as sparingly soluble uric acid. High plasma levels of uric acid can lead to deposition of uric acid crystals in joints, most frequently the first metatarsophalangeal joint (great toe). Acute attacks of gout cause intense pain but typically occur infrequently. A number of rational therapies exist for the treatment of gout. These therapies are broadly divided into two groups: those that treat acute gout attacks and those that prevent recurrent attacks. Drugs that suppress the immune response to crystal deposition or limit the extent of inflammation may be used for both indications, although they are more commonly used to treat acute attacks. Agents that reduce uric acid synthesis or increase the renal excretion of uric acid prevent monosodium urate crystal formation and are useful for prevention of recurrent attacks. These pharmacologic interventions provide effective therapy for most cases of gout.

 PHYSIOLOGY OF PURINE METABOLISM Gout is a disease caused by imbalances in purine metabolism. To understand the cause and treatment of gout, it is necessary to recall the principles of nucleotide biochemistry. Although pyrimidines such as cytosine, thymidine, and uracil are straightforward for the body to metabolize and excrete, it is a challenge to metabolize purines (most notably

837

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842 Principles of Inflammation and Immune Pharmacology N O2

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Oxypurinol

FIGURE 48-5. Interaction between 6-mercaptopurine and allopurinol. 6-Mercaptopurine and azathioprine (a prodrug) are metabolized and eliminated from the body via the same pathways as other purines. Allopurinol and its metabolite, oxypurinol, inhibit xanthine oxidase, thereby inhibiting the breakdown of 6-mercaptopurine. Decreased degradation causes plasma levels of 6mercaptopurine to rise. When co-administering 6-mercaptopurine and allopurinol (for example, in cancer chemotherapy), the dose of 6-mercaptopurine should be substantially reduced.

drug, such as mycophenolic acid (see Chapter 45, Pharmacology of Immunosuppression), is another option. Although allopurinol is generally well tolerated, several important adverse effects should be considered when prescribing this agent. A small percentage of patients taking allopurinol may develop a hypersensitivity reaction characterized by a rash that, in rare instances, can progress to Stevens–Johnson syndrome. For this reason, all patients who develop a cutaneous reaction to allopurinol should discontinue the drug. Rarely, allopurinol may also cause leukopenia, eosinophilia, and/or hepatic necrosis. Febuxostat is a nonpurine small-molecule inhibitor of xanthine oxidase that has recently been approved for the treatment of chronic gout. In a large clinical trial, febuxostat was as effective as allopurinol in preventing recurrent flares of gout. Unlike allopurinol, febuxostat undergoes extensive hepatic metabolism, and it may not require dose adjustment in renal insufficiency. Because of its nonpurine structure, febuxostat use might not be associated with development of cutaneous reactions. As with allopurinol, initiation of febuxostat therapy should be accompanied by a suppressive medication such as colchicine in order to reduce the risk of gout flares in the first several months after initiation of uratelowering therapy. Agents That Increase Uric Acid Excretion Because the kidney reabsorbs a substantial amount of filtered uric acid, a pharmacologic agent that blocks tubular reabsorption increases uric acid excretion. Such drugs are called uricosuric agents. Probenecid was one of the first drugs used to increase urate excretion. Individuals lacking the URAT1 anion transporter protein have very low serum uric acid levels and do not respond to uricosuric agents, including probenecid, indicating that URAT1 is the molecular target for this class of drugs. Probenecid is not specific for URAT1; it also inhibits other transporters, including some of the renal organic anion

transporters (OATs) responsible for penicillin secretion. Decades ago, when penicillin was in limited supply, it was co-administered with probenecid to prolong the half-life of the antibiotic and decrease the penicillin dose required to achieve therapeutic levels of the drug. In patients with gout, probenecid is useful for the treatment of chronic hyperuricemia. Probenecid shifts the balance between renal excretion and endogenous production of urate, thereby lowering plasma urate levels. Uric acid levels lower than 6.0–6.5 mg/dL support dissolution of urate crystals, thereby reversing the process of crystal deposition in synovial joints. However, increasing renal urate excretion can predispose to formation of urate stones in the kidney or ureter. The likelihood of this complication can be diminished by recommending that patients increase their fluid intake and make their urine less acidic, commonly by co-administration of oral calcium citrate or sodium bicarbonate: uric acid has a pKa of 5.6, and it remains predominantly in the more soluble neutral form if the urine pH is above 6.0. Because probenecid inhibits the secretion of many organic anions, the dose of other drugs excreted by this pathway should be reduced when probenecid is co-administered. Low-dose aspirin may antagonize probenecid action; the mechanism of this antagonism is unknown. Sulfinpyrazone is a uricosuric agent that acts by the same mechanism as probenecid. It is more potent than probenecid, and it is effective in mild-to-moderate renal insufficiency. In addition to acting as a uricosuric, sulfinpyrazone has antiplatelet effects; it should therefore be used with caution in patients taking other antiplatelet agents or anticoagulants. Benzbromarone is a uricosuric agent with a mechanism of action similar to that of probenecid and sulfinpyrazone. Benzbromarone may have greater uricosuric efficacy than probenecid and sulfinpyrazone, particularly in patients with impaired renal function. However, the frequent incidence of hepatotoxicity has limited widespread use of the drug, and it is currently not available in the United States. Losartan is an angiotensin II receptor antagonist (see Chapter 21, Pharmacology of Vascular Tone) that has a modest uricosuric effect. Losartan may be a logical therapeutic choice in patients with concomitant hypertension and gout, although no controlled studies have been performed to prove that losartan reduces the incidence of acute gout attacks. Agents That Enhance Uric Acid Metabolism Most mammals other than humans express the enzyme uricase. This enzyme oxidizes uric acid to allantoin, a compound that is easily excreted by the kidney. In cancer chemotherapy, the rapid lysis of tumor cells can liberate free nucleotides and greatly increase plasma urate levels. By this mechanism, tumor lysis syndrome can lead to massive renal injury. Exogenous uricase can be co-administered with cancer chemotherapy to reduce plasma urate levels rapidly, and thereby to prevent renal damage. Allopurinol can also be used to prevent this component of tumor lysis syndrome. Currently, uricase is available in Europe as a protein purified from the fungus Aspergillus flavus. A recombinant version of the Aspergillus uricase, rasburicase, is available in the United States. A small percentage of patients have allergic reactions to the foreign protein and antidrug antibodies are common. Pegloticase, a pegylated formulation of recombinant porcine uricase, was recently approved for treatment of gout refractory to conventional therapy.

CHAPTER 48 / Integrative Inflammation Pharmacology: Gout 843

 CONCLUSION AND FUTURE DIRECTIONS Gout can be thought of as a disorder of purine metabolism and excretion. An imbalance between urate synthesis and excretion leads to hyperuricemia; in some individuals, hyperuricemia progresses to gout. Acute therapeutic interventions are aimed at symptomatic treatment of gout attacks; these treatments interrupt inflammatory pathways by inhibiting neutrophil and monocyte activation. Treatments for chronic gout lower plasma urate levels by reestablishing the balance between urate synthesis and excretion. Allopurinol and febuxostat inhibit urate synthesis; probenecid increases renal urate excretion. Recombinant uricase rapidly decreases plasma urate levels by converting uric acid to allantoin, thereby preventing the adverse renal consequences of tumor lysis syndrome. New therapies are under development; for example, IL-1 antagonists such as anakinra, canakinumab, and rilonacept are being studied for

the treatment of acute gout flares unresponsive to standard therapies or for patients in whom standard therapies are contraindicated.

Suggested Reading Chohan S, Becker MA. Update on emerging urate-lowering therapies. Curr Opin Rheumatol 2009;21:143–149. (Provides clinical details on febuxostat and uricases.) Eggebeen AT. Gout: an update. Am Fam Physician 2007;76:801–808. (Excellent clinical summary of gout, including criteria for diagnosis and clinical guidelines.) Martinon F. Mechanisms of uric acid crystal-mediated autoinflammation. Immunol Rev 2010;233:218–232. (Detailed review of uric acid-induced inflammation and inflammasome biology.) Neogi T. Gout. N Engl J Med 2011;364:443–452. (Recent clinical practice review of gout.) So A, Busso N. A magic bullet for gout? Ann Rheum Dis 2009;68:1517–1519. (Reviews advances in gout pathophysiology, including the role of IL-1 and development of IL-1 antagonists.)

VII Fundamentals of Drug Development and Regulation

49 Drug Discovery and Preclinical Development John L. Vahle, David L. Hutto, Daniel M. Scott, and Armen H. Tashjian, Jr.

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . 847-848 THE DRUG DISCOVERY PROCESS . . . . . . . . . . . . . . . . . . . . 848 Compound-Centered Drug Design . . . . . . . . . . . . . . . . . . 849 Natural and Synthetic Compounds . . . . . . . . . . . . . . . 849 Analogues of Natural Ligands . . . . . . . . . . . . . . . . . . . 849 Target-Centered Drug Design. . . . . . . . . . . . . . . . . . . . . . 851 High-Throughput Screening . . . . . . . . . . . . . . . . . . . . 851 Combinatorial Chemistry. . . . . . . . . . . . . . . . . . . . . . . 852 Structure-Based Drug Design . . . . . . . . . . . . . . . . . . . 853 Lead Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853 PHASES OF DRUG DEVELOPMENT . . . . . . . . . . . . . . . . . . . 853

KEY DISCIPLINES IN DRUG DISCOVERY AND DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854 Discovery Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854 Discovery Biology: Biochemical Assays, Cellular Assays, and Animal Models . . . . . . . . . . . . . . . . . 855 Absorption, Distribution, Metabolism, and Excretion (ADME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856 Development Chemistry: Chemical Synthesis, Scale-Up, and Manufacturing . . . . . . . . . . . . . . . . . . . . . . 857 Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 858 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 859 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 859

 INTRODUCTION

Increased attention has recently been focused on the inability of the biomedical research community to produce innovative new therapies. The challenges involved in drug discovery and development were highlighted (along with potential solutions) in the 2004 report of the FDA Critical Path Initiatives (see Suggested Reading). This report noted that both the National Institutes of Health (NIH) budget and pharmaceutical company research and development spending approximately doubled over a 10-year period beginning in 1993. The added investment did not increase the rate of development of new medicines, however, as evidenced by a decline in major drug and biological product submissions to the FDA. While several potential solutions have been offered to address this issue, it is important to note that a joint report by the FDA and the Association of American Medical Colleges highlighted the critical role of physician-scientists in improving the effectiveness of drug discovery and development. This chapter describes the phases of drug discovery and development and the scientific disciplines that are involved in these phases. Drug discovery spans the period from the identification of a potential therapeutic target to the selection of a single molecule for testing in humans. Drug development is generally defined as the period from the preclinical studies that support initial clinical trials through approval of the drug by regulatory authorities. The process of drug discovery and development is complex, requiring contributions from many otherwise disparate scientific disciplines.

Over the past decade, the U.S. Food and Drug Administration (FDA) has approved approximately 240 new drugs and biologics for therapeutic use. Many such drugs have enabled treatments for diseases that were previously untreatable. Others have yielded expanded treatment options because they are more efficacious and/or less toxic than previously available treatments. In the fight against infectious diseases, for example, pharmaceutical and biotechnology companies, university laboratories, and others have continued to develop new agents to treat diseases that have become resistant to existing treatments. With the availability of new technologies, knockout animal models, and information from the human genome project, it is anticipated that important new classes of drugs will continue to be discovered and developed in the coming decades. The development of a new drug is difficult and costly. Very few molecules that reach the development stage are ultimately approved as drugs: of 10,000 compounds considered promising from the results of initial screening assays, fewer than 10 make it to clinical trials and only two are eventually approved. Furthermore, the costs associated with discovering and developing a new drug averages approximately 800 million U.S. dollars. Although the development of new drugs is a risky venture, successful drugs can be quite profitable for those willing to take such risks. The most commercially successful drugs, such as atorvastatin, have annual sales of more than 12 billion dollars each.

847

CHAPTER 49 / Drug Discovery and Preclinical Development 849

Drug discovery Phase

Target-centered Compound-centered

Drug development

Lead optimization

Preclinical development

Phase I

Phase II

Phase III

Discovery chemistry Discovery biology

ADME

Toxicology

Target identification

Assay development and screening In vitro metabolism

Animal models of disease

Metabolism

Pharmacokinetics (animal)

(human)

Drug-drug interactions Development Carcinogenesis and reproduction

Screening

Preclinical

GLP toxicology

Development chemistry

Medical

Safety

Efficacy

Exposure

Dose selection

IND

Registration trials

NDA

FIGURE 49-2.

Sequence of phases of drug discovery and development. The important points to note are the general sequence of activities and the considerable overlap of functions with time. The process is highly interactive among several disciplines in an attempt to obtain the molecule with the greatest efficacy, fewest adverse effects, and greatest safety. The clinical trials and regulatory approval phases are described in Chapter 50. The entire process from hit to drug approval can take 8–12 years and cost more than $1 billion. IND, investigational new drug application; NDA, new drug application; ADME, absorption, distribution, metabolism, excretion; GLP, good laboratory practices.

Compound-Centered Drug Design Natural and Synthetic Compounds Traditionally, drugs were discovered using a compoundcentered approach. Many of the earliest drugs discovered were natural products isolated from plants, molds, or other organisms. Often, the discoveries were made serendipitously. For example, penicillin (see Chapter 34, Pharmacology of Bacterial and Mycobacterial Infections: Cell Wall Synthesis) was discovered when Alexander Fleming observed that spores of the contaminant mold Penicillium notatum inhibited bacterial growth in a petri dish. Other natural products that have been transformed into successful drugs include paclitaxel, a chemotherapeutic agent derived from the Pacific yew tree, morphine, an opioid analgesic obtained from the opium poppy, streptokinase, a thrombolytic agent obtained from streptococcal bacteria, and cyclosporine, an immunosuppressive agent obtained from a fungus. Table 49-1 lists a number of drugs obtained from natural products. There are several advantages to examining natural products as a source for potential drugs. First, natural products have a reasonable likelihood of biological activity. Second, it may be easier to isolate a compound from its natural source than to synthesize a compound de novo, especially if the structure of the compound is complex or requires difficult synthetic manipulations. Paclitaxel, for example, has a complex structure that contains four fused rings, one of which contains eight carbons. A chemical synthesis of the

compound took over 50 steps to complete and had a total yield of less than 1%. Third, it may be feasible to use the natural compound as a starting point for synthetic fine-tuning, i.e., to form a semisynthetic product. Of course, natural products also have disadvantages: it often takes significant effort to isolate a natural product, without a guarantee of success. Although natural products are more likely than many synthetic compounds to have biological activity, it may be difficult to predict which assay system would be optimal for testing the function of these molecules. Even if it is found to be pharmacologically active, a natural product can be expensive to isolate and modify. Synthetic compounds are now frequently used to search for new drugs. Researchers can construct a library consisting of thousands of compounds with differing structural characteristics, tailored for a particular type of investigation. A library could, for example, consist of numerous compounds that have a phenylalanine–proline bond or that are likely agonists or antagonists of a particular class of receptors. Analogues of Natural Ligands An alternative compound-centered approach uses the natural ligand (often an agonist) of a receptor as the starting point for drug development. For example, because lack of dopamine is associated with Parkinson’s disease (see Chapter 13, Pharmacology of Dopaminergic Neurotransmission), one of the first effective treatments involved administering the drug levodopa (L-DOPA), a metabolic precursor of dopamine.

Drug

Clinical Use and Chapter Reference

Cyclosporine O

O

Source

Immunosuppressant (Chapter 45)

Beauveria nivea (fungus)

Antiarrhythmic, cardiac inotrope (Chapters 23, 34)

Digitalis lanata (white foxglove), Digitalis purpurea (purple foxglove), numerous other plants

Analgesic (Chapter 17)

Papaver somniferum (poppy plant)

Cancer chemotherapeutic (Chapter 38)

Taxus brevifolia (Pacific yew tree)

Antibacterial (Chapter 34)

Penicillium chrysogenum (mold)

Antihypertensive (Chapter 25)

Rauwolfia serpentina (plant)

Thrombolytic (Chapter 22)

Beta-hemolytic streptococci (bacteria)

OH

H OH

H H

O HO

O

O

O

O

O

OH

OH

OH

Digoxin N

H O

HO

H

OH

Morphine O O

O

NH

O

OH

O O OH

O

H O O

OH O

O

Paclitaxel H N O

H S N

O

Penicillin G

COOH

H3CO

N H

N H

H O H

H3CO

OCH3

O O

OCH3

Reserpine Streptokinase

OCH3 OCH3

O O

O

Br

Br

O

O O

O

O

H N

H N H2N

H2N

O

O O

N H

N

OH

OH O

O N

OH

O

N H

OH

H

O

OH

H

O Simple

Complex

OH

Linear synthesis

B

C

A

D

A-B

A-B-C

A-B-C-D

Convergent synthesis

B A

A-B A-B-C-D

D C

C-D

CHAPTER 49 / Drug Discovery and Preclinical Development 859

hemolysis. The solution must also be sterile for intravenous injection. Finally, a drug is often less stable in solution than as a solid, so formulation chemists must test its stability in solution. If the drug is unstable, it may be prepared as a lyophilized powder that can be dissolved in water or buffer immediately before administration.

 CONCLUSION AND FUTURE DIRECTIONS The discovery and development of new drugs is a complex, interdisciplinary process that often requires 10 or more years and hundreds of millions of dollars. Researchers start by searching for a biologically active compound. This may involve a compound-centered approach or a target-centered approach. New pharmacologic targets are currently being identified by gene sequencing, by analysis of genetic factors that predispose to disease, by gene knockout experiments in laboratory animals, and by other techniques. For example, it is now possible to target proteins that enable the expression of genes rather than the gene products themselves. In addition, information about genetic polymorphisms may enable the products of specific, mutant genes to be the targets of new drugs (see Chapter 6, Pharmacogenomics). Finally, new methods to discover compounds that interact with these targets are also becoming available.

Suggested Reading Drews J. Drug discovery: a historical perspective. Science 2000;287:1960– 1964. (Historical description of the major methods of drug discovery.) International Conference on Harmonization: guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals 2009. http://www.ich.org/cache/compo/276-254-1. html (Describes the types of animal studies required by regulatory authorities to support clinical testing and registration of pharmaceuticals.) Levine RR. Pharmacology, drug actions and reaction. 6th ed. New York: Parthenon Publishing; 2000. (Explains how new drugs are discovered and describes the drug development process through clinical development.) Pritchard JF, Jurima-Romet M, Reimer ML, et al. Making better drugs: decision gates in nonclinical drug development. Nat Rev Drug Discov 2003;2:542–553. (Explores the key scientific questions that are addressed during drug discovery and preclinical development.) Rademann J, Günther J. Integrating combinatorial synthesis and bioassays. Science 2000;287:1947–1948. (Novel techniques for screening large libraries of compounds.) Sams-Dodd F. Strategies to optimize the validity of disease models in the drug discovery process. Drug Discov Today 2006;11:355–363. (Discusses how to optimize animal models of human disease to allow selection of better drug candidates.) United States Food and Drug Administration, United States Department of Health and Human Services. Innovation or stagnation: challenge and opportunity on the critical path to new medical products. 03/16/04. http:// www.fda.gov/oc/initiatives/criticalpath/whitepaper.pdf. (Discusses current challenges and opportunities in the development of new drugs, biologic products, and medical devices.)

50 Clinical Drug Evaluation and Regulatory Approval Mark A. Goldberg, Alexander E. Kuta, and John L. Vahle

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . 860-861 HISTORY OF THE U.S. FOOD AND DRUG LAW . . . . . . . . . . . 861 ETHICS IN CLINICAL DRUG INVESTIGATION . . . . . . . . . . . . 862 DRUG EVALUATION AND CLINICAL DEVELOPMENT . . . . . . 863 Authorizations to Initiate Clinical Trials . . . . . . . . . . . . . . . 863 Clinical Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863 Target Product Profile . . . . . . . . . . . . . . . . . . . . . . . . . 864 Development of a Clinical Trial . . . . . . . . . . . . . . . . . . 864 Phase 1 Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 865 Phase 2 Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 866 Phase 3 Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 866 Clinical Pharmacology. . . . . . . . . . . . . . . . . . . . . . . . . 866 Challenges in the Development of Drugs to Treat Rare Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 866 Successful Drug Development: Design and Execution . . . 867

DRUG APPROVAL PROCESS . . . . . . . . . . . . . . . . . . . . . . . . 867 FDA Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 867 FDA Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868 Approval in Other Countries . . . . . . . . . . . . . . . . . . . . . . . 869 Compassionate Use Protocols . . . . . . . . . . . . . . . . . . . . . 869 Drug Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 Drug Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 Additional Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870 REGULATORY ASPECTS OF DRUG PRODUCTION AND QUALITY CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870 GENERIC DRUGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870 NONPRESCRIPTION DRUGS AND SUPPLEMENTS . . . . . . . . 870 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 871 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871

 INTRODUCTION

must be well designed not only to appropriately demonstrate safety and clinical efficacy, but also, through the use of appropriate biomarkers, pharmacodynamic markers, and safety monitoring, to allow for the early discontinuation of development of drugs that are destined to fail. This is an exciting and challenging time to be involved in drug development. Major advances in the biological sciences have yielded a much greater understanding of the molecular basis of many diseases and provide the opportunity to have an unprecedented impact on the alleviation of human suffering. However, translating these scientific advances into new and more effective therapies for human diseases has proven to be daunting. In the 10 years between 1993 and 2003, there was a steady decline in the number of new small-molecule drugs and biologic agents submitted for approval to regulatory agencies around the world. This decline has translated into fewer new drugs being approved and made available to patients. To close this apparent gap between innovative basic-science discoveries and the stagnating approvals of innovative new therapies will require diligent clinical drug development programs that include rigorous, well-controlled trials, integrated clinical development plans, the use of novel statistical methods, and the inclusion of novel pharmacodynamic and other biomarkers at the various stages of drug

Controlled clinical trials provide the scientific and legal basis by which regulatory authorities around the world evaluate new prescription drugs and approve them for sale. In the United States, the regulatory review of drugs and devices is the responsibility of the U.S. Food and Drug Administration (FDA). Over the past 50 years, improved methods for large-scale clinical studies have precipitated a shift toward evidence-based medicine and helped to accelerate the pace of drug development. This greater emphasis on clinical trials to appropriately assess the safety and efficacy of new drugs has resulted in a dramatic rise in the costs associated with drug development. Various reports have estimated that the cost of successfully developing a new drug product from discovery, through preclinical and clinical development, to regulatory filing and approval costs in the range of $0.5 to $2.0 billion. The time for the clinical development phase of drug development alone averages 7–8 years. Moreover, only about one in ten drugs that enters the clinic for testing ultimately receives regulatory approval and is marketed. Given the tremendous cost and duration of clinical drug development, it is imperative that every effort is made to plan carefully and execute effectively. Drug development programs 860

Phase II trials

Develop manufacturing Develop QA/QC program, GMP practices

Manufacturing

Legal

Phase I trials

Patent application

Patent granted

Phase III trials

FDA approval

Phase IV

Phase IV

Generics available

IND filed

Clinical

Patent expires

Toxicology studies

Toxicology

ANDA filed

Biological characterization

NDA filed

Compound identification and optimization

Post-approval regulation

Manufacturing begins

Chemistry and biology

Drug development (5-9 years) End of phase II meeting

Drug discovery (2-5 years)

CHAPTER 50 / Clinical Drug Evaluation and Regulatory Approval 863

• Contains safeguards for vulnerable populations, such as children and the mentally disabled IRB/IEC oversight and approval begins before the commencement of human trials and continues for the duration of clinical trials. The membership of an IRB/IEC consists of five or more experts and laypersons from various backgrounds. Federal regulations stipulate that IRB membership must include at least one member whose primary expertise is in a scientific area, one member whose primary expertise is in a nonscientific area, and one member who is not affiliated with the institution overseeing the clinical research protocol. In addition, the other members’ qualifications must be such that the IRB is able to evaluate research proposals in terms of institutional requirements, applicable law, standards of professional practice, and community attitudes. Thus, many IRBs include clergy, social workers, and attorneys as well as physicians, scientists, and other health care professionals. Clinical trials must be appropriately designed and rigorously executed in order to optimize the ratio of benefit to risk and to satisfactorily answer the scientific questions under study. Scientific clinical trial design must include appropriate control or comparator arm(s), randomization and blinding, and sample size, among other elements (see below). Some institutions have a scientific review committee that must approve all protocols involving human subjects to ensure that the protocol is appropriately designed to answer the questions being asked. To further assure that the findings of clinical trials are accurate and credible and that the rights of clinical trial subjects are protected, regulatory agencies require that clinical trials leading to the approval of new drugs be conducted according to good clinical practices (GCP). Guidelines for GCP have been developed by the International Conference on Harmonization (ICH) to provide a standard for the design, conduct, recording of findings, monitoring of data, analysis, auditing, and reporting of results of clinical trials.

 DRUG EVALUATION AND CLINICAL DEVELOPMENT The investigation of a new drug candidate comprises several phases, beginning with preclinical evaluation and proceeding through phase 3 clinical studies. At the conclusion of this process, the FDA may consider the molecule for approval as a new drug.

Authorizations to Initiate Clinical Trials Preclinical research establishes the potential efficacy and safety of a compound for use in human trials. During this stage of testing, described in Chapter 49, a compound is studied to determine its biological actions, chemical properties, and metabolism, and a process is developed for its synthesis and purification. A major focus of preclinical testing is determining whether the molecule has an acceptable safety profile in animals prior to initiating testing in humans. The International Conference on Harmonization has established requirements for the animal studies used to support different types of clinical trials. The primary studies used to support clinical drug development are animal toxicity studies and investigations on the absorption, distribution, metabolism, and excretion (ADME) of the compound. As described in Chapter 49, the duration of animal studies is determined by

the length of the clinical trials being undertaken. For this reason, it is essential that there is close coordination among the preclinical and clinical scientists on the drug development team. Many potential drug candidates either do not proceed to human trials or are removed from clinical testing due to adverse safety findings in animal studies. The preclinical research phase is also an important time to explore potentially important pharmacodynamic markers and other biomarkers that could help facilitate clinical development. The mechanism for seeking approval to initiate clinical trials in the United States is the submission of an Investigational New Drug application (IND) to the FDA. The IND contains data from the preclinical studies, data from prior clinical investigations (if available), the proposed protocol for human trials, and other background information. The IND also contains a document referred to as the Investigator’s Brochure (IB). The IB is provided to regulators, clinical investigators, and IRBs/IECs; it represents a summary of all available information on the investigational drug and may be several hundred pages in length. The IND must also contain information on the composition and stability of the drug and evidence that the drug can be manufactured in consistent batches for clinical trials. Commercial INDs are submitted by sponsors with the ultimate goal of obtaining approval for marketing and sale of a new drug product. Noncommercial filings, such as Investigator, Emergency Use, and Treatment INDs, are used for different purposes, as described below. The FDA must review the IND within 30 days and decide whether human trials may begin. Figure 50-2 is a flowchart representing the process used by the FDA to review an IND. The areas of review include a chemistry review, a pharmacology/toxicology review, and a medical review. If the IND review does not identify any safety concerns, the IND is considered open or active after the 30-day wait period. If the review reveals the potential for unreasonable risk to participants, the FDA contacts the sponsor, and a clinical hold is issued, preventing initiation of human studies. The sponsor must address any issues in question before the clinical hold is lifted. A clinical hold may be issued at any time during clinical drug development; such a hold can be based on new findings from animal studies, clinical data indicating an unacceptable risk profile, or a finding that a sponsor did not accurately disclose the risk of the study to investigators or subjects.

Clinical Development Given the time, cost, and risks associated with clinical drug development, it is imperative to plan carefully and execute meticulously. The goals of clinical drug development include: • Assessment of the dose–response profile • Assessment of the toxicity profile for a given dosing regimen • Assessment of pharmacokinetic/pharmacodynamic relationships • Establishment of the safety and efficacy profile in wellcontrolled studies in well-defined patient populations These goals are accomplished through the conduct of clinical trials. Each clinical trial must be designed to answer specific questions. In turn, each trial should be part of an integrated development plan leading to the ultimate demonstration of safety and efficacy in well-controlled trials.

Applicant (drug sponsor)

IND

Medical

Chemistry

Sponsor submits new data

Safety review

Safety acceptable for study to proceed?

Pharmacology/ toxicology

NO

Clinical hold decision

YES

Study starts Notify sponsor Complete reviews

Notify sponsor of review results

866 Fundamentals of Drug Development and Regulation

drug’s volume of distribution and clearance enables study designers to determine an appropriate maintenance dose and dosing frequency for phase 2 and 3 trials (see Chapter 3, Pharmacokinetics). Although phase 1 trials focus on safety and tolerability, biomarkers of the desired pharmacologic effect are increasingly being used to provide data early in drug development on the potential effectiveness of the molecule. One example of a simple marker would be the phenotyping of peripheral blood lymphocytes in a trial of an agent designed to inhibit B cells; more generally, biochemical or cell-based assays are used to detect whether the drug has effectively regulated the targeted enzyme, cell type, or tissue. Phase 2 Studies Phase 2 studies may involve up to several hundred subjects with the medical condition of interest. Phase 2 clinical trials have multiple objectives, including the acquisition of preliminary data regarding the effectiveness of the drug for treatment of a particular condition. Like phase 1 trials, phase 2 trials continue to monitor safety. Because phase 2 studies enroll more patients, they are capable of detecting less common adverse events. Phase 2 studies also evaluate dose–response and dosing regimens, which are critically important in establishing the optimum dose or doses and frequency of administration of the drug. A typical phase 2 design may involve either single-blind or double-blind trials in which the drug of interest is evaluated against placebo and/or an existing therapy. The trial usually compares several dosing regimens to obtain optimum dose range and toxicity information. The results of phase 2 studies are critically important in establishing a specific protocol for phase 3 studies. Specifically, phase 2 studies should be designed to obtain a reasonable estimate of the size of the treatment effect of the experimental therapy. This critical information will then be used to determine the appropriate sample size for the phase 3 studies. Phase 2 results can also be used to pinpoint additional data that must be collected in phase 3 trials, such as monitoring of liver function tests if phase 2 data suggest possible hepatotoxicity. Throughout the process of drug development, the sponsors of the program have the opportunity to consult with the regulatory agencies through formal meetings. After the completion of phase 2 studies and before the initiation of phase 3 (pivotal) studies, the sponsor will typically request a meeting with the FDA to discuss the results obtained to date and to outline for the FDA reviewers the plans for the phase 3 program. Given the time and expense of phase 3 clinical trials, it is critical that there is agreement between the FDA and the sponsor on the appropriate trial design(s) before the trial is initiated. Phase 3 Studies Phase 3 studies involve several hundred to several thousand patients and are conducted at multiple sites and in settings similar to those in which the drug will ultimately be used. Phase 3 studies utilize specific clinical endpoints as the primary endpoints of the trial. Examples of accepted clinical endpoints include survival, improvement in patient functional status, or improvement in how patients feel (e.g., quality of life assessments). Occasionally, surrogate endpoints for clinical benefit may be used. Examples of surrogate endpoints include markers for decreased disease burden, such

as a reduction in the plasma levels of biochemical markers (e.g., glucose and LDL cholesterol), an increase in cardiac output, or a reduction in size of a tumor. Surrogate endpoints that have been validated in prior clinical trials (e.g., reduction in serum LDL cholesterol as a surrogate for clinically meaningful improvement in cardiac outcomes) may be acceptable endpoints in phase 3 pivotal trials. In situations of life-threatening diseases for which no acceptable therapy is available, surrogate endpoints that are reasonably likely to predict clinical benefit (but that are not yet validated) may be used as endpoints in pivotal trials. In such instances, the FDA may grant accelerated approval. Accelerated approval allows the drug to be approved more quickly and to be made available to patients in need in a more timely manner. Drugs being considered for accelerated approval may often be given priority review status, in which case the review period is 6 months rather than the standard review period of 10 months. This approach has been used to approve drugs for the treatment of acquired immunodeficiency syndrome (AIDS) and several types of cancer, among other indications. However, under accelerated approval, the sponsor is required to conduct postapproval phase 4 studies to verify and confirm the clinical benefit of the drug. In the introductory case, imatinib was granted accelerated approval based on surrogate clinical endpoints and was then granted full approval upon the successful completion of postapproval studies. Clinical Pharmacology Many pharmaceutical and biotechnology companies have developed groups dedicated to studying the clinical pharmacology of their products in development. These groups may be called by names such as Experimental Medicine, Molecular Medicine, or Clinical Pharmacology. The groups typically investigate aspects of the drug’s clinical pharmacology, including: fasting and fed single-dose and repeat-dose pharmacokinetics; drug–drug interactions, with a special emphasis on the role of cytochrome P450 isoforms on drug metabolism; and the impact of renal or hepatic impairment on drug metabolism. They perform comprehensive QT studies to assess the impact of the drug on cardiac electrophysiological function. The groups make careful assessments of immunogenicity, particularly if the drug is a protein therapeutic, and of the drug’s clinical pharmacology in pediatric patients and in specific ethnic groups such as Asian populations. Clinical pharmacology groups also work closely with preclinical scientists to develop appropriate biomarkers to better assess the impact of the drug at the earliest stages of clinical development. Biomarker assessments may take a variety of forms, including exploration of the population of patients most likely to benefit or most likely to be susceptible to toxicity as well as pharmacodynamic markers of drug activity. The groups may attempt to correlate gene polymorphisms or expression profiles with responsiveness. These and other clinical pharmacology studies are performed throughout the clinical development program and are incorporated into phase 1, 2, and 3 studies. Challenges in the Development of Drugs to Treat Rare Diseases Historically, pharmaceutical companies had typically been disinterested in developing products for diseases with small patient populations, since the cost of developing drugs for small markets was similar to that for developing drugs for

CHAPTER 50 / Clinical Drug Evaluation and Regulatory Approval 869 Applicant (drug sponsor)

NDA

Application fileable?

NO

Refuse to file letter issued

YES

Medical

Biopharmaceutical

Pharmacology

Statistical

Chemistry

Microbiology

to the type and amount of data required in product labeling. In Europe, many drugs are first evaluated by the European Medicines Evaluation Agency and then approved by the European Union. In Canada, Health Canada administers the regulations embodied in the Canadian Food and Drugs Act. In Japan, approval of new drugs is granted by the Ministry of Health and Welfare. Importantly, Japanese regulatory authorities require studies to be performed in ethnic Japanese patients in order to demonstrate that the pharmacokinetic and safety profiles observed in a Japanese population are similar to those observed in a western population. Demonstration of efficacy in Japanese patients may also be required.

Compassionate Use Protocols

Advisory committee meeting

Sponsor revises

Meetings with sponsor

Reviews complete and acceptable? YES

NO

NO

Additional info or revisions requested or submitted (amendment)

YES

Labeling review acceptable?

The FDA has created compassionate use protocols, also known as treatment investigational new drug applications (treatment INDs), to expand access to investigational drugs. These protocols permit promising investigational therapies to be used, before general approval, for extremely sick patients who are not eligible for an ongoing clinical trial. Three conditions must be satisfied in order for an investigational drug to be eligible for a compassionate use protocol: (1) the drug must show preliminary evidence of efficacy; (2) patients must be likely to die or suffer rapid disease progression within several months, or to die prematurely without treatment; and (3) there must be no comparable approved therapy to treat the disease at that stage.

Drug Labeling

Inspection of sites acceptable? NO YES

Pending satisfactory results NDA action

FIGURE 50-3.

Process of new drug application (NDA) review. When a new drug application is filed, the drug sponsor provides data regarding the drug’s medical, pharmacologic, chemical, biopharmaceutical, statistical, and microbiological characteristics; these data are reviewed by separate committees at the FDA. The FDA or an FDA Advisory Committee (optional) may meet with the sponsor. If the review is complete and acceptable, then the drug application is reviewed for acceptable labeling (official instructions for use). The manufacturing sites and sites where significant clinical trials were performed also undergo inspections and audits. Blue boxes correspond to actions by the drug sponsor; white boxes correspond to actions by the FDA.

Approval in Other Countries Before drugs may be sold in countries outside the United States, they must first be evaluated and approved by the appropriate regulatory authorities in that country. In some countries, this may include a comprehensive review of all the data, similar to the NDA/BLA review. In other countries, a more limited review may occur if the drug has already been approved in one of the major foreign markets (United States, Europe, Japan). During these reviews, a regulatory authority may require additional types of studies that were not required for U.S. approval. In addition, different regulatory agencies may have different approaches

Each country’s regulatory bodies establish a standard format and organization for labeling an approved drug in that country. A drug label must include the drug’s proprietary and chemical name, formula and ingredients, clinical pharmacology, indications and usage, contraindications, warnings, precautions, adverse reactions, drug abuse/dependence potential, overdosage, dosage, rate and route of administration, and how the drug is supplied. In the United States, this information is also known as the drug’s package insert. When a new drug approaches approval, the FDA reviews and negotiates the final package insert with the sponsor to ensure that the labeling is justified by the data submitted in the NDA. To provide more accessible and informative drug information, the FDA has instituted structured product labeling, which provides key information important to prescribers in a standardized format. Regulatory agencies may use additional methods to ensure that the important attributes of the drug are clearly communicated. For example, in the United States, package inserts for drugs that have certain safety risks include a “black box” warning, in which key safety information is prominently displayed. In addition, the FDA may require sponsors to create Medication Guides for mandatory distribution to patients; these guides communicate critical safety information in language that is readily understandable.

Drug Naming Another facet of drug approval involves the determination of a drug’s name. A drug is known by two principal names, the generic name and the brand name (or trade name). A drug’s generic name is based on its chemical name and is unprotected by a trademark. In contrast, a drug’s brand

870 Fundamentals of Drug Development and Regulation

name refers to the exclusive name of a substance or drug product owned by a company under trademark law. For example, imatinib mesylate is the generic name, while Gleevec is the brand name, of the drug discussed in the introductory case.

Additional Indications Once a drug is approved, physicians and certain other health care professionals are permitted to prescribe the drug in various doses or dosage regimens. Providers may also prescribe the drug for additional clinical indications, known as “off-label” use. Physicians are also permitted to conduct investigational studies with the drug, provided that they follow the rules of informed consent and obtain IRB approval for the studies. Although health care providers are permitted to use a drug off-label, such use may nonetheless subject them to medical malpractice liability, just as any other treatment decision could. Pharmaceutical companies, however, may not market the drug for any indications other than those for which it has been approved by the FDA. Current regulations prohibit pharmaceutical companies from providing any marketing materials, including scientific articles, on the off-label use of a drug, unless such materials are requested by a physician. In order to market a drug for a new indication, a pharmaceutical company must conduct an additional program of development to prove that the drug is safe and efficacious for that new indication. These data are then submitted to regulatory authorities as a supplemental NDA (sNDA) and subjected to additional review prior to the granting of approval for the new indication.

 REGULATORY ASPECTS OF DRUG PRODUCTION AND QUALITY CONTROL In addition to demonstrating a drug’s safety and efficacy, manufacturers must also comply with FDA regulations for manufacturing as a requirement for drug approval. The Good Manufacturing Practice (GMP) guidelines govern quality management and control for all aspects of drug manufacturing, and the FDA has the authority to inspect manufacturing facilities in order to determine compliance. FDA regulations specify impurity tolerance levels, quality control procedures, and testing of sample batches. A company must obtain prior FDA approval before implementing any manufacturing change that is determined by the FDA to have substantial potential to affect the safety or effectiveness of a drug through alterations in its identity, strength, quality, purity, or potency. Other changes may be implemented either with or without submission of a supplemental NDA. Changes not requiring a supplement may be noted in the report filed annually with the FDA or on another date determined by the agency.

 GENERIC DRUGS The FDA also oversees approval of generic drugs, which the agency defines as drugs that are comparable to innovator drugs in dosage form, safety, strength, route of administration, quality, performance characteristics, and intended use. Under the Drug Price Competition and Patent Term Restoration Act of 1984, also known as the Hatch–Waxman

Act, a company may submit an Abbreviated New Drug Application (ANDA) before the patent governing the brand-name drug expires. However, the company must wait for the original drug’s patent to expire before it can market a generic version. The first company to file an ANDA has the exclusive right to market the generic drug for 180 days. ANDAs for generic drugs are not required to provide data establishing safety and efficacy, because this has been established in the NDA for the innovator drug. To establish bioequivalence, which is required in the ANDA, sponsors may submit a formulation comparison, comparative dissolution testing (where there is a known correlation between in vitro and in vivo effects), in vivo bioequivalence testing (comparing the rate and extent of absorption of the generic with that of the reference product), and, for nonclassically absorbed products, a head-to-head evaluation of comparative effectiveness based on clinical endpoints. In addition, an ANDA sponsor must provide evidence that its manufacturing processes and facilities, as well as any outside testing or packaging facilities, are in compliance with federal GMP regulations. “Generic” versions of biologic drugs, primarily proteins, present much greater challenges than generic versions of small-molecule drugs. Whereas small molecules can readily be shown to be comparable to the innovator drug as described above, this is not so easy with recombinant proteins, which usually have many post-translational modifications. Small changes in post-translational modifications may result in marked differences from the innovator drug in safety and efficacy. Changes in cell lines used to manufacture such proteins and changes in any step of the production process may alter post-translational modifications. As a result, the precise regulatory path for the development of “biosimilars” is currently being actively debated in Congress and within the FDA.

 NONPRESCRIPTION DRUGS AND SUPPLEMENTS The 1951 Durham-Humphrey Amendment to the Food, Drug, and Cosmetic Act defined prescription drugs as drugs that are unsafe for use except under professional supervision. In determining which drugs do not require a prescription, the FDA examines a drug’s toxicity and the facility with which a condition may be self-diagnosed. Because over-the-counter (OTC) drugs are sold in lower doses than their prescription counterparts and are used primarily to treat symptoms of disease, the FDA requires their labels to contain the following: • Intended uses of the product, as well as the product’s effects • Adequate directions for use • Warnings against unsafe use • Adverse effects Although OTC products present a potential danger of misuse or misdiagnosis in the absence of physician oversight, the increased availability of these products has provided many U.S. citizens with access to effective and relatively inexpensive treatments. The Dietary Supplement Health and Education Act of 1994 defines a dietary supplement as any product intended for ingestion as a supplement to the diet, including vitamins, minerals, herbs, botanicals, other plant-derived substances,

CHAPTER 50 / Clinical Drug Evaluation and Regulatory Approval 871

amino acids, concentrates, metabolites, and constituents and extracts of these substances. The FDA oversees the safety, manufacturing, and health claims made by dietary supplements. The FDA does not, however, evaluate the efficacy of supplements as it does for drugs. The FDA may restrict or halt the sale of unsafe supplements, but it must demonstrate that such supplements are unsafe before taking action. This occurred in December 2003, when the FDA announced a rule banning dietary supplements containing ephedrine alkaloids (ephedra) after reviewing the substantial number of adverse events (including deaths) associated with these products.

 CONCLUSION AND FUTURE DIRECTIONS Specific laws and regulations have been established to provide for the development of new drugs, while at the same time assuring privacy and safety for the individuals participating in clinical trials. Regulatory approval of new drugs follows a lengthy process of preclinical and clinical studies. Each phase of development provides critical information that defines the study protocol for subsequent investigations. However, no amount of animal and clinical data can guarantee complete safety for all future patients. Thus, the FDA and drug manufacturers continue to monitor the adverse effects, manufacturing processes, and overall safety of a drug for its lifetime (see Chapter 51, Systematic Detection of Adverse Drug Events). In the future, there will be an increased focus on evaluating the safety of new medicines, both during clinical trials and after the drug is approved and introduced into larger, more diverse patient populations.

Acknowledgment We thank Armen H. Tashjian, Jr. for his valuable contribution to this chapter in the Second Edition of Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy.

Suggested Reading Adams CP, Brantner VV. Estimating the cost of new drug development: is it really $802 million? Health Aff 2006;25:420–428. (Finds that developing a new drug costs between $500 million and $2 billion, depending on the indication.) Center for Drug Evaluation and Research, Food and Drug Administration, United States Department of Health and Human Services. The CDER Handbook. Revised 03/16/98. Available at http://www.fda.gov/ downloads/AboutFDA/CentersOffices/CDER/UCM198415.pdf. (Describes the processes by which the FDA evaluates and regulates drugs, including new drug evaluation and postmarketing monitoring of drug safety and effectiveness.) Cohen MH, Williams G, Johnson JR, et al. Approval summary for imatinib mesylate capsules in the treatment of chronic myelogenous leukemia. Clin Cancer Res 2002;8:935–942. (Summarizes the approval of imatinib mesylate, the drug discussed in the introductory case.) DiMasi JA, Grabowski HG. The cost of biopharmaceutical R&D: is biotech different? Manag Decis Econ 2007;28:469–479. (First paper to estimate costs of biopharmaceutical development compared to costs of traditional pharmaceutical development.) Dixon JR. The International Conference on Harmonization Good Clinical Practice guideline. Qual Assur 1999;6:65–74. (Guidelines for standard design of drug development.) Innovation/stagnation: challenge and opportunity on the critical path to new medical products. U.S. Department of Health and Human Services, Food and Drug Administration. March 2004. (An FDA report that addresses the recent slowdown in innovative drug development.)

51 Systematic Detection of Adverse Drug Events Jerry Avorn

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . 872-873 CHALLENGES IN THE ASCERTAINMENT OF DRUG SAFETY . . . 872 Study Size and Generalizability . . . . . . . . . . . . . . . . . . . . 872 Surrogate Outcomes and Comparators . . . . . . . . . . . . . . 873 Duration and Postapproval Studies. . . . . . . . . . . . . . . . . . 874 PHARMACOEPIDEMIOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . 874 Sources of Pharmacoepidemiologic Data . . . . . . . . . . . . . 875 Spontaneous Reports . . . . . . . . . . . . . . . . . . . . . . . . . 875 Automated Databases . . . . . . . . . . . . . . . . . . . . . . . . . 875 Patient Registries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875 Ad Hoc Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876 Study Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876 Cohort and Case-Control Studies . . . . . . . . . . . . . . . . 876 Evaluation of Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876

Issues in Study Design and Interpretation . . . . . . . . . . . . 877 Confounding by Indication. . . . . . . . . . . . . . . . . . . . . . 877 Selection Bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 The “Healthy User” Effect . . . . . . . . . . . . . . . . . . . . . . 877 Interpreting Statistical Significance . . . . . . . . . . . . . . . 878 ADVERSE DRUG EFFECTS AND THE HEALTH CARE SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878 Balancing Benefits and Risks . . . . . . . . . . . . . . . . . . . . . . 878 Role of the FDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879 Legal and Ethical Issues. . . . . . . . . . . . . . . . . . . . . . . . . . 879 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 879 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879

 INTRODUCTION

in informatics and analytic techniques in this field hold promise for enhancing our understanding of drug risks so that they can be better understood and managed, with the goal of putting a drug’s benefits into context and guiding clinical decision-making and regulatory action.

Because medications act by interfering with one or more aspects of molecular and cellular function, it is difficult to do so without also causing an undesirable effect either by that perturbation or by another (perhaps unexpected) drug action. Because all drugs have risks, the goal of pharmacotherapy cannot be to prescribe a risk-free regimen. Instead, it is to ensure that the risks of drug therapy are as low as possible and are acceptable in the context of a medication’s clinical benefit. Some adverse effects of a drug are apparent during its early development and often result from the same on-target mechanism responsible for its therapeutic effect (e.g., cytotoxic cancer chemotherapy). Even in such situations, however, it is necessary to know how those expected adverse effects will be manifested when the drug is in routine use—in terms of both their frequency and their severity. After a drug has been approved for clinical use, the goal becomes detecting and quantifying the risks as quickly and rigorously as possible. Serious or even life-threatening adverse effects have led to the withdrawal of widely used drugs. This has heightened the sensitivity of clinicians and patients to the emerging field of pharmacoepidemiology—the measurement of drug effects in large “real-world” populations of patients. Advances 872

 CHALLENGES IN THE ASCERTAINMENT OF DRUG SAFETY The randomized controlled trial (RCT) is the gold standard for determining the efficacy of a drug and is the sole criterion used by regulatory agencies, such as the U.S. Food and Drug Administration (FDA), in deciding whether to approve a new medication for use. But this valuable tool also has limits, and it is important to understand those limits when assessing the benefits and risks of a given agent.

Study Size and Generalizability Compared to the number of patients who eventually use a drug, the number of subjects in clinical trials supporting the approval of that drug is relatively modest. Approval decisions are generally made on the basis of trials that include 2,000–4,000 participants, or fewer for rare conditions. If a particular adverse event occurs just once in every

876 Fundamentals of Drug Development and Regulation

Ad Hoc Studies Some important questions in pharmacoepidemiology cannot be addressed by these methods, but must instead be answered by collecting data de novo on particular groups of patients with a given disease, or patients taking a particular class of medications. One example is the definition of sudden uncontrollable somnolence (sometimes called “sleep attacks”) in patients taking dopamine agonists for Parkinson’s disease (PD). Such events were not systematically documented in most large clinical trials of these drugs, and were not likely to be recorded as a new diagnosis in an office visit. Determining whether some drugs cause this problem more than others required interviewing a large sample of PD patients using different classes of medications.

Case-control study

= filled prescription for drug X

Controls (no M.I.) Time

Study Strategies Once a source of pharmacoepidemiologic data has been identified, statistical methods are used to evaluate those data and reach conclusions about the associations between a drug and possible adverse effects. The two most common types of analyses used to evaluate these observational data are cohort studies and case-control studies. Each is designed to evaluate statistically the risk of a particular adverse outcome associated with exposure to a given drug. Cohort and Case-Control Studies In cohort studies, one identifies a group of patients exposed to a given drug (e.g., patients with arthritis treated with a particular NSAID), and another group of patients who are as similar as possible to the exposed group but who did not take the drug of interest (e.g., patients with arthritis of comparable severity who were treated with another NSAID). Both groups are then followed over time to determine how many in each group develop an adverse effect of interest (e.g., myocardial infarction; Fig. 51-1). While this can be done on a real-time basis, more commonly, exposure (or nonexposure) that occurred in the past is defined from an existing database, so that subsequent events can be analyzed retrospectively. Cohort studies make it possible to measure actual incidence rates (i.e., the likelihood of a given outcome following use of a particular drug) and to track multiple outcomes. In case-control studies, one specifies the case-defining outcome event (e.g., myocardial infarction) and identifies a group of patients in a population who have experienced that event; these are the cases. The controls are patients in the same population who are as similar as possible to the cases but have not had the outcome of interest (e.g., patients of similar age and gender, and with similar cardiac risk factors, who have not had a myocardial infarction). One then looks back in time prior to the occurrence (or nonoccurrence) of the event of interest to review all the medications that were taken by cases and by controls, to determine whether use of a given drug was higher than expected among cases than among controls (Fig. 51-1). The case-control design is more efficient than the cohort design if the outcome of interest is rare and one has to interview all study participants, because it is possible to focus on a selected group of patients known to have had the outcome of interest. Evaluation of Risk At the most basic level, cohort and case-control studies yield data that comprise a 2 ⫻ 2 table defining the presence or absence of exposure to the drug of interest as well as the

Cases (e.g., M.I.)

Cohort (follow-up) study

Exposed (taking drug X)

= outcome event (e.g., M.I.)

Unexposed (not taking drug X) Time

FIGURE 51-1.

Schematic design of case-control and cohort studies. Top. In a case-control study, cases are identified as patients in a population who have experienced the case-defining outcome event (e.g., myocardial infarction); controls are patients in the same population who are as similar as possible to the cases but have not had the outcome of interest. All medications taken by cases and controls are reviewed retrospectively to determine whether use of a given medication was higher among cases than among controls. Bottom. In a cohort study, two groups of patients are identified: those who were exposed to a given drug and another group who are as similar as possible to the exposed group but did not take the drug of interest. All patients are followed over time to determine how many in each group develop a specified outcome event of interest (e.g., myocardial infarction).

presence or absence of the adverse outcome. The data can be arranged in four cells, as shown in Figure 51-2: patients who took the drug of interest and had the outcome (A); patients who took the drug but did not have the outcome (B); patients who did not take the drug but had the outcome anyway (C); and patients who did not take the drug and did not have the outcome of interest (D). Cells A and D are concordant for the drug–outcome relationship, and cells B and C are discordant for this association. The product A ⫻ D divided by the product B ⫻ C reflects the strength of such an association. For cohort studies, this is referred to as the relative risk; for case-control studies (as long as the case outcome is not common), this is known as the odds ratio. A relative risk (or odds ratio) of 2 means that patients using the drug are twice as likely to have the outcome as patients not using that drug; a relative risk or odds ratio of 0.5 means that users of the drug are half as likely as nonusers to experience that outcome (i.e., the drug has a protective effect for that outcome).

Adverse outcome

A

B Exposure + Outcome +

Drug exposure

C No drug exposure

No adverse outcome

Exposure + Outcome –

D Exposure – Outcome +

Exposure – Outcome –

878 Fundamentals of Drug Development and Regulation

Alzheimer’s disease in patients taking statins. Such studies often seem to be flawed by what has been called the “healthy user effect.” Patients who are regular users of any preventive medication appear to be different from those who do not exhibit this behavior: they are more likely to visit their doctor seeking preventive therapy, or are at least open to receiving it, and their physicians are sufficiently prevention-oriented to write such a prescription. Such patients are probably also more likely to engage in other health-promoting behaviors, such as tobacco avoidance, weight control, exercise, and adherence to their other prescribed drug regimens. Several large randomized trials have proven a similar point: patients randomized to placebo who comply with their dummy-pill regimen have better outcomes (including mortality) than patients who do not comply with their placebo “regimen.” Because the content of the placebo could not have produced this effect, these findings provide clear evidence that patients who consistently behave in a healthpromoting manner are more likely to have better clinical outcomes, apart from the therapeutic effect of a specific drug in their regimen. To address this issue in observational studies, some research groups have begun to use only “active controls” as comparator groups—for example, comparing patients adherent to statin regimens with patients adherent to regimens of other preventive drugs, rather than simply comparing such patients with patients who are not statin users. Interpreting Statistical Significance In evaluating the results of both observational studies and randomized trials, it is conventional to use a p value of 0.05 as a threshold or benchmark for statistical significance. This criterion is often mistakenly interpreted to mean that a finding is “real” if the p value for the difference between groups is below that value, and “not real” if it is above. However, more sophisticated readers of the literature understand that such a cut-point is largely arbitrary (compared to, for example, a p value of 0.03 or 0.07), and that attention must also be paid to the magnitude of the difference. For example, a p ⬍0.05 difference between a new drug and placebo may be clinically meaningless if there is only a 2% difference in effect size. The situation is even more critical in assessing the statistical significance of data about adverse events, whether from a randomized trial or from an observational analysis. It is useful to recall that the p value is determined by both sample size and the magnitude of an observed difference. Most clinical trials are powered to be large enough to detect a difference between a study drug and its comparator in producing a clinical outcome that is relatively common (e.g., reduction in blood pressure or LDL level). As a result, however, such studies are not likely to have adequate statistical power to find a “significant” difference between groups for outcomes that are much more rare (e.g., reduction in renal function). Adherence to a “p ⬍0.05” standard for uncommon adverse effects can lead to dismissal of important risks that a study may not have been powered to detect. The solution is not to embrace all differences in adverse effect rates regardless of their statistical properties. Instead, it is to consider such rate differences thoughtfully and to seek additional evidence to clarify worrisome relationships even if they are not “significant” in p value terms. For example, when the FDA was evaluating the risk of suicidal thoughts and actions in adolescents and children taking

SSRI antidepressants in placebo-controlled trials, the rates of these relatively rare outcomes were generally higher in the treated patients than in those randomized to placebo. Each individual study did not find a p ⬍0.05 level of significance for these differences. However, when the FDA aggregated the data from all such trials (in some cases, years after the studies were completed), it became clear that the risk across all studies was clear and consistent (and also met the conventional p ⬍0.05 level). The opposite problem arises when considering the statistical significance of data from large population-based epidemiological studies. Here, sample size (power) is not a limitation, especially when studies employ data on several hundred thousand patients through use of an automated claims database. A 4% or 5% difference in rates of a given effect (either therapeutic or adverse) may achieve a p value ⬍0.001, simply because of the huge size of the population studied. But here, even if the finding appears to have statistical significance, a difference of such small magnitude may have little or no clinical importance.

 ADVERSE DRUG EFFECTS AND THE HEALTH CARE SYSTEM The series of safety-related withdrawals of commonly used drugs in the 1990s and early 2000s led to renewed interest in developing ways to prevent such problems, or at least to limit the number of patients exposed to risk by identifying adverse effects earlier. As a result, the concept of “risk management” has become an important theme in drug development and regulation.

Balancing Benefits and Risks As noted above, new products are often not compared with existing alternatives when they are evaluated for approval, and such studies are not commonly performed after approval either. For drugs with known risks, it is therefore difficult to know whether an adverse effect occurs more commonly with a new drug than with another drug in the same class (e.g., gastrointestinal hemorrhage with NSAIDs, or rhabdomyolysis with statins). A higher rate of a given adverse event might be acceptable for a particular drug if it were accompanied by substantially greater efficacy. In this case, however, the absence of head-to-head clinical trials makes it difficult to make such an evaluation. Thus, in most instances, the individual clinician is left to make therapeutic decisions without the data needed to make such choices rigorously. A recent development designed to remedy this problem is the movement toward comparative effectiveness research—a program of publicly funded studies that systematically evaluates therapies against one another. It was initiated in 2009 with a $1.1 billion federal investment and is expected to be an important ongoing component of the research agendas of several federal agencies. The clinical use of medications is heavily influenced by the $30 billion spent annually by the pharmaceutical industry to market its products. This expenditure is heavily “front-end loaded,” with vast sums spent soon after a drug is launched in order to maximize sales for as many years as possible while the company’s patent is still in effect. Ironically, this means that the heaviest promotion of a drug occurs during the period in which there is least experience with its use and effects in

VIII Environmental Toxicology

A

O N N

O

O

C N

N

Fe2+

Fe2+

N

N

Oxyhemoglobin

N N

Carboxyhemoglobin

Hemoglobin oxygen saturation (%)

B 100

75

Normal O2 delivery

50

25

Decreased O2 delivery

0 0

20

40

60

80

100

120

Partial pressure oxygen (torr) Normal hemoglobin

50% carboxyhemoglobin

CHAPTER 52 / Environmental Toxicology 883

Cyanide 

The cyanide ion (C⬅N ) is a highly toxic and frequently lethal poison. It may be inhaled, ingested, or absorbed through the skin from sources as diverse as hydrogen cyanide gas, cyanide salts, apricot pits, peach pits, cherry pits, cassava, fire smoke, and vapors from industrial metal plating operations. Cyanide is also a metabolite of nitriles and nitroprusside. Cyanide binds to the ferric iron in the heme a3/CuB center of cytochrome c oxidase, thereby blocking aerobic respiration and preventing cellular use of oxygen. This causes a shift to anaerobic metabolism and a resulting metabolic acidosis. As with CO poisoning, cyanide poisoning damages tissues with high oxygen demand, such as the brain and heart. Signs and symptoms of cyanide poisoning depend on dose and route of exposure, and are somewhat nonspecific: headache, confusion, altered mental status, hypertension (early) or hypotension (late), nausea, and other symptoms are all possible. Pallor or cyanosis is not present (assuming no co-exposure to carbon monoxide). Unless cyanide exposure is reported or witnessed, or is likely to have occurred given the patient’s occupation or recent activities, diagnosis may be difficult. Sometimes, an odor of bitter almonds may be noted. Because cyanide is cleared rapidly from the blood, and because of technical challenges, measurements of cyanide in the blood may be both time-consuming and misleading. Moreover, some endogenous production of cyanide occurs in healthy individuals, and smokers’ blood contains elevated cyanide concentrations. There is some debate about the blood concentrations of cyanide deemed toxic or potentially lethal, but 1 mg/L (39 mol/L) is typically regarded as a potentially toxic level. Treatment for acute cyanide poisoning may include decontamination, supportive therapy, and administration of an antidote. Decontamination may entail simply the removal of contaminated clothes, and care should be taken to avoid inadvertent exposure of responders to the cyanide-containing material. Supportive therapy, including supplemental oxygen, should aim to avoid organ failure and may be needed to address toxicities such as coma, lactic acidosis, hypotension, and respiratory failure. The traditional antidotal treatment for acute cyanide poisoning in the United States is a cyanide antidote “kit” (CAK) that contains amyl nitrite, sodium nitrite, and sodium thiosulfate. The nitrites act by oxidizing hemoglobin to methemoglobin to provide a substrate that can compete with heme a3 in cytochrome c oxidase for cyanide molecules. Amyl nitrite is usually given by inhalation and acts (and is cleared from the bloodstream) rapidly, while sodium nitrite is administered intravenously and has a longer duration of action. The methemoglobin-bound cyanide is oxidized to the relatively nontoxic thiocyanate by the enzyme rhodanese (also known as transsulfurase) and excreted in urine. Sodium thiosulfate provides a ready source of sulfur for the detoxication reaction and enhances cyanide metabolism. Importantly, use of the CAK may pose significant risk to the patient, since a substantial fraction of hemoglobin must be converted to methemoglobin to compete effectively for the cyanide ion. Smoke inhalation victims (who have high levels of exposure to carbon monoxide) are sometimes presumed to have been poisoned by cyanide gas. Such patients may already be suffering from hypoxia before CAK treatment is begun. Exacerbation of hypoxia by forcing conversion of hemoglobin

to methemoglobin may be detrimental to such patients. CAK should also be avoided in pregnant women and infants, who may carry fetal hemoglobin and have immature methemoglobin reductase activity. Furthermore, the CAK may cause severe hypotension and lead to cardiovascular collapse. Concerns regarding possible terrorist use of cyanide led to the relatively recent (2006) approval by the U.S. Food and Drug Administration (FDA) of an alternative antidote, hydroxocobalamin (Cyanokit®). This member of the vitamin B12 family is an endogenous compound that was already in use at lower doses for treatment of vitamin B12 deficiency. The mechanism of action of this drug differs from that of the compounds in the CAK: the cobalt moiety in hydroxocobalamin has a strong affinity for cyanide and competes directly with the ferric iron in cytochrome c oxidase for cyanide, forming nontoxic cyanocobalamin that is excreted in the urine. Hydroxocobalamin is generally well tolerated, but anaphylactic reactions are possible. The compound also causes urine to have a bright red color for about a week and may discolor the skin at the site of injection. Interference with spectrophotometric tests and assays for oxyhemoglobin, carboxyhemoglobin, and methemoglobin may also occur.

Lead Lead is ubiquitous in the environment because of its persistence, its formerly widespread and unnecessary use as a gasoline additive, and its use in pigments, paints, plumbing, solder, and other products. Lead is toxic to the central nervous system, making exposure a particular concern for fetuses and children up to the age of about 7 years. Young children are also at risk because they are more likely than adults to ingest lead-contaminated paint dust and other nonfood materials. Despite a five-fold decrease in the exposure to lead in the United States and elsewhere since the mid-twentieth century, children today may still be at risk of developing lead-induced neurocognitive deficits. This is especially true for children who live near active, poorly controlled lead mines or smelters, or in countries where leaded fuels are, or recently were, used. (Leaded gasoline was not banned in China until 2000, for example.) Lead-glazed clay cookware and solder remain common in some areas, and some of this lead contaminates food and water. Exposures to lead that present no overt symptoms may nonetheless be toxic, and testing young children’s blood lead levels is essential. Although the half-life of lead in soft tissues is relatively short, its half-life in bone is more than 20 years; a substantial exposure in early childhood can result in elevated bone lead levels for decades. Lead causes a disruption of the blood–brain barrier, allowing both lead and other potential neurotoxins to reach the CNS. There, lead can block voltage-dependent calcium channels, interfere with neurotransmitter function, and, most importantly, interfere with cell–cell interactions in the brain; the latter effect causes permanent changes in neuronal circuitry. Overt lead encephalopathy, which is rare in the United States today, results in lethargy, vomiting, irritability, and dizziness, and can progress to altered mental status, coma, and death. Low- and moderate-level exposures to young children are believed to result in IQ deficits of two to four points for every 10 g/dL increase in blood lead concentration. Whether some blood lead levels are so low as to present essentially no risk of neurobehavioral deficit is a matter of debate and ongoing research.

884 Environmental Toxicology

Lead interferes with the synthesis of hemoglobin at multiple steps, causing a microcytic, hypochromic anemia. Specifically, lead inhibits the action of delta-aminolevulinic acid dehydratase (ALA-D), which catalyzes the synthesis of porphobilinogen, a heme precursor. Lead also inhibits the incorporation of iron into the porphyrin ring. In the kidney, lead causes both reversible and irreversible toxicity. Lead can interfere reversibly with energy production in proximal tubular cells by interfering with mitochondrial function, resulting in decreased energy-dependent reabsorption of ions, glucose, and amino acids. Chronic exposure to lead results in interstitial nephritis, with the eventual development of fibrosis and chronic kidney disease. When indicated clinically, body burdens of metals such as lead, mercury, or cadmium can be reduced using electron donors such as an amine, hydroxide, carboxylate, or mercaptan to form metal–ligand complexes. A chelator, which in Greek means “claw,” is a multidentate structure with multiple binding sites (Fig. 52-2). Binding the metal at multiple sites shifts the equilibrium constant in favor of metal ligation. High-affinity metal–ligand binding is critical because the chelator must compete with tissue macromolecules for binding. In addition, the chelator should be nontoxic and watersoluble, and the complex should be readily cleared. Finally, an ideal chelator should have a low binding affinity for endogenous ions such as calcium. To prevent the depletion of tissue calcium, many chelators are administered as their calcium complexes. The target metal is then exchanged for calcium, and the body’s calcium stores are not depleted. The most important heavy metal chelators are edetate disodium (the calcium, disodium complex of EDTA), which can be used to bind lead; dimercaprol (also known as British antiLewisite or BAL), which binds gold, arsenic, lead, and mercury to its two thiol groups; and succimer (2,3-dimercaptosuccinic acid), which has supplanted dimercaprol for the removal of lead, cadmium, mercury, and arsenic. Deferoxamine is used for the removal of toxic levels of iron, such as would occur in accidental overdoses of iron-containing supplements or in patients with transfusion-dependent anemias. Deferasirox is an orally bioavailable iron chelator that has recently been approved by the U.S. FDA; this agent may supplant deferoxamine for many conditions associated with chronic iron overload. Removal of copper, typically in patients with Wilson’s disease, is accomplished with penicillamine or, for patients who do not tolerate penicillamine, trientine.

A

B H2N

L M L

Na+O-

O-Na+

SH

Dimercaprol

OH

Ca2+

O

OH

O

O-

O-

O

O

Calcium disodium edetate (EDTA)

Mercury complex

NH2

N

N

Lead complex

SH

O

Penicillamine

Copper complex

O

OH H N

H2N

NH2 N H2

L L

C

HS

N Cu+2

N

N OH

O

2

O

Deferoxamine

Iron complex

FIGURE 52-2. Heavy metal chelators. A. A ligand (L) is a compound containing a Lewis base (such as amine, thiol, hydroxyl, or carboxylate groups) that can form a complex with a metal (M). B. A chelator is a multidentate ligand, that is, a ligand that can bind to a metal through multiple atoms, as in this example of a tetra-amino ligand bound to copper (Cu 2) via its four amine groups. C. The structures of dimercaprol, calcium EDTA, penicillamine, and deferoxamine are shown; the atoms that form bonds with the metal are identified in blue. Three-dimensional structures of the mercury complex of dimercaprol, the lead complex of EDTA, the copper complex of penicillamine, and the iron complex of deferoxamine are also shown. Here, the heavy metal is highlighted in red. For simplicity, hydrogen atoms are not shown.

Food Contaminants An estimated one in four Americans experience significant foodborne illnesses each year. The mechanisms of food poisoning involve either infection, which typically manifests 1 to several days after exposure, or intoxication from a preformed microbial or algal toxin, with symptoms occurring within a few hours of exposure. Infectious food poisoning is typically caused by species of Salmonella, Listeria, Cryptosporidium, or Campylobacter. Less common but quite virulent are poisonings by enteropathogenic Escherichia coli, which can cause sometimes fatal hemorrhagic colitis and hemolytic-uremic syndrome (HUS), likely through the uptake of pathologic bacterial proteins by host cells. Food intoxication is often caused by toxins elaborated by Staphylococcus aureus or Bacillus cereus, or by marine algal toxins ingested via seafood. S. aureus produces a variety of

toxins; the staphylococcal enterotoxins (SE) cause emesis by stimulating receptors in the abdominal viscera. Improper food handling after cooking, followed by poor refrigeration, contaminates high-protein foods such as meats, cold cuts, and egg and dairy products. B. cereus is a common contaminant of cooked rice. It produces several toxins that cause vomiting and diarrhea. Of particular concern is the production of cerulide, a small, cyclic peptide that stimulates intestinal 5-HT3 receptors, resulting in emesis. The peptide is heat-stable to 259°F for up to 90 minutes, so reheating of contaminated cooked rice will typically not prevent intoxication. Most algal toxins are neurotoxic and heat-stable, so, again, cooking leaves the toxins intact. Algal toxins such

CHAPTER 52 / Environmental Toxicology 885

as saxitoxins are a group of approximately 20 heterocyclic guanidine derivatives that bind with high affinity to the voltage-gated sodium channel, thus inhibiting neuronal activity and causing tingling and numbness, loss of motor control, drowsiness, incoherence, and, with sufficient doses (greater than about 1 mg), respiratory paralysis. Many foodborne illnesses appear to be caused by pathogens that are not yet characterized. Moreover, novel pathogens can emerge because of changing ecologies or technologies, or can arise via transfer of mobile virulence factors such as bacteriophages.

Toxic Plants and Fungi Acute illness can also be caused by mistaken ingestion of nonfood items, such as poisonous mushrooms collected by amateur mycologists or any number of poisonous plants. The highly toxic “death cap” mushroom, for instance, Amanita phalloides, produces numerous cyclopeptide toxins that are not destroyed by cooking or drying, have no distinctive taste, and are taken up by hepatocytes. The amatoxins bind strongly to RNA polymerase II, substantially slowing RNA and protein synthesis and leading to hepatocyte necrosis. The somewhat less toxic phallotoxins and virotoxins interfere with F- and G-actins in the cytoskeleton. Consumption of Amanita species or their relatives can thus cause severe liver dysfunction, even hepatic (and renal) failure and death. Initial symptoms of poisoning, such as abdominal pain, nausea, severe vomiting and diarrhea, fever, and tachycardia, may occur 6–24 hours after consumption of the mushrooms. Hepatic and renal function may deteriorate even while the initial symptoms abate, leading to jaundice, hepatic encephalopathy, and fulminant liver failure; death may occur 4–9 days after consumption. There is no specific antidote. An anticholinergic syndrome may be caused by deliberate or accidental ingestion of jimson weed, a plant belonging to the Datura family. All parts of the plant are toxic, but the seeds and leaves, in particular, contain atropine, scopolamine, and hyoscyamine. These compounds are rapidly absorbed and produce anticholinergic symptoms such as mydriasis, dry, flushed skin, agitation, tachycardia, hyperthermia, and hallucinations. The mnemonic for anticholinergic effects, “blind as a bat, dry as a bone, red as a beet, mad as a hatter, and hot as a hare,” is applicable to jimson weed poisoning. Some plants in the families Umbelliferae (such as parsley, parsnip, dill, celery, and giant hogweed), Rutaceae (such as limes and lemons), and Moraceae (such as figs) contain psoralen isomers (furocoumarins) in leaves, stems, or sap that can be absorbed into the skin after contact. Subsequent exposure to ultraviolet (UV) A radiation of wavelength 320 nm (generally via sunlight) can excite furocoumarins, resulting in epidermal tissue damage. Within 2 days, burning, redness, and blistering are observed in areas of contact with the plant and light; after healing, hyperpigmentation may persist for months. The response is greater with increasing plant contact, humidity, and duration and intensity of radiation exposure. This nonallergic phytophototoxic mechanism is the basis of psoralen  UV-A (PUVA) therapy for eczema and other dermatological disorders.

Acids and Bases Strong acids, alkalis (caustic agents), oxidants, and reducing agents damage tissue by altering the structure of proteins,

lipids, carbohydrates, and nucleic acids so severely that cellular integrity is lost. These substances, such as potassium hydroxide in drain cleaners and sulfuric acid in car batteries, produce chemical burns by hydrolyzing, oxidizing, or reducing biological macromolecules or by denaturing proteins. High concentrations of detergents can also cause nonspecific tissue damage by disrupting and dissolving the plasma membrane of cells. Although some of these agents may target particular macromolecules, direct tissue-damaging agents tend to be relatively nonspecific. Thus, the systems most commonly affected are those most exposed to the environment. Skin and eyes are frequently affected by splashes or spills. The respiratory system is affected when toxic gases or vapors are inhaled, and the digestive system is affected by accidental or deliberate ingestion of toxic substances. Many agents can cause damage to deep tissues after breaking through the barrier formed by the skin. Other agents are able to pass through the skin causing relatively little local damage, but destroy deeper tissues such as muscle or bone. For example, hydrofluoric acid (HF; found in, among other products, grout cleaner) causes milder skin burns than an equivalent amount of hydrochloric acid (HCl). However, once HF reaches deeper tissue, it destroys the calcified matrix of bone. In addition to the direct effects of the acid, the release of calcium stored in bone can cause life-threatening cardiac arrhythmias. For this reason, HF can be more dangerous than an equivalent amount of HCl. Three characteristics determine the extent of tissue damage: the compound’s identity, its concentration/strength, and its buffering capacity, or its ability to resist change in pH or redox potential. As mentioned above, HF is more injurious than an equivalent amount of HCl. In general, a stronger acid or base (measured by pH) or oxidant or reductant (measured by redox potential) will cause more damage than an equivalent compound at a more physiologic pH or redox potential. A solution of 102 M sodium hydroxide in water has a pH of 12 but has a low capacity to cause tissue damage, because it has a small buffering capacity and is rapidly neutralized by body tissue. In contrast, a buffered solution of pH 12, such as that found in wet ready-set concrete [made with buffered Ca(OH)2], can cause more serious alkali burns because tissues cannot readily neutralize the material’s extreme pH.

Pesticides Pesticides include insecticides, herbicides, rodenticides, and other compounds designed to kill unwanted organisms in the environment. By their nature, pesticides—of which there are hundreds (vastly more natural than synthetic)—are biologically active; however, the degree of their specificity toward target organisms varies, and many of these compounds cause toxicity in humans and other nontarget organisms. Some of the more common acute poisonings involve organophosphate and pyrethroid insecticides and rodenticides. Organophosphate insecticides, derived from phosphoric or thiophosphoric acid, include parathion, malathion, diazinon, fenthion, chlorpyrifos, and many other chemicals. These widely used compounds are acetylcholinesterase (AChE) inhibitors due to their ability to phosphorylate AChE at its esteratic active site (Fig. 52-3). Inhibition of AChE, and consequent accumulation of acetylcholine at cholinergic junctions in nerve tissue and effector organs, produces acute

886 Environmental Toxicology A O

O

R1

P R1

N H

R3

R2 Organophosphonate

R2

O

Carbamate

B O

O

O

P

P

P

N

O

O

Sarin

P

O

O

F

CN

F

O

Tabun

N S

Soman

VX

O

C NO2

S

S

O

P

P O

O

O

O S

O

O

O Malathion

Parathion

O

D H N

O

OR2

R1

N+

Acetylcholinesterase active site (serine)

P X

P

HO

O R1

O

O

OR2

N

Organophosphate

Organophosphate-bound pralidoxime 1

2

O

3

O

O

H N

P

H N

R1

O

O

OH

OR2

4

O

P O

N+

O OH

R1 After aging

HOR2

P O

OH N

OR2 R1

Organophosphate-bound acetylcholinesterase

Pralidoxime

FIGURE 52-3.

Structures and mechanisms of acetylcholinesterase inhibitors. A. Structures of typical acetylcholinesterase inhibitors, an organophosphonate on the left and a carbamate on the right. B. Structures of the principal nerve gases sarin, tabun, soman, and VX, which are potent inhibitors of human acetylcholinesterase. C. Structures of the organophosphate insecticides parathion and malathion. The thiophosphate bonds between sulfur and phosphorus are oxidized more efficiently by arthropod oxygenases than by mammalian oxygenases, so the compounds are less toxic to humans than the structurally related nerve gases. D. Organophosphates attack the serine active site in acetylcholinesterase, forming a stable phosphorus–oxygen bond (1). Pralidoxime abstracts the organophosphate from serine, restoring active acetylcholinesterase (2). Organophosphate-bound pralidoxime is unstable and spontaneously regenerates pralidoxime (3). Organophosphate-bound acetylcholinesterase can lose an alkoxy group, in a process called aging. The end product of aging is more stable and cannot be detoxified by pralidoxime (not shown).

muscarinic, nicotinic, and central nervous system (CNS) effects such as bronchoconstriction, increased bronchial secretions, salivation, lacrimation, sweating, nausea, vomiting, diarrhea, and miosis (muscarinic signs), as well as twitching, fasciculations, muscle weakness, cyanosis, and elevated blood pressure (nicotinic signs). CNS effects can include anxiety, restlessness, confusion, and headache. Symptoms usually occur within minutes or hours of exposure and resolve within a few days in nonlethal poisonings.

Toxic exposures may occur by inhalation, ingestion, or dermal contact, depending on the product formulation and manner of use or misuse. Toxic secondary exposures have occasionally occurred in people coming into close contact with the victim of direct exposure; for example, emergency responders and emergency department staff have suffered organophosphate toxicity after contacting—or simply being near—contaminated clothing, skin, secretions, or gastric contents.

888 Environmental Toxicology

and nonionizing), and occupational exposures to specific fibers, dusts, and chemicals. Carcinogenesis due to the toxic byproducts of oxygen and other endogenous or unavoidable causes (such as spontaneous errors in DNA replication and repair) also accounts for a presumably sizable share of cancers that arise in humans and all other animals. All aerobic organisms, including bacteria, have developed defenses against oxidative and other damage to DNA, and these defenses work to counter at least low-level exposures to endogenous and many exogenous mutagens and carcinogens. As shown in Figure 52-4, carcinogens vary widely in their modes of action. Many organic chemical carcinogens are not genotoxic per se, but only via one or more electrophilic metabolites that form addition-products—adducts— with one or more bases of DNA. These adducts can cause mutations, some of which lead ultimately to tumors. Interestingly, some such electrophiles have very short half-lives, and so are mutagenic only in the organ, such as the liver or kidney, in which they are formed; others are stable enough to migrate to other tissues and organs, increasing risks of cancers at these distal sites. Carcinogenic metals can be directly Carcinogen exposure Excretion

Metabolism

Genes Genotoxic mechanisms DNA adducts Chromosome breakage, fusion, deletion, mis-segregation, non-disjunction

Cell cycle DNA repair Differentiation Apoptosis

Genomic damage

Non-genotoxic mechanisms Inflammation Immunosuppression Reactive oxygen species Receptor activation Epigenetic silencing

Altered signal transduction Hypermutability Genomic instability Loss of proliferation control Resistance to apoptosis

Cancer

FIGURE 52-4.

Overview of genotoxic and nongenotoxic effects of carcinogens. When chemical carcinogens are internalized by cells, they are often metabolized, and the resulting metabolic products are either excreted or retained. Retained carcinogens or their metabolic products can directly or indirectly affect the regulation and expression of genes involved in cell-cycle control, DNA repair, cell differentiation, and apoptosis. Some carcinogens act by genotoxic mechanisms, such as forming DNA adducts or inducing chromosome breakage, fusion, deletion, mis-segregation, and nondisjunction. Others act by nongenotoxic mechanisms such as induction of inflammation, immunosuppression, formation of reactive oxygen species, activation of receptors such as aryl hydrocarbon receptor (AhR) or estrogen receptor (ER), and epigenetic silencing. Together, these genotoxic and nongenotoxic mechanisms can alter signal transduction pathways, thus leading to hypermutability, genomic instability, loss of proliferation control, and resistance to apoptosis—some of the characteristic features of cancer cells.

toxic, or toxic via metabolism such as methylation, and can alter chromosomal structure via hypermethylation of DNA and deacetylation of histone. Carcinogenic viruses and the carcinogenic bacterium, Helicobacter pylori, may act via many mechanisms, including induction of inflammation, itself a risk factor for cancer. Worldwide, chronic infections contribute to an estimated 15% of all cancers. Carcinogenesis occurs via progressive stages broadly characterized as tumor initiation, promotion, and progression (Fig. 52-5). The sequence involves multiple rounds of stochastic mutations and selection, notably in proto-oncogenes and tumor suppressor genes. Infrequent mutations in other genes and cancer pathways are also involved, and determining which mutations are “cancer-drivers” and which are mere passengers is a subject of active research. The evolution from a normal cell to a clinically apparent tumor typically occurs over decades, so that cancer risk increases with age for the majority of cancers. Cigarette smokers, for example, develop lung cancer on average 30 years after first exposures. This explains why people who successfully quit smoking (a notoriously difficult task) reduce but do not eliminate their increased cancer risks relative to lifelong nonsmokers. Cancer deaths in dogs, cats, and laboratory rodents also occur largely in old age—and occur despite the animals’ lack of deliberate exposures to exogenous chemical carcinogens. Exceptions to long latencies include cancers of childhood and acute myelogenous leukemias that develop secondary to treatment of another cancer with alkylating agents: such leukemias may arise in as little as 2–5 years after therapy.

Tobacco It is difficult to overstate the toxicity of tobacco. Worldwide, tobacco kills 5 million people annually. Cigarette smoke is the most significant cause of cancer known: 30% of cancer deaths in developed countries are caused by cigarettes; and the burden of deaths due to cigarettes in developing nations is expected to rise in proportion to increasing prevalence of cigarette use. Smoking also causes nonmalignant pulmonary disease (such as COPD) and increases smokers’ risks of cardiovascular disease and death such that about half of all people addicted to tobacco die of tobacco-related diseases. The carcinogenicity of cigarette smoke is likely due to the combined actions of at least 60 carcinogens and countless free radicals. Among the former are two “tobacco-specific” (that is, derived from nicotine) nitrosamines, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN). Other carcinogenic components of cigarette smoke include polycyclic aromatic hydrocarbons (PAHs), aromatic amines, benzene, aldehydes and other volatile organic compounds, and various metals. Benzo(a)pyrene (Fig. 52-6) is among the carcinogenic PAHs in tobacco smoke, and is also believed to account, in part, for the carcinogenicity of soots and coal tars. The important carcinogens and other toxins in tobacco smoke appear to be both in the solid “tar” phase of the smoke and in the gases and vapors. Thus, “low tar” cigarettes are apparently no less potent as carcinogens or causes of cardiovascular disease than are “regular” cigarettes. Smokeless tobacco, which is variously “dipped,” used as snuff, or chewed (alone, or with betel quid or other substances), contains significant concentrations of carcinogenic nitrosamines (and nicotine), and causes oral cancer as well

CHAPTER 52 / Environmental Toxicology 889

A Tumor initiation BP

Men+ (e.g. Ni2+)

O

OH

Benzo[a]pyrene

OH OH

Benzo[a]pyrene-4,5-epoxide

NER

Decoy

Mutations in cancersusceptibility genes Glutathione S

Alterations in p53 or RAS

OH

B Tumor promotion CYP1A1, CYP1B1, CYP1A2

TCDD AhR

Glucuronate O OH

O

Benzo[a]pyrene-7,8-epoxide

Conjugated products (noncarcinogenic)

ARNT

AhR ARNT XRE O

Extracellular signalling PAI1

HO

Proliferation

Apoptosis

Cell cycle

GEF

TNF

Cyclin B2

MT-II

COT

HSP40

NEK2

HEF1

KRAS2

FIGURE 52-5.

Tumor initiation and tumor promotion. Genotoxic carcinogens can induce damage in tumor suppressor genes or oncogenes in various ways, some of which contribute to the transformation of normal cells into tumor cells: this is known as tumor initiation. Some chemical carcinogens can also promote the outgrowth of transformed cell clones: this is termed tumor promotion. A. Tumor initiation typically occurs via mutations. For example, the benzo(a)pyrene (BP)–DNA adduct can cause mutations in cancer-susceptibility genes such as p53 or RAS. The potency of such adducts can be increased due to inhibition of nucleotide excision repair (NER) by metals such as nickel (Ni2) or as a result of NER factor immobilization at repair-resistant DNAadduct sites, also known as decoy adducts. B. Chemical compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can serve as tumor promoters through aryl hydrocarbon receptor (AhR)-mediated signal transduction. Binding of TCDD to AhR leads to activation and translocation of the complex into the nucleus. After heterodimerization with the AhR nuclear translocator (ARNT), the complex binds to xenobiotic-responsive elements (XREs) and induces the expression of a variety of different genes involved in carcinogen metabolism, including cytochrome P450 (CYP) isoforms 1A1, 1B1, and 1A2. It also changes the expression pattern of factors involved in cellular growth and differentiation, such as plasminogen-activator inhibitor type 1 (PAI1), metallothionein II (MT-II), human enhancer of filamentation 1 (HEF1), guanine nucleotide exchange factor (GEF), COT (a serine/threonine protein kinase), and K-RAS (KRAS2). Pro-apoptosis factors such as tumor necrosis factor (TNF) and heat shock protein 40 (HSP40) are down-regulated, and cellcycle genes can either be up-regulated (such as cyclin B2) or down-regulated (such as NEK2, another serine/threonine protein kinase).

OH

Benzo[a]pyrene-7,8-diol9,10-epoxide (carcinogen)

FIGURE 52-6. Metabolism of benzo[a]pyrene. Benzo[a]pyrene is metabolized into several products (not all shown). Epoxidation at carbons 4 and 5, followed by conjugation with glutathione or glucuronate, leads to nontoxic derivatives that are readily excreted. In contrast, oxidation at the “bay region” generates the proximate carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide, which goes on to form a repair-resistant adduct with guanine. Subsequent DNA replication, in the presence of this “bulky” polycyclic aromatic adduct, leads to G to T base pair transversions, including in the cancer genes p53 and RAS.

as gum disease. The fraction of oral cancer that is attributable to smokeless tobacco in any given population depends on the prevalence of the habit, the potency of the local tobacco products (such as tobacco in betel quid and tobacco-areca nut mixtures), and competing causes of oral cancer. Thus, in the United States, smokeless tobacco accounts for 7% of oral cancer cases, while in India, more than 50% of oral cancers, in both men and women, are attributable to smokeless tobacco.

Ethanol Excessive consumption of ethyl alcohol is a common and complex problem. Binge drinking occurs in a sizable minority of adolescents and young adults, at least in some cultures. In adults with coronary artery disease, binge drinking can cause myocardial ischemia and angina. Acutely, alcohol is a sedative and causes psychomotor retardation. A sizable fraction of morbidity and mortality from alcohol intoxication results from injuries suffered (and inflicted) while impaired.

890 Environmental Toxicology

Chronic excess drinking results in fatty liver disease in almost 100% of such consumers. Some 30% of those afflicted go on to develop fibrosis; 10–20% progress to hepatic cirrhosis; and a significant fraction of those with cirrhosis develop and die of liver cancer (hepatocellular carcinoma). As expected, risk of liver disease increases with increasing dose-rates of alcohol: on average, chronic drinkers increase their risk of hepatocellular carcinoma by two-fold; this risk increases to six-fold for people drinking five drinks or more per day. Ethanol metabolism produces several reactive species, including acetaldehyde, hydroxyl radicals, superoxide anions, and hydrogen peroxide. Acetaldehyde in particular is genotoxic and believed to be the proximate carcinogen in some cases of alcohol-associated cancer. Chronic excess drinking up-regulates the production of CYP2E1, which generates not only acetaldehyde from ethanol but also transforms various nitrosamines and polycyclic aromatic hydrocarbons into their proximate genotoxic metabolites. Although acetaldehyde can be further metabolized to nontoxic acetate, the requisite enzymes, aldehyde dehydrogenases, may be inherently impaired or inactive in certain groups of people (notably East Asians), leading to substantially increased cancer risks from alcoholic beverages. Ethanol is a teratogen as well; it causes fetal alcohol syndrome (FAS), which is characterized by retardation of craniofacial growth, both pre- and postnatally, and neurocognitive impairment. General physical growth is also retarded in children with FAS, affecting boys more severely than girls. Children with FAS present with a range of disabilities, presumably due to both the amount and the timing of maternal alcohol ingestion. With regard to the latter, women may drink immoderately in the early stages of pregnancies of which they are as yet unaware. Thus, the simplest prevention strategy (elimination of exposure) may not always be practical. FAS can be generated in laboratory rats and mice, and mechanistic studies using animal models have led to several working hypotheses. The facial abnormalities of FAS are believed to be due to apoptosis of neural crest cells during gastrulation or neurulation. Embryonic exposure to alcohol can result in reduced production of retinoic acid, and retinoic acid is essential for normal morphogenesis. Other postulated mechanisms involve ethanol-induced free-radical formation, altered gene expression, disruption of cell membrane lipid bilayers, and interference with the activity of growth factors. Excess drinking also increases the risks of pancreatitis, hemorrhagic stroke, and heart failure. The pathophysiology of alcoholic cardiomyopathy is complex and appears to involve cell death and pathologic changes in myocyte function. Light-to-moderate ingestion of alcohol, on the other hand, appears to protect against cardiovascular disease. Red wine is believed by some to be particularly protective, perhaps because it contains not only ethanol but also resveratrol and other polyphenols; various cardioprotective mechanisms have been hypothesized, including improvements in endothelial function and effects on hemostasis. However, to the extent that evidence of the benefits of moderate drinking derives from observational rather than experimental studies, one must be mindful of the possibility that moderate drinkers are, by genetics or other habits or factors, less susceptible to cardiovascular disease in general, so that the association with moderate drinking and health protection is confounded rather than causal. If so, then encouraging nondrinkers to start may do them no good.

Aflatoxins In 1960, a mysterious disease killed more than 100,000 farmed turkeys in England; other birds and farm animals also succumbed. The deaths were linked to specific batches of peanut meal, and, eventually, to secondary metabolites of the mold Aspergillus flavus: these compounds are termed aflatoxins. The most important form, aflatoxin B1 (AFB1, where “B” denotes blue fluorescence under ultraviolet light), causes both acute liver toxicity and liver cancer (hepatocellular carcinoma) in myriad mammalian and other species. The proximate metabolite is an unstable, exocyclic epoxide that reacts with DNA, forming a potent mutagenic adduct on the N-7 position of guanine. Minuscule concentrations of AFB1 in the feed of laboratory rodents induce liver tumors. Co-administration of drugs that induce glutathione S-transferases, such as the antihelminthic oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol3-thione], renders rodents resistant to AFB1-tumorigenesis. Trials to determine whether such chemoprevention works in humans are ongoing. As suggested above, various aflatoxin metabolites are nontoxic, including the glutathione conjugate and a hydrolysis product that binds to lysine residues on proteins such as serum albumin. Aflatoxin adducts and other biomarkers in blood and urine reflect a person’s exposures to dietary aflatoxins over the prior 2–3 months and, in tropical regions where aflatoxin contamination is endemic and rural diets contain few foods, over many years. Epidemiological studies using these markers in populations in Africa and China have demonstrated that aflatoxin causes hepatocellular carcinoma, both directly and acting synergistically with liver damage due to hepatitis B virus (HBV). Both vaccination against HBV and reduction in aflatoxin exposure reduce the risk of liver cancer, which worldwide accounts for approximately 500,000 deaths annually.

Arsenic In several regions of the world, such as parts of Bangladesh, Taiwan, West Bengal, Chile, Argentina, and the United States, the groundwater contains naturally high concentrations of inorganic arsenic (up to thousands of micrograms per liter, g/L). Some of this water is tapped by underground wells and, inadequately treated, serves as drinking water. Safer water supplies may sometimes be found and utilized, but various constraints have resulted in hundreds of thousands of people developing chronic arsenic poisoning—arsenicosis— and millions being at risk of arsenic-induced cancers. Arsenicosis is characterized by skin lesions, peripheral vascular disease, cerebrovascular disease, cardiovascular disease, and other chronic conditions. Arsenic-induced skin lesions and peripheral vascular diseases are well recognized and include abnormal pigmentation, keratoses, “blackfoot” disease, and Raynaud’s syndrome of fingers and toes. Both hyper- and hypopigmentation may result, typically on the soles of the feet, the palms, and the torso. Hyperkeratosis also occurs on the soles and palms. Epidemiologic study suggests that these skin lesions may develop at lower arsenic concentrations (tens of g/L) than do other arsenicinduced toxicities. Blackfoot disease used to be endemic in southwestern Taiwan, where arsenic occurred at high concentration in artesian wells, and reached its highest incidence in the late 1950s before the introduction of tap water from safer sources. The disease has a typical progression:

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the first signs are preclinical peripheral vascular disease, followed by progressive discoloration of the skin from the toes toward the ankles. Feelings of numbness or coldness develop in the legs, followed by intermittent claudication, and eventually gangrene, ulceration, and surgical or spontaneous amputation. It is not clear why blackfoot disease is not seen in other regions with high, chronic oral exposures to arsenic. Epidemiologic studies have indicated associations between exposure to high concentrations of arsenic (hundreds of g/L) and various cardiovascular diseases such as hypertension and ischemic heart disease, and between urinary levels of arsenic and levels of circulating markers of inflammation and endothelial damage, such as soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule-1 (sVCAM-1), both of which correlate with cardiovascular disease risk. In addition, recent studies of ApoE-knockout mice (which are vulnerable to development of atherosclerosis) exposed to inorganic arsenic support an association between this environmental contaminant and cardiovascular disease. Mechanisms remain to be elucidated, however, as does the degree of cardiovascular risk posed by drinking water containing lesser concentrations of arsenic. Inorganic arsenic is a recognized human carcinogen that is causally associated with cancers of the skin, bladder, and lung. Associations with other cancers (e.g., liver, prostate) are less certain. The links to cancer were established in communities with extensive exposure to arsenic in drinking water, particularly in Taiwan and Chile, with clear dose–response patterns. Skin cancers may, but do not always, arise at sites of nonmalignant keratotic lesions and tend to be nonmelanomas. Interestingly, no adequate animal models have been identified for arsenic-induced cancers. However, experimental models have been used (and human cohorts have been observed) to shed light on the potential carcinogenic mechanisms: arsenic has indirect genotoxicity, effects on cell-cycle control, and the ability to cause oxidative damage, and it interferes with DNA repair or methylation. Other factors, such as nutritional status, genetic polymorphisms, and co-exposure to other toxins, may also influence the risk of arsenic-induced cancer.

Workplace Exposures Various occupational exposures increase workers’ risks of developing cancer and other diseases. As a general matter, exposure levels in industry are much higher than those in the general environment. To the extent that important and harmful occupational exposures have been characterized and minimized, the toll of occupational carcinogenesis has decreased. Needless to say, enforcement or even presence of occupational exposure limits is not guaranteed, and groups of workers in certain industries or nations continue to be at excess risk of developing one or more forms of cancer. An eighteenth-century English surgeon, Percivall Pott, was among the first to recognize occupational carcinogenesis, deducing that “lodgment of soot in the rugae of the scrotum” caused scrotal cancer in young men employed as chimney sweeps (who typically worked naked, to avoid soiling their clothes). In the nineteenth and twentieth centuries, industrial workers overexposed (1) to benzene were found to develop bone marrow disease, including aplastic anemia and acute myelogenous leukemia; (2) to 2-naphthylamine in dye-

making were at high risk for bladder cancer; (3) to various metals were susceptible to lung cancer; and (4) to asbestos developed lung cancer and mesothelioma. Other occupational carcinogens (including specific chemicals, industries, and industrial processes) were also identified. Asbestos, Silica, Dusts, and Metals Numerous cases of occupational lung injury are (or were) caused by inhalation of fibers or dusts, such as asbestos, crystalline silica, talc, and coal dust, and various metals. Asbestos is carcinogenic to the lung and mesothelium after long-term exposure to respirable fibers of specific dimensions. The formerly widespread use of asbestos-containing products in shipbuilding, construction, textiles, and other industries caused perhaps 200,000 cancer deaths in industrialized countries; because of latency, such deaths continue to occur. Current occupational exposures to asbestos are problematic in parts of India and elsewhere in Asia. Asbestos and cigarette smoking act synergistically, such that the risk of lung cancer (although, interestingly, not of mesothelioma, which is not caused by smoking) due to co-exposure is much larger than the risk from either factor alone. The toxic and carcinogenic potency of asbestos fibers and fiber-types varies with their dimensions, surface chemistry, and biopersistence. The mechanisms by which asbestos fibers damage the lung or pleura involve production of reactive oxygen and nitrogen species by macrophages attempting to destroy the fibers. Asbestos also causes severe nonmalignant respiratory disease, asbestosis, characterized by fibrotic lesions in the lung parenchyma that limit gas exchange. Black lung, or coal worker’s pneumoconiosis (CWP), is another nonmalignant (but potentially fatal) fibrotic lung disease induced by excessive exposure to coal dust. The simple form of CWP may not markedly limit respiration and may affect only small areas of the lung, whereas progressive CWP can develop and worsen even in the absence of continued exposure, leading to severe emphysema. Interestingly, coal dust does not appear to increase the risk of lung cancer. Although worker exposures to coal dust have been limited by U.S. regulations in recent decades and underground mining is less common than in the past, thousands of coal miners in other countries, especially China, are at risk for CWP and related illnesses. Occupational exposures to metals such as arsenic, cadmium, chromium (VI), and nickel increase workers’ risks of cancers of the lung and, in some cases, nasal cavity and paranasal sinuses. A large number of mechanisms have been identified, both genetic and epigenetic. Overexposures to certain metals can also cause nonmalignant disease. Chronic exposure to cadmium, for example, causes kidney disease. Abnormal renal function, characterized by proteinuria and decreased glomerular filtration rate (GFR), was first reported in cadmium workers in 1950 and has been confirmed in numerous investigations. The proteinuria consists of low-molecular-weight proteins such as 2-microglobulin, retinol binding protein, lysozyme, and immunoglobulin light chains; these proteins are normally filtered in the glomerulus and reabsorbed in the proximal tubule. Cadmium-exposed workers also have a higher rate of kidney stone formation, perhaps due to disruption of calcium metabolism as a consequence of renal damage. Renal tubular dysfunction appears only after a threshold concentration of cadmium is reached in the renal cortex. The threshold varies among

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individuals but has been estimated to be approximately 200 g/g wet weight. Several studies of the prevalence of proteinuria in worker populations suggest that inhalation exposure in excess of about 0.03 mg/m3 for 30 years is associated with increased risk of tubular dysfunction. Unfortunately, removal from exposure does not necessarily halt disease in workers with cadmium-induced kidney damage, and progressive decreases in GFR and end-stage renal disease may occur. Progression of disease may depend on both the body burden of cadmium and the severity of proteinuria at last exposure. Unless renal damage is significant, urinary cadmium concentration reflects the body burden of the metal. Although renal damage is clearly due to accumulation of cadmium in the kidney, the molecular mechanism of this damage is unclear. Metallothionein may be involved; this cadmium-binding protein, which is synthesized in the liver and kidney, appears both to facilitate transport of cadmium to the kidney and to promote retention of cadmium in the kidney. Chlorinated Hydrocarbons Low-molecular-weight chlorinated hydrocarbons are widely used in industrial and other settings. Vinyl chloride, for example, is a gas used to make the plastic polyvinylchloride (PVC). Vinyl chloride gas is neither irritating nor acutely toxic (except at extremely high, narcotizing concentrations), and PVC workers were initially exposed to quite high concentrations. In the 1970s, exposures to vinyl chloride were found to cause angiosarcoma, a rare form of liver cancer, in both laboratory rats and workers; strict workplace exposure limits have since been imposed in most settings. Carcinogenesis is due to the epoxide metabolite of vinyl chloride. Some 98% of the DNA adducts formed from vinyl chloride epoxide are benign, but the other 2% are highly mutagenic etheno-adducts with guanine and cytosine. Interestingly, these adducts are the same as those formed from everyday oxidative stress and lipid peroxidation. These etheno-adducts are normally eliminated by base excision repair, but at sufficiently high rates of DNA damage, repair fails to be 100% effective. Thus, high-level exposures to vinyl chloride and similar genotoxins are demonstrably carcinogenic, while low-level exposures may not be. For example, laboratory rats exposed to low doses of vinyl chloride develop precancerous changes (altered hepatic foci) at rates indistinguishable from those seen in unexposed laboratory controls. Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene) are solvents used for degreasing and dry cleaning. All of us are exposed to trace concentrations of TCE and perchloroethylene in ambient air. Exposures to high concentrations of TCE cause kidney tumors, but moderate and low-level exposures apparently do not. This is because at low doses, trichloroethylene is converted to nontoxic metabolites that are readily eliminated, whereas at high doses, the detoxification pathway becomes overwhelmed and a second pathway becomes operative. The latter pathway forms a nephrotoxic metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and the subsequent kidney damage appears to be a necessary precursor to TCE-induced kidney tumors. Nontoxic exposures to trichloroethylene up-regulate genes associated with stress, DCVC metabolism, cell proliferation and repair, and apoptosis, affording protection against renal tubular cell damage. Perchloroethylene does not appear to cause tumors in people, probably because virtually all of it is eliminated without metabolic activation.

Air Pollution Toxicity due to ambient air pollution depends on both the types and concentrations of pollutants. As with other environmental exposures, air pollution takes much of its toll in regions lacking adequate environmental protection or resources. Combustion of fuels is an important source of air pollution; in most cities and suburbs, exhaust from gasolineand diesel-powered vehicles is the largest source of pollutants. New and recently manufactured motor vehicles burn much more cleanly than vehicles made prior to the 1970s, but the number of vehicles in use continues to grow, and tailpipe emissions are, of course, close to ground level, limiting dilution into cleaner air. Combustion of low-quality fuels indoors is not uncommon in some settings. For example, soft coal, charcoal, or dried cow dung are burned for cooking and heating in poorly ventilated homes in China, Nepal, Mexico, and elsewhere. Measurements indicate indoor pollutant levels that exceed outdoor concentrations by two orders of magnitude. As a result of these residential exposures, women and children in particular are at risk of developing chronic bronchitis, dyspnea, and, eventually, interstitial lung disease. Moreover, the carcinogenic potency of soft coal smoke is 1,000 times greater than cigarette smoke (in a mouse skin-tumor assay). Women in China who use soft coal indoors have extraordinarily high body burdens of benzo(a)pyrene-adducted guanine, and their rates of death due to lung cancer are eight times higher than the national average for women. Combustion generates thousands of chemicals, some of which depend on the material burned, and others of which are inherent to combustion. These include carbon monoxide, organic irritants such as formaldehyde and acrolein, nitrogen oxides, sulfur dioxide, ammonia, hydrogen cyanide, and hydrogen fluoride, among other potentially toxic substances. Semi-volatile and nonvolatile chemicals also form in abundance, and adsorb to the particle phase of smoke. Metals present in the combusted material are, of course, not destroyed by combustion, and so may add to the acute and chronic toxicity of inhaled smoke. Under certain meteorological and chemical conditions, polluted air may become unusually acidic, and inhalation of acidic aerosols can induce bronchoconstriction and reduce the efficacy of mucociliary clearance. The action of ultraviolet radiation (sunlight) on reactive hydrocarbons and nitrogen oxides results in the formation of smog, containing significant concentrations of oxidizing chemicals such as ozone, peroxides, and peroxyacetyl nitrate. Acute and subchronic exposures to toxic levels of such oxidants can cause inflammation and irritation, sloughing of epithelium, and loss of cilia. Chronic overexposures can result in fibrosis or chronic obstructive pulmonary disease, perhaps via altered metabolism of collagen and elastin. The pulmonary effects of air pollutants depend in part on their water solubility. Sulfur dioxide, for example, dissolves readily in the mucous membranes of the upper airways and so does not typically reach the lung. Dissolution of the gas is not instantaneous, however, so that exercising or otherwise hyperventilating permits some of the inhaled sulfur dioxide to reach the lower respiratory tract where, at sufficient concentrations, it can induce bronchoconstriction. Asthmatics are particularly sensitive to this effect.

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 CONCLUSION AND FUTURE DIRECTIONS Much of the treatment for toxic exposures focuses on the acutely poisoned patient. Much of the morbidity associated with environmental factors, however, is caused by chronic exposures and may be clinically apparent only years or decades after the initial insult. In fact, there is generally no specific treatment for injury caused by chronic toxic exposures, and treatment modalities for cancers are independent of the underlying causes. In theory, cancers and other chronic diseases caused by habits such as tobacco smoking and excessive drinking are entirely preventable, and although progress has been made in this regard, more remains to be done, and complete eradication of these threats seems unrealistic. Occupational exposures are well controlled in most of the developed world but remain problematic in industrializing nations. Epidemiologic evidence indicates that some specific foods—such as Chinese-style salted fish (containing high concentrations of the carcinogen dimethylnitrosamine) and foods contaminated with aflatoxins—increase people’s risk of cancer, and that consumption of fruits and vegetables in general decreases risks of cancer, but the specific dietary components or characteristics that modify risk remain areas of active research. Obesity (and perhaps sedentary lifestyle) is an increasingly important risk factor for cancer, presumably in combination with environmental exposures or other factors. Environmental exposures typically involve complex mixtures of only partially characterized chemicals and characteristics. Traditional toxicological testing of individual chemicals or simple mixtures may yield results of incomplete or uncertain relevance. Additional information may be generated via microarray technologies and other tools of genomics, proteomics, and metabolomics applied to toxicological inquiry. Our “microbiomes”—that is, the microorganisms within us that, in toto, outnumber our own cells ten to one—presumably affect our responses to environmental exposures in many ways yet to be elucidated. More broadly, basic, mechanistic, and applied research is expected to continue to unravel the interconnections between and among genetic, environmental, and random factors involved in disease causation, with the hope that safer environments will lead to healthier lives.

Suggested Reading Busl KM, Greer DM. Hypoxic-ischemic brain injury: pathophysiology, neuropathology and mechanisms. NeuroRehabilitation 2010;26:5–13. (Reviews the pathophysiologic and molecular basis of hypoxic and cytotoxic brain injury.) Hall AH, Saiers J, Baud F. Which cyanide antidote? Crit Rev Toxicol 2009;39:541–552. (Reviews mechanisms, clinical efficacy, safety and tolerability, and supporting toxicology for antidotes to cyanide poisoning in use in the United States and elsewhere.)

Hecht SS. Progress and challenges in selected areas of tobacco carcinogenesis. Chem Res Toxicol 2008;21:160–171. (Review by a major researcher in the field.) International Agency for Cancer Research (IARC). Continuing series of monographs. Available at http://monographs.iarc.fr/. (As part of ongoing efforts since 1971, IARC convenes panels of experts charged with evaluating published evidence relevant to the determination of the established, probable, or possible carcinogenic effects of various chemical, biological, and physical agents and exposures. To date, some 107 substances and exposures have been characterized by IARC as carcinogenic to humans.) Klaassen CD, ed. Casarett & Doull’s toxicology: the basic science of poisons. 7th ed. New York: McGraw-Hill; 2007. (A comprehensive textbook of toxicology, this resource provides a solid foundation for the understanding of toxicology. It includes sections on general principles, toxicokinetics, nonspecific toxicity, organ-specific toxicity, toxic agents, environmental toxicology, and applications of toxicology, including a chapter on clinical toxicology.) Lang CH, Frost RA, Summer AD, et al. Molecular mechanisms responsible for alcohol-induced myopathy in skeletal muscle and heart. Int J Biochem Cell Biol 2005;37:2180–2195. (Reviews cellular and molecular mechanisms by which alcohol impairs skeletal and cardiac muscle function, with special emphasis on alterations in signaling pathways that regulate protein synthesis.) Luch A. Nature and nurture—lessons from chemical carcinogenesis. Nat Rev Cancer 2005;5:113–125. (Reviews mechanisms of chemical carcinogenesis.) Sant’Anna LB, Tosello DO. Fetal alcohol syndrome and developing craniofacial and dental structures—a review. Orthod Craniofacial Res 2006;9:172–185. (Reviews clinical and experimental studies, discusses treatment strategies, and suggests avenues for research.) Schumacher-Wolz U, Dieter HH, Klein D, Schneider K. Oral exposure to inorganic arsenic: evaluation of its carcinogenic and non-carcinogenic effects. Crit Rev Toxicol 2009;39:271–298. (Emphasizes findings with respect to risk of disease following relatively low exposures to arsenic.) Seitz HK, Stickel F. Risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress. Biol Chem 2006;387:349–360. (Review by major researchers in the field.) States JC, Srivastava S, Chen Y, Barchowsky A. Arsenic and cardiovascular disease. Toxicol Sci 2009;107:312–323. (Reviews epidemiologic and experimental data.) Tauxe RV. Emerging foodborne pathogens. Int J Food Microbiol 2002;78:31–41. (Overview of common sources of food poisoning.) Tseng C-H. Blackfoot disease and arsenic: a never-ending story. J Environ Sci Health 2005;23:55–74. (Review by a principal researcher in the field.) Toxnet. Available at http://toxnet.nlm.nih.gov/. (This government resource, sponsored by the National Library of Medicine, contains a vast database of both toxic substances and articles in the field of toxicology.) Tzipori S, Sheoran A, Akiyoshi D, et al. Antibody therapy in the management of Shiga toxin-induced hemolytic uremic syndrome. Clin Microbiol Rev 2004;17:926–941. (Reviews the structure and mechanism of action of Shiga toxins, produced by E. coli O157:H7 and other enteropathic bacteria, the manifestations and treatment of hemolytic-uremic syndrome, and the potential utility of antibody therapy.) Weaver LK, Hopkins RO, Chan KJ, et al. Hyperbaric oxygen for acute carbon monoxide poisoning. N Engl J Med 2002;347:1057–1067. (Although hyperbaric oxygen had been postulated to help treat carbon monoxide poisoning and has been used since 1960, this study established its clinical efficacy in reducing cognitive deficits at 6 weeks and 12 months.) Wogan GN, Hecht SS, Felton JS, et al. Environmental and chemical carcinogenesis. Semin Cancer Biol 2004;14:473–486. (Review by major researchers in the field.)

IX Frontiers in Pharmacology

53 Protein Therapeutics Quentin J. Baca, Benjamin Leader, and David E. Golan

INTRODUCTION & CASE . . . . . . . . . . . . . . . . . . . . . . . . 895-896 USES OF PROTEINS IN MEDICINE . . . . . . . . . . . . . . . . . . . . 897 Group I: Enzymes and Regulatory Proteins . . . . . . . . . . . . 897 Group II: Targeted Proteins . . . . . . . . . . . . . . . . . . . . . . . . 904 Group III: Protein Vaccines . . . . . . . . . . . . . . . . . . . . . . . . 911 Group IV: Protein Diagnostics . . . . . . . . . . . . . . . . . . . . . . 912

CHALLENGES FOR PROTEIN THERAPEUTICS . . . . . . . . . . . 912 CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . 915 Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

 INTRODUCTION

approval time of protein therapeutics may be faster than that of small-molecule drugs. A study published in 2003 showed that the average clinical development and approval time was more than 1 year faster for 33 protein therapeutics approved between 1980 and 2002 than for 294 small-molecule drugs approved during the same time period. Last, because proteins are unique in form and function, companies are able to obtain far-reaching patent protection for protein therapeutics. The last two advantages make proteins attractive from a financial perspective compared with small-molecule drugs. A relatively small number of protein therapeutics are purified from their native source, such as pancreatic enzymes from hog and pig pancreas and ␣-1-proteinase inhibitor from pooled human plasma. Instead, most therapeutic proteins are now produced by recombinant DNA technology and purified from a wide range of organisms. Production systems for recombinant proteins include bacteria, yeast, insect cells, mammalian cells, and transgenic animals and plants. The system of choice can be dictated by the cost of production or the modifications of the protein (for example, glycosylation, phosphorylation or proteolytic cleavage) that are required for biological activity. For example, bacteria do not perform glycosylation reactions, and each of the other biological systems listed above produces a different type or pattern of glycosylation. Protein glycosylation patterns can have a dramatic effect on the activity, half-life and immunogenicity of the recombinant protein in the body. For example, the half-life of native erythropoietin, a growth factor important in erythrocyte production, can be lengthened by increasing the glycosylation of the protein. Darbepoetin-␣ is an erythropoietin analogue that is engineered to contain two additional amino acids that are substrates for N-linked glycosylation reactions. When expressed in Chinese hamster ovary cells, the analogue is synthesized with five rather than three N-linked carbohydrate chains; this modification causes the half-life of darbepoetin to be three times longer than that of erythropoietin.

Proteins have the most dynamic and diverse role of any macromolecule in the body, catalyzing biochemical reactions, constituting receptors and channels in membranes, providing intracellular and extracellular scaffolding support, and transporting molecules within a cell or from one organ to another. It is currently estimated that there are approximately 25,000 protein-coding genes in the human genome, and with alternative splicing of genes and post-translational modification of proteins (for example, by cleavage, phosphorylation, acylation and glycosylation), the number of functionally distinct proteins is likely much greater. Viewed from the perspective of disease mechanisms, these estimates pose an immense challenge to modern medicine, as disease may result when any one of these proteins contains mutations or other abnormalities, or is present in an abnormally high or low concentration. Viewed from the perspective of therapeutics, however, these estimates represent a tremendous opportunity in terms of harnessing protein therapeutics to alleviate disease. At present, more than 145 different proteins or peptides are approved for clinical use by the U.S. Food and Drug Administration (FDA), and many more are in development. Protein therapeutics have several advantages over smallmolecule drugs. First, proteins often serve a highly specific and complex set of functions that cannot be mimicked by simple chemical compounds. Second, because the action of proteins is highly specific, there is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects. Third, because the body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses. Fourth, for diseases in which a gene is mutated or deleted, protein therapeutics can provide effective replacement treatment without the need for gene therapy, which is not currently available for most genetic disorders. Fifth, the clinical development and FDA

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provide immunity in an individual without exposing the individual to the risks of infection or toxic reaction. Proteins in Group IIIa are used to generate protection against infectious diseases or toxins. One successful example is the hepatitis B vaccine. This vaccine was created by producing recombinant hepatitis B surface antigen (HBsAg), a noninfectious protein of the hepatitis B virus. When immunocompetent humans are challenged and rechallenged with this protein, significant immunity results in the large majority of individuals. Similarly, the noninfectious lipoprotein on the outer surface of Borrelia burgdorferi has been engineered into a vaccine for Lyme disease (OspA). A recently approved vaccine against human papillomavirus (HPV) combines the major capsid proteins from four HPV strains that commonly cause genital warts (strains 6 and 11) and cervical cancer (strains 16 and 18). In addition to generating protection against foreign invaders, recombinant proteins can induce protection against an overactive immune system that attacks its own body or “self.” One theory is that administration of large amounts of this selfprotein causes the body’s immune system to develop tolerance to that protein by eliminating or deactivating cells that react against the self-protein. Proteins in Group IIIb are used to treat patients with disorders that arise from this type of autoimmune phenomenon. Immunological acceptance of a fetus during pregnancy represents a special situation with respect to vaccine use. Occasionally, a pregnant woman can reject a fetus after she has been immunized against certain antigens carried by a fetus from a previous pregnancy. Administration of an anti-Rhesus D antigen Ig prevents the sensitization of an Rh-negative mother at the time of delivery of an Rh-positive neonate. Because the woman fails to develop antibodies directed against the fetal Rh antigens, immune reactions and pregnancy loss do not occur in subsequent pregnancies, even when the new fetus carries the Rh antigens. Proteins in Group IIIc could be used as therapeutic anticancer vaccines. Although there are currently no FDA-approved recombinant anticancer vaccines, there are promising clinical trials that use patient-specific cancer vaccines. For example, a vaccine for B-cell non-Hodgkin’s lymphoma uses transgenic tobacco plants (Nicotiana benthamiana). Each patient with this type of lymphoma has a malignant proliferation of an antibody-producing B cell that displays a unique antibody on its surface. By subcloning the idiotype region of this tumorspecific antibody and expressing the region recombinantly in tobacco plants, a tumor-specific antigen is produced that can be used to vaccinate a patient. This process requires only 6–8 weeks from biopsy of the lymphoma to a ready-to-use, patient-specific vaccine. As the genomes of infectious organisms and the nature of autoimmune diseases and cancer are more fully elucidated, more recombinant proteins will undoubtedly be developed for use as vaccines.

Group IV: Protein Diagnostics Proteins in Group IV are not used to treat disease, but purified and recombinant proteins used for medical diagnostics (both in vivo and in vitro) are mentioned here because they are invaluable in the decision-making process that precedes the treatment and management of many diseases. Table 53-7 provides selected examples. A classic example of an in vivo diagnostic is the purified protein derivative (PPD) test, which determines whether

an individual has been exposed to antigens from Mycobacterium tuberculosis. In this example, a noninfectious protein component of the organism is injected under the skin of an immunocompetent individual. An active immune reaction is interpreted as evidence that the patient has been previously infected by M. tuberculosis or exposed to the antigens of this organism. Several stimulatory protein hormones are used to diagnose endocrine disorders. Growth hormone releasing hormone (GHRH) stimulates somatotroph cells of the anterior pituitary gland to secrete growth hormone. Used as a diagnostic, GHRH can help to determine whether pituitary growth hormone secretion is defective in patients with clinical signs of growth hormone deficiency. Similarly, the recombinant human protein secretin is used to stimulate pancreatic secretions and gastrin release, and thereby aid in the diagnosis of pancreatic exocrine dysfunction or gastrinoma. In patients with a history of thyroid cancer, recombinant thyroidstimulating hormone (TSH) is an important component of the surveillance methods used to detect residual thyroid cancer cells. Before the advent of recombinant TSH, patients with a history of thyroid cancer were required to stop taking replacement thyroid hormone in order to develop a hypothyroid state to which the anterior pituitary would respond by releasing endogenous TSH. TSH-stimulated cancer cells could then be detected by radioactive iodine uptake. Unfortunately, this method required patients to experience the adverse consequences of hypothyroidism. Use of recombinant TSH instead of endogenous TSH not only allowed patients to remain on replacement thyroid hormone but also resulted in the improved detection of residual thyroid cancer cells. Imaging agents are a broad group of protein diagnostics that can be used to help identify the presence or localization of a pathologic condition. For example, apcitide is a technetiumlabeled synthetic peptide that binds glycoprotein IIb/IIIa receptors on activated platelets and is used to image acute venous thrombosis. Capromab pendetide is an indium-111-labelled anti-PSA (prostate-specific antigen) antibody that can be used to detect prostate cancer. Protein-based imaging agents are often used to detect otherwise hidden disease so it can be treated early, when treatment is most likely to succeed. Imaging agents are currently used to detect cancer, image myocardial injury, or identify sites of occult infection; these agents are presented in more detail in Table 53-7. There are numerous in vitro protein diagnostics, and two are presented here as examples of a much larger class. Natural and recombinant HIV antigens are essential components of common screening (enzyme immunoassay) and confirmatory (western blot) tests for HIV infection. In these tests, the antigens serve as “bait” for specific antibodies to HIV gag, pol, and env gene products that have been elicited in the course of infection. Hepatitis C infection is diagnosed by using recombinant hepatitis C antigens to detect antibodies directed against this virus in the serum of potentially infected patients.

 CHALLENGES FOR PROTEIN THERAPEUTICS There are now many examples in which proteins have been used successfully in therapy. Nonetheless, potential protein therapies that have failed far outnumber the successes so far,

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must be synthesized in a genetically engineered cell type for large-scale production. The host system must produce not only biologically active protein but also a sufficient quantity of this protein to meet clinical demand. Also, the system must allow purification and storage of the protein in a therapeutically active form for extended periods of time. The protein’s stability, folding, and tendency to aggregate may be different in large-scale production and storage systems than in those used to produce the protein for animal testing and clinical trials. Some have proposed engineering host systems that co-express a chaperone or foldase with the therapeutic protein of interest, but these approaches have had limited success. Potential solutions could include the development of systems in which entire cascades of genes involved in protein folding are induced together with the therapeutic protein; the impetus for this work is the observation that plasma cells, which are natural protein production “facilities,” use such gene cascades to produce large quantities of monoclonal antibody. Although bacteria and yeast are generally considered easy to culture, certain mammalian cell types can be more difficult and more costly to culture. Other methods of production—such as genetically engineered animals and plants—could provide a production advantage. Transgenic cows, goats, and sheep have been engineered to secrete protein in their milk, and transgenic chickens that lay eggs filled with recombinant protein are anticipated in the future. Transgenic plants can inexpensively produce vast quantities of protein without waste or bioreactors, and potatoes can be engineered to express recombinant proteins and thereby make edible vaccines. Finally, by using fluid-shaking bioreactors, microliter-sized culture systems might be able to predict the success of large-scale culture systems and thereby provide substantial cost savings by focusing investment on systems that are more likely to succeed. A fourth important challenge is the costs involved in developing protein therapies. For example, switching to recombinant methodology from laborious purification of placentally derived protein has allowed the production of sufficient ␤-glucocerebrosidase to treat Gaucher’s disease in many patients. Even so, the cost of the recombinant protein can be greater than $100,000 per patient per year. The example of Gaucher’s disease also illustrates aspects of a fifth issue associated with protein therapeutics: ethics (although these ethical issues are not exclusive to protein therapeutics). For example, the possibility of efficacious but expensive protein therapeutics for small but severely ill patient populations, such as patients with Gaucher’s disease, can present a dilemma with respect to allocation of financial resources of health care systems. In addition, the definition of illness or disease could be challenged by protein therapeutics that can “improve upon” conditions previously viewed as variants of normal. For example, the definition of short stature may begin to change with the possibility of using growth hormone to increase the height of a child. Finally, the regulatory landscape that governs protein therapies will likely continue to have a significant impact on the development of new therapies and their cost. As the field of protein therapeutics matures and certain therapies lose patent protection, the role of follow-on or generic protein therapies in medicine will be decided. As of 2010, there

is not a clear regulatory pathway in the United States that addresses the development of generic versions of protein therapeutics (so-called biosimilars). Due to the complexity of protein manufacture and the costs and risks associated with development and testing, relatively small changes in the regulatory landscape for protein therapeutics may have strong impacts on the investment in and development of protein therapeutics.

 CONCLUSION AND FUTURE DIRECTIONS Medicine is entering a new era in which approaches to manage disease are being used at the level of the genetic and protein information that underlies all biology, and protein therapeutics are playing an increasingly important role. Already, recombinant human proteins make up the majority of FDA-approved biotechnology medicines, which include monoclonal antibodies, natural interferons, vaccines, hormones, modified natural enzymes, and various cell therapies. The future potential for such therapies is huge, given the thousands of proteins produced by the human body and the many thousands of proteins produced by other organisms. Furthermore, recombinant proteins not only provide alternative (or the only) treatments for particular diseases, but can also be used in combination with small-molecule drugs to provide additive or synergistic benefit. Treatment of EGFR-positive colon cancer is illustrative of this point: combination therapy with the small-molecule drug irinotecan, which prevents DNA repair by inhibiting DNA topoisomerase, and the recombinant monoclonal antibody cetuximab, which binds to and inhibits the extracellular domain of the EGFR, results in increased survival in patients with colorectal cancer. The therapeutic synergy between irinotecan and cetuximab may be due to the fact that both drugs inhibit the same EGFR signaling pathway, with one drug (cetuximab) inhibiting the initiation of the pathway and the other drug (irinotecan) inhibiting a target downstream in the pathway. The early success of recombinant insulin production in the 1970s created an atmosphere of enthusiasm and hope, which was unfortunately followed by an era of disappointment when the vaccine attempts, nonhumanized monoclonal antibodies, and cancer trials in the 1980s were largely unsuccessful. Despite these setbacks, significant progress has been made recently. As well as the major successes with protein therapeutics described in this chapter, new production methods are changing the scale, cost, and even route of administration of recombinant protein therapeutics. With the large number of protein therapeutics both in current clinical use and in clinical trials for a range of disorders, one can confidently predict that protein therapeutics will have an expanding role in medicine for years to come.

Acknowledgment We thank Armen H. Tashjian, Jr. for many helpful discussions. Portions of this chapter have been published as a review article (Leader B, Baca QJ, Golan DE. Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov 2008;7:21–39) and are adapted with permission.

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Suggested Reading Hansel TT, Kropshofer H, Singer T, et al. The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov 2010;9:325–338. (Reviews mechanisms, safety, and adverse effects of therapeutic monoclonal antibodies.) Keen H, Glynne A, Pickup JC, et al. Human insulin produced by recombinant DNA technology: safety and hypoglycaemic potency in healthy men. Lancet 1980;2:398–401. (A milestone in the use of a recombinantly produced protein therapeutic.) Mascelli MA, Zhou H, Sweet R, et al. Molecular, biologic, and pharmacokinetic properties of monoclonal antibodies: impact of these parameters on

early clinical development. J Clin Pharmacol 2007;47:553–565. (Discusses trends in antibody formulation and how specific properties of candidate drugs guide early drug development.) Walsh CT. Posttranslational modification of proteins: expanding nature’s inventory. Greenwood Village, CO: Roberts & Company; 2005. (Reviews mechanisms and biological roles of covalent modifications of proteins.) Woodcock J, Griffin J, Behrman R, et al. The FDA’s assessment of follow-on protein products: a historical perspective. Nat Rev Drug Discov 2007;6:437–442. (Discusses challenges of developing protein therapeutics, including difficulties in demonstrating bioequivalence in follow-on protein therapeutics.)

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B Polymer capsule

C

Polymer backbone

Polymer matrix

Drug

Drug dissolved or dispersed in polymer

Drug in reservoir

D

E Polymer matrix

F Semipermeable membrane

Water Water

Drug dissolved or dispersed in polymer

Water or enzyme

Drug dissolved in polymer

Swollen polymer from which drug is being released

Osmotic delivery orifice

Osmotic core containing drug

Water

Drug solution leaves

FIGURE 54-1. Polymer release mechanisms. In all panels except C, the simplified diagrams represent polymeric systems in cross section. The most common release mechanism is diffusion, whereby the drug migrates from its initial location in the polymer system to the polymer’s outer surface and then to the body. A, B. Diffusion can occur from a reservoir, in which a drug core is surrounded by a polymer film, or from a matrix, where the drug is uniformly distributed through the polymeric system. C, D. Drugs can also be released by chemical mechanisms such as cleavage of the drug from a polymer backbone or hydrolytic degradation of the polymer. E. Exposure to a solvent can also activate drug release. For example, the drug can be retained in place by polymer chains; upon exposure to environmental fluid, the outer polymer regions begin to swell, allowing the drug to diffuse outward. F. An osmotic system in the form of a tablet with a laser-drilled hole in the polymer surface can provide constant drug release rates. Water diffuses through the semipermeable membrane into the tablet along its osmotic gradient, swelling the osmotic core inside the tablet and forcing drug solution out through the hole. Combinations of the previously described approaches are possible. Release rates can be controlled by the nature of the polymeric material and the design of the system. In one common matrix system design, the drug is contained in a series of interconnecting pores within the polymer, rather than in one large reservoir (Fig. 54-1B). This system is less limited by the size of the drug molecules because each pore can accommodate molecules with molecular weights of several million daltons. The rate of diffusion between the pores—and thus through the matrix and out of the system—is controlled architecturally; tight constrictions and tortuous connections between pores prevent rapid release of the stored drug. One such system is used clinically to administer gonadotropin-releasing hormone (GnRH) analogues. GnRH analogues are peptide hormones that, when administered continuously, inhibit anterior pituitary gland production of gonadotropins (LH and FSH) and are useful in the treatment of sex-hormone-dependent diseases such as prostate cancer. A major previous limitation of this therapeutic approach was the short in vivo half-life of GnRH analogues following intramuscular injection. When the drug is incorporated into polymer microcapsules, and the capsules are injected intramuscularly, the half-life of GnRH is extended significantly, so that therapeutic concentrations are maintained over a period of 1–4 months. Drug delivery by the microcapsule system utilizes two mechanisms: first, the drug diffuses out of the microcapsules; and second, the polymer matrix itself degrades slowly. The second mechanism of polymer-based drug delivery involves a chemical reaction between the polymer and water (see below).

Chemical Reaction In chemical reaction-based systems, part of the system is designed to degrade over time. Degradation can involve either a chemical or enzymatic reaction. In some designs, covalent bonds that connect the drug to a polymer are cleaved in the body by endogenous enzymes (Fig. 54-1C). Such polymer– drug complexes are typically administered intravenously, and the use of water-soluble polymers such as polyethylene glycol (PEG) increases the biological half-life of the drug considerably. For example, PEG-Intron®, a pegylated form of interferon-␣2b, has been approved by the U.S. Food and Drug Administration (FDA) for weekly administration; this treatment for hepatitis C infection previously required injections three times as often. In the case of the intramuscular GnRH microcapsules discussed above, the polymer itself is degraded in a reaction with water (Fig. 54-1D). Most insoluble polymers considered for these applications exhibit bulk erosion (i.e., the entire matrix dissolves at the same rate), which results in larger pores and a more spongelike and unstable structure. This pattern of degradation makes constant release rates difficult to achieve and creates the potential risk of undesirable “dose dumping.” Novel polymers have been designed to overcome this problem by optimizing degradation for controlled drug delivery (i.e., through surface erosion). For example, a polymer with desirable erosion properties can be engineered by using hydrophobic monomers connected by anhydride bonds. The hydrophobic

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monomers exclude water from the interior of the polymer matrix, eliminating bulk erosion. In contrast, the anhydride bonds are highly water reactive, allowing surface erosion in the aqueous environment of the body. This design allows the polymer to degrade from the outside only (Fig. 54-2). The rate of degradation can be controlled by using a combination of monomers, one more hydrophobic than the other. The length of time over which the polymer persists is specified by the ratio of monomers used, and a drug that is uniformly distributed within such a polymer matrix will be released constantly over time. Based on these principles, Gliadel® has become the first local controlled-release system for an anticancer drug to receive FDA approval. After surgeons remove glioblastoma multiforme, an aggressive form of brain cancer, they place up to eight small polymer–drug wafers at the tumor site. As the polymer surface erodes over 1 month, the drug carmustine (an alkylating agent; see Chapter 38, Pharmacology of Cancer: Genome Synthesis, Stability, and Maintenance) is slowly released. The concentration of carmustine at the tumor site is maintained at a level high enough to kill many of the remaining tumor cells, while adverse effects of systemic delivery are avoided. This treatment significantly prolongs the lives of patients with this cancer. Solvent Activation The third mechanism for polymer-based drug delivery is solvent activation, in which the solvent does not react with the polymer chemically, but rather initiates drug release via swelling (Fig. 54-1E) or osmosis (Fig. 54-1F) of the system.

A

Surface erosion

B

O

O

Bulk erosion

O

R

O

O

O

R

O

O

O

R

O

Polyanhydride H2O

O

HO

O

R

OH

Breakdown products

FIGURE 54-2.

Surface erosion using polyanhydride polymers. A. Surface erosion of degradable polymer delivery devices allows for more accurately controlled release rates and is therefore preferable to bulk erosion. B. Polyanhydrides are used to promote surface erosion. They have hydrophobic monomers that exclude water from the interior of the polymer matrix and prevent bulk erosion. However, the monomers are linked by water-soluble anhydride bonds, allowing breakdown at exposed surfaces.

One widely used example of such a system is an extended release oral formulation of nifedipine, a calcium channel blocker (see Chapter 21, Pharmacology of Vascular Tone). The drug is mixed with an osmotically active agent, such as a salt, and coated with a membrane that is permeable to water but not the drug. A small hole is then drilled in the capsule membrane with a laser. After ingestion, the constant osmotic influx of water through the membrane forces the drug out of the pill through the hole, thereby controlling release. This delivery technique, when compared to conventional (immediate release) oral formulations, provides patients greater relief from ischemic events with fewer adverse effects. Concerta®, an extended release formulation of methylphenidate, uses a similar system to treat children with attention deficit hyperactivity disorder (ADHD).

Intelligent Delivery There are situations in which pulsatile delivery is desirable to mimic the body’s natural pattern of producing chemicals. In the case of Mr. F, the insulin pump he wore provided a constant, basal rate of insulin to maintain his blood glucose levels between meals. When Mr. F ate, he could set the pump to provide an additional bolus of insulin and thereby prevent a sudden, excessive rise in blood glucose concentration. Several innovative approaches have been taken to incorporate such versatility in polymer-based drug delivery systems, which have traditionally been designed to deliver drugs at constant or decreasing release rates. In one early design, magnetic beads were incorporated in the polymer matrix together with a 2-year supply of insulin. The system was then implanted subcutaneously in rats, where the insulin was slowly released by diffusion out of the matrix, as discussed above. When an oscillating magnetic field was applied externally, movement of the magnetic beads within the matrix caused alternating expansion and contraction of the drug-carrying pores. The insulin could thus be effectively squeezed out of the matrix, resulting in higher dose delivery for as long as the oscillating magnetic field was applied. This system significantly lowered blood glucose levels in the treated rats compared to control rats and may eventually become a viable method of insulin delivery. In Mr. F’s hypothetical future, the implanted magnetic oscillator allowed him to administer a rapid bolus of insulin simply by selecting the appropriate program on his wristwatch controller, which sent the instructions to the implanted device via a radiofrequency signal. Other methods of increasing the rate of drug diffusion from a polymer matrix include the application of either ultrasound or electric current. Ultrasound delivered at an appropriate frequency can have an effect similar to that of the magnetic bead system. Ultrasound causes cavitation (the formation of tiny air pockets) in the polymer, disrupting the porous architecture to facilitate faster drug release. Applying an electric current to certain polymers can induce electrolysis of water at the polymer surface, lowering local pH and disrupting hydrogen bonding within the complex. The polymer subsequently degrades at a faster than normal rate, allowing transient release of larger drug doses. Pulsatile delivery can also be achieved via local environmental stimuli. For example, hydrogels (materials composed of polymers and water) can be designed to sense changes in temperature, pH, and even specific molecules by virtue of their structure.

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A silicon microchip delivery system that offers even more control over release rates has also been designed. The microchip contains up to 1,000 tiny drug reservoirs, each covered with a thin gold film. Applying a small external voltage to an individual implanted reservoir dissolves the gold film electrochemically, releasing the drug stored in that reservoir. Because the reservoirs can be loaded and opened individually, almost limitless possibilities exist for both dosing of single drugs and combining multiple drugs.

Targeting Accurate targeting allows for larger, more effective doses to reach the tissues of interest without risking the toxic effects of systemic delivery. The first variable that can be controlled is the anatomic placement of the polymer-based drug delivery system; the carmustine wafer delivery system discussed earlier makes use of this basic consideration. Other notable examples include Estring®, a vaginal ring that delivers estradiol for vaginal dryness; Vitrasert®, an eye implant that delivers ganciclovir for the treatment of cytomegalovirus retinitis in AIDS patients (see Chapter 37, Pharmacology of Viral Infections); and drug-eluting stents that deliver sirolimus, everolimus, zotarolimus, or paclitaxel for the prevention of in-stent restenosis in coronary angioplasty (see Chapter 45, Pharmacology of Immunosuppression). Many tissues are accessed practically only via the bloodstream, however, making targeted delivery more difficult. Both passive and active targeting techniques have been developed to direct polymer-based systems to specific tissues following intravenous administration. Passive targeting exploits vascular differences between the target tissue and other tissues to deliver drugs selectively. For example, high molecular mass polymer–drug complexes accumulate in some tumor tissues to a greater extent than in normal tissues because the tumor has more permeable capillary beds. Therefore, rather than using lower doses of low molecular mass anticancer drugs, which rapidly pass through all cell membranes and distribute throughout the body, larger and more effective doses of high molecular mass polymer– drug conjugates can be used to target tumors. In addition, the polymer–drug conjugates can be constructed in such a way as to allow enzymatic cleavage of the drug after the complex has left the bloodstream and been taken up by tumor cells (Fig. 54-1C). In one example of such a system, the anticancer drug doxorubicin (see Chapter 38) is conjugated to a water-soluble, nonimmunogenic polymer through a peptidyl linker. The polymer–drug complex accumulates in mouse melanoma tumors at concentrations up to 70 times greater than in normal tissue because of the relatively leaky microvasculature in the tumor. Once inside the tumor cells, the peptidyl linker is cleaved by lysosomal proteases, releasing the cytotoxic drug. The polymer portions of the complex either degrade or are eliminated by the kidneys. In active targeting, the polymer–drug conjugate is linked to a molecule that is recognized specifically by cell surface receptors in the tissue of interest. For example, a human IgM antibody directed against a tumor-associated antigen can be used to target a polymer–doxorubicin complex to malignant tissues. Linked to the polymer with an acid-labile bond, the doxorubicin is selectively released in the acidic environment of the tumor. In another system, galactose is used to target a polymer–drug complex to the liver via the hepatocyte cellsurface asialoglycoprotein receptor.

 LIPOSOME-BASED DELIVERY SYSTEMS Drugs attached to a single polymer chain are stable structures that can remain in the circulation for long periods of time; the drug–polymer complexes discussed above in the context of tissue targeting are examples of such systems. However, these polymer chains can accommodate only small amounts of drug, thus limiting the dose per unit volume administered. The potentially high drug-carrying capacity of liposomes, small vesicles with lipid bilayer membranes, makes them an attractive option for a circulating drug delivery system. Important considerations in the design of liposomebased delivery systems include tissue targeting and protection from the immune system. Highly specific antibodies, analogous to those used for active targeting of polymer– drug complexes, can be used to improve tissue targeting. For example, antibodies against the Her2 proto-oncogene, implicated in the progression of breast cancer and other cancers, are being explored for tumor targeting. Similarly, antibodies against E-selectin, an endothelial-specific surface molecule, can be used to target vascular endothelial cells. Protection of liposomes from the immune system can be accomplished by the addition of water-soluble polymers to the liposomal surface. As discussed above, moieties such as PEG increase the hydrophilicity of the structures to which they are attached; in this case, liposomes are made more hydrophilic in the blood and are therefore less liable to be taken up by the reticuloendothelial system. Because liposomes with the PEG moiety (“stealth liposomes”) have a prolonged circulation time (days), larger doses can be administered without the risk of drug toxicity. These principles have been used to develop liposomes loaded with daunorubicin and doxorubicin for the treatment of several tumors, including HIV-associated Kaposi’s sarcoma. Liposomal amphotericin B, used to treat fungal infections, has also been approved for clinical application (see Chapter 35, Pharmacology of Fungal Infections). Liposomal cyclosporine is being studied for targeted immunosuppression following transplant surgery (see Chapter 45).

 CONCLUSION AND FUTURE DIRECTIONS The delivery modalities described in this chapter represent selected novel approaches to optimizing absorption, distribution, metabolism, and excretion of drugs. There are several advantages of improved drug delivery: • Drug levels can be continuously maintained in a therapeutically desirable range. Sustained release oral formulations, large particles that can be inhaled, and many polymerbased designs have this desirable property. • Harmful adverse effects can be reduced by preventing transient high peak blood levels of drug. Designs that alter absorption kinetics, targeted delivery systems (e.g., antibody-labeled polymer–drug complexes), and systems that avoid first-pass liver metabolism (e.g., transdermal delivery of drugs normally taken orally) achieve this goal. • The total amount of drug required can be reduced, as with advanced inhaler designs. Both a decrease in the number of required dosages and a less invasive administration

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route contribute to improved patient adherence. Mr. F’s case illustrates the influence of lifestyle factors on patient adherence. • Pharmaceuticals with short half-lives, such as peptides and proteins, can be successfully delivered using controlledrelease polymer-based delivery systems. Advanced drug delivery technologies also introduce new concerns that must be considered in their design. For example, each material put in the body, as well as its degradation products, must be evaluated for toxic effects; this factor is especially important for synthetic materials such as polymers. Other potential dangers must be avoided, such as unwanted rapid release of the drug from a system intended for sustained release. Discomfort caused by the delivery system or its insertion is another potential disadvantage: Mr. F’s insulin pump, while providing better control of his diabetes, was uncomfortable to him. Finally, advanced technology is often accompanied by increased cost, which can be a problem for patients, their insurance companies, and hospitals. Despite these obstacles, advanced drug delivery technologies play an increasingly valuable role in making the

pharmacologic management of disease safer, more effective, and more agreeable to patients.

Suggested Reading Edwards DA, Ben-Jabria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 1998;84:379– 385. (Review of aerodynamic diameter principles and the potential advantages and applications of large, porous inhaled particles.) Langer R. Drug delivery and targeting. Nature 1998;392:5–10. (Review of drug delivery techniques, with emphasis on polymer and liposome-based systems as well as novel use of delivery routes.) Langer R. Where a pill won’t reach. Sci Am 2003;April:50–57. (Broad overview of concepts in drug delivery.) Leong KW, Brott BC, Langer R. Bioerodible polyanhydrides as drug-carrier matrices: I. Characterization, degradation, and release characteristics. J Biomed Mater Res 1985;24:1463–1481. (Good starting point for learning more about polymer matrix design.) Prausnitz M, Langer R. Transdermal drug delivery. Nat Biotech 2008;26:1261–1268. (Reviews advances in transdermal drug delivery.) Santini JT Jr, Cima MJ, Langer R. A controlled-release microchip. Nature 1999;397:335–338. (More detailed information about intelligent drug delivery using silicon microchips with arrays of drug reservoirs.)

Credit List Figure 1-1: Adapted from an illustration (www .genome.gov/Glossary/resources/protein.pdf) on the National Human Genome Research Institute website: www.nhgri.nih.gov. Figure 1-2: Data used to render the image in panel A were deposited in the RCSB Protein Data Bank (www.rcsb.org/pdb, PDB ID: 1FPU) by Schindler T, Bornmann W, Pellicena P, et al. Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science. 2000;289:1938–1942, Figure 1. Panels B and C were adapted with permission from Schindler, et al. (ibid., Figures 1 and 2). Figure 2-7A: Adapted with permission from Stephenson RP. A modification of receptor theory. Brit J Pharmacol. 1956;11:379–393, Figure 10. Figure 2-7B: Data used to generate the dose–response curves for morphine and buprenorphine were published in Cowan A, Lewis JW, Macfarlane IR. Agonist and antagonist properties of buprenorphine, a new antinociceptive agent. Brit J Pharmacol. 1977;60:537–545. Figure 3-1: Adapted with permission from Hardman JG, Limbird LE, eds. Goodman & Gilman’s the pharmacological basis of therapeutics (10th ed.). New York: The McGraw-Hill Companies; 2001:3, Figure 1-1. Figure 3-7: Adapted with permission from Katzung BG, ed. Basic & clinical pharmacology (7th ed.). New York: Lange Medical Books/The McGraw-Hill Companies, Inc.; 1998:38, Figure 3-2. Figure 4-2A: Adapted with permission from Katzung BG, ed. Basic & clinical pharmacology (7th ed.). New York: Lange Medical Books/The McGraw-Hill Companies, Inc.; 1998:52, Figure 4-3. Figure 5-3A: Adapted with permission from Grattagliano I, Bonfrate L, Diogo CV, et al. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J Gastroenterol. 2009;15:4865–4876, Figure 1. Figure 5-3B: Adapted with permission from Grattagliano I, Bonfrate L, Diogo CV, et al. Biochemical mechanisms in drug-induced liver injury: certainties and doubts. World J Gastroenterol. 2009;15:4865–4876, Figure 2. Figure 6-1A: Adapted with permission from Bertilsson L, Lou YQ, Du YL, et al. Pronounced differences between native Chinese and Swedish populations in the polymorphic hydroxylations of debrisoquin and S-mephenytoin. Clin Pharmacol Ther. 1992;51:388–397 [Erratum, Clin Pharmacol Ther. 1994;55:648].

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Figure 6-1B: Photo of the AmpliChip CYP450 array was provided by Roche Diagnostics. Figure 6-2A: Adapted with permission from Jin Y, Desta Z, Stearns V, et al. CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant breast cancer treatment. J Natl Cancer Inst. 2005;97:30–39. Figure 6-2B: Adapted with permission from Goetz MP, Knox SK, Suman VJ, et al. The impact of cytochrome P450 2D6 metabolism in women receiving adjuvant tamoxifen. Breast Cancer Res Treat. 2007;101:113–121. Figure 6-3: Adapted with permission from Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Human Genet. 1980;32:651–662, and Weinshilboum R, Wang L. Pharmacogenomics: Bench to bedside. Nature Rev Drug Discovery. 2004;3:739–748. Figure 6-5: Adapted with permission from The SEARCH Collaborative Group, Link E, Parish S, et al. SLCO1B1 variants and statin-induced myopathy—a genomewide study. N Engl J Med. 2008;359:789–799. Figure 7-9: Adapted with permission from Rizo J, Rosenmund C. Synaptic vesicle fusion. Nat Struct Mol Biol. 2008;15:665–674. Figure 8-14: Adapted with permission from Goldstein GW, Laterra J. Appendix B: Ventricular organization of cerebrospinal fluid: blood– brain barrier, brain edema, and hydrocephalus. In: Kandel ER, Schwartz JH, Jessell TM, eds. Principles of neural science (4th ed.). New York: The McGraw-Hill Companies; 2000:1291, Figure B-4. Figure 9-2: Adapted with permission from Changeux JP. Chemical signaling in the brain. Sci Am. 1993;269:58–62. Figure 9-4: Adapted with permission from Kandel ER, Schwartz JH, Jessell TM, eds. Principles of neural science (4th ed.). New York: The McGrawHill Companies; 2000:188, Figure 11-1. Table 9-5: Adapted with permission from Hardman JG, Limbird LE, eds. Goodman & Gilman’s the pharmacological basis of therapeutics (10th ed.). New York: The McGraw-Hill Companies; 2001:159, Table 7-1. Table 10-1: Adapted with permission from Hardman JG, Limbird LE, eds. Goodman

& Gilman’s the pharmacological basis of therapeutics (10th ed.). New York: The McGrawHill Companies; 2001:137, Table 6-3. Table 11-1: Adapted with permission from Carpenter RL, Mackey DC. Local anesthetics. In: Barash PG, Cullen BF, Stoelting RK, eds. Clinical anesthesia (2nd ed.). Philadelphia: Lippincott; 1992:509–541. Figure 12-2B: Adapted with permission from Cooper JR, Bloom FE, Roth RN. Biochemical basis of neuropharmacology (7th ed.). New York: Oxford University Press; 1996: Figures 6-1 and 6-11. Figure 12-4: Adapted with permission from Neelands TR, Greenfield J, Zhang J, et al. GABAA receptor pharmacology and subtype mRNA expression in human neuronal NT2-N cells. J Neurosci. 1998;18:4993–5007, Figure 1a. Box 13-1: With permission from American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, DC, American Psychiatric Association, 1994. Figure 13-4: Adapted with permission from Hardman JG, Limbird LE, eds. Goodman & Gilman’s the pharmacological basis of therapeutics (10th ed.). New York: The McGraw-Hill Companies; 2001:554, Figure 22-5. Figure 13-5: Adapted with permission from Seeman P. Dopamine receptor sequences. Therapeutic levels of neuroleptics occupy D2 receptor, clozapine occupies D4. Neuropsychopharmacology. 1992;7:261–284, Figure 2. Figure 13-9: Adapted with permission from Seeman P. Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse. 1987;1:133–152. Box 14-1: With permission from American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, DC, American Psychiatric Association, 1994. Box 14-2: With permission from American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, DC, American Psychiatric Association, 1994. Figure 15-3: Adapted with permission from Lothman EW. Pathophysiology of seizures and epilepsy in the mature and immature brain: cells, synapses and circuits. In: Dodson WE, Pellock

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JM, eds. Pediatric epilepsy: diagnosis and therapy. New York: Demos Publications; 1993:1–15. Figure 15-4: Adapted with permission from Lothman EW. The neurobiology of epileptiform discharges. Am J EEG Technol. 1993;33:93–112. Figure 15-5A: Adapted with permission from Kandel ER, Schwartz JH, Jessell TM, eds. Principles of neural science (4th ed.). New York: The McGrawHill Companies; 2000:899, Figure 45-9. Figure 16-2: Adapted from Miller KW. General anesthetics. In: Wolff ME, ed. Burger’s medicinal chemistry and drug discovery, Volume 3: therapeutic agents (5th ed.). Hoboken, NJ: John Wiley & Sons; 1996: Figure 36-2. This material used by permission of John Wiley & Sons, Inc. Figure 16-6: Adapted with permission from Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figure 4-7. Figure 16-7: Adapted from Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia (5th ed.). Philadelphia: Churchill Livingstone; 2000: Figure 4-2. With permission from Elsevier. Figure 16-9: Adapted with permission from Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figures 7-1 and 7-8. Figure 16-10: Adapted from Eger EI. Uptake and distribution. In: Miller RD, ed. Anesthesia (5th ed.). Philadelphia: Churchill Livingstone; 2000: Figure 4-10. With permission from Elsevier. Figure 16-12: Adapted with permission from Eger EI. Anesthetic uptake and action. Baltimore: Williams & Wilkins; 1974: Figure 14-8. Figure 16-13: Adapted with permission from Trevor AJ, Miller RD. General anesthetics. In: Katzung BG, ed. Basic & clinical pharmacology (7th ed.). New York: Lange Medical Books/The McGraw-Hill Companies, Inc.; 1998:421, Figure 25-6. Box 18-1: With permission from American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, DC, American Psychiatric Association, 1994. Figure 18-6: Adapted from Jones RT. The pharmacology of cocaine smoking in humans. In: Chiang CN, Hawks RL, eds. NIDA research monograph 99 (research findings on smoking of abused substances). Washington, DC: U.S. Department of Health and Human Services; 1990:30–41. Figure 19-1: Adapted from Larsen PR, Kronenberg HM, Melmed S, et al., eds. Williams textbook of endocrinology (10th ed.). Philadelphia: WB Saunders; 2003: Figure 34-5. With permission from Elsevier. Figure 19-2: Adapted with permission from Scapa EF, Kanno K, Cohen DE. Lipoprotein metabolism. In: Benhamou JP, Rizzetto M, Reichen J, et al., eds. The textbook of hepatology: from basic science to clinical practice (3rd ed.). Oxford, UK: Blackwell; 2007: Figure 2. Figure 19-6B: Adapted with permission from Mahley RW, Ji ZS. Remnant lipoprotein metabolism: Key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res. 1999;40:1–16. Figure 19-8: Adapted with permission from Quinn MT, Parthsarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a

potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci USA. 1987;84:2995–2998, Figure 1. Figure 19-9B: Adapted with permission from Scapa EF, Kanno K, Cohen DE. Lipoprotein metabolism. In: Benhamou JP, Rizzetto M, Reichen J, et al., eds. The textbook of hepatology: from basic science to clinical practice (3rd ed.). Oxford, UK: Blackwell; 2007: Figure 6B. Figure 19-11: Adapted from Vaughan CJ, Gotto AM Jr, Basson CT. The evolving role of statins in the management of atherosclerosis. J Am Coll Cardiol. 2000;35:1–10. With permission from Elsevier. Table 19-1: Adapted from Jonas A. Lipoprotein structure. In: Vance DE, Vance JE, eds. Biochemistry of lipids, lipoproteins and membranes (4th ed.). Amsterdam: Elsevier; 2002:483–504. With permission from Elsevier. Table 19-3: Adapted from Grundy SM, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J Am Coll Cardiol. 2004;44:720–732. With permission from Elsevier. Figure 20-10: Adapted from Skorecki KL, Brenner BM. Body fluid homeostasis in congestive heart failure and cirrhosis with ascites. Am J Med. 1982;72:323–338, Figure 1. With permission from Elsevier. Figure 20-11: Adapted with permission from Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology (3rd ed.). Philadelphia: Lippincott Williams & Wilkins; 2000:1494, Figure 54-8. Figure 20-12: Adapted with permission from Katzung BG, ed. Basic & clinical pharmacology (8th ed.). New York: Lange Medical Books/ The McGraw-Hill Companies, Inc.; 2001:173, Figure 11-6. Figure 21-1: Adapted with permission from Greineder K, Strichartz GR, Lilly LS. Basic cardiac structure and function. In: Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:9, Figure 1.7, and adapted from Berne RM, Levy MN. Control of cardiac output: coupling of heart and blood vessels. In: Cardiovascular physiology. St. Louis: Mosby Year Book; 1997: Figure 9.2. With permission from Elsevier.

(6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 5-7. With permission from Elsevier. Figure 22-11: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 5-12. With permission from Elsevier. Figure 22-15: Adapted from Lefkovits J, Topol EJ. Direct thrombin inhibitors in cardiovascular medicine. Circulation. 1994;90:1522–1536, Figure 1. Figure 23-1: Adapted from Ackerman M, Clapham DE. Normal cardiac electrophysiology. In: Chien KR, Breslow JL, Leiden JM, et al., eds. Molecular basis of cardiovascular disease: a companion to Braunwald’s heart disease. Philadelphia: WB Saunders; 1999:282, Figure 12-1. With permission from Elsevier. Figure 23-2: Adapted from Ackerman M, Clapham DE. Normal cardiac electrophysiology. In: Chien KR, Breslow JL, Leiden JM, et al., eds. Molecular basis of cardiovascular disease: a companion to Braunwald’s heart disease. Philadelphia: WB Saunders; 1999:284, Figure 12-2. With permission from Elsevier. Figure 23-3: Adapted from Ackerman M, Clapham DE. Normal cardiac electrophysiology. In: Chien KR, Breslow JL, Leiden JM, et al., eds. Molecular basis of cardiovascular disease: a companion to Braunwald’s heart disease. Philadelphia: WB Saunders; 1999:282,284, Figures 12-1 and 12-2. With permission from Elsevier. Figure 23-5: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:241, Figure 11.7. Figure 23-6: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:241, Figure 11.8. Figure 23-7: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:243, Figure 11.9. Figure 23-9A: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:371, Figure 17.11B.

Figure 21-10: Adapted with permission from Benowitz NL. Antihypertensive agents. In: Katzung BG, ed. Basic & clinical pharmacology (7th ed.). New York: Lange Medical Books/The McGrawHill Companies, Inc.; 1998:168, and Kalkanis S, Sloane D, Strichartz GR, Lilly LS. Cardiovascular drugs. In: Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:360, Figure 17.7.

Figure 23-10: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:371, Figure 17.11A.

Figure 22-1A–D: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 5-5. With permission from Elsevier.

Figure 23-12: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:377, Figure 17.13.

Figure 22-1E: Courtesy of James G. White.

Figure 23-13: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:380, Figure 17.14.

Figure 22-2: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 5-7. With permission from Elsevier. Figure 22-3: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease

Figure 23-11: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:376, Figure 17.12.

Figure 24-1: Adapted with permission from Katz AM. Congestive heart failure: role of altered myocardial cellular control. N Engl J Med. 1975;293:1184–1191, and Lilly LS, ed.

926 Credit List Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:11, Figure 1.9. Figure 24-2: Adapted with permission from Katz AM. Physiology of the heart (2nd ed.). New York: Raven Press; 1992:187, Figure 8.4. Figure 25-1: Adapted with permission from Deshmukh R, Smith A, Lilly LS. Hypertension. In: Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:270, Figure 13.3. Figure 25-2: Adapted with permission from Deshmukh R, Smith A, Lilly LS. Hypertension. In: Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:286, Figure 13.10. Figure 25-5: Adapted with permission from Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:141, Figure 6.5. Figure 25-6: Adapted from Gould KL, Lipscomb K. Effects of coronary stenoses on coronary flow reserve and resistance. Am J Cardiol. 1974;34:48–55, Figure 2. With permission from Elsevier. Figure 25-7: Adapted with permission from Libby P. Current concepts of the pathogenesis of acute coronary syndromes. Circulation. 2001;104:365–372. Figure 25-8: Adapted with permission from Libby P. Current concepts of the pathogenesis of acute coronary syndromes. Circulation. 2001;104:365–372. Figure 25-10: Adapted with permission from Frankel SK, Fifer MA. Heart failure. In: Lilly LS, ed. Pathophysiology of heart disease (2nd ed.). Baltimore: Williams & Wilkins; 1998:199, Figure 9.5. Figure 25-11: Adapted with permission from Harvey RA, Champe PC, eds. Lippincott’s illustrated reviews: pharmacology. Philadelphia: Lippincott Williams & Wilkins; 1992:157, Figure 16-6. Table 25-1: Data (available at hin.nhlbi.nih.gov/ Nhbpep_slds/jnc/jncp2_2.htm) from National Heart Lung and Blood Institute. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Washington, DC: National Institutes of Health; 2003 (see Chobanian AV, Bakris GL, Black HR, et al. The seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. JAMA. 2003;289:2560–2571, Table 1). Table 25-4: Adapted from Kaplan NM. Systemic hypertension: therapy. In: Zipes DP, Libby P, Bonow RO, Braunwald E, eds. Braunwald’s heart disease (7th ed.). Philadelphia: Elsevier Saunders; 2005: Table 38-4. With permission from Elsevier. Figure 28-2: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 26-27. With permission from Elsevier. Figure 28-8: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 26-27. With permission from Elsevier. Figure 29-5: Adapted with permission from Thorneycroft IH, Mishell DR Jr, Stone SC, et al. The relation of serum 17-hydroxyprogesterone and estradiol-17 levels during the human menstrual cycle. Am J Obstet Gynecol. 1971;111:947–951.

Figure 29-8: Structures were deposited in the Protein Data Bank [www.rcsb.org/pdb/; structures 1ERE and 1ERR] by Brzozowski AM, Pike ACW, Dauter Z, et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature. 1997;389:753–758, and are reproduced with permission. Figure 30-4: Adapted with permission from Braunwald E, Fauci AS, et al, eds. Harrison’s principles of internal medicine (15th ed.). New York: The McGraw-Hill Companies; 2001: Figure 33-34. Figure 32-5: Adapted from Haskell CM, ed. Cancer treatment (3rd ed.). Philadelphia: WB Saunders Company; 1990:5, Figure 1.2. With permission from Elsevier. Figure 33-2C: Data used to render the image were deposited in the RCSB Protein Data Bank (www .rcsb.org/pdb; PDB ID: 1AFZ) by Zegar IS, Stone MP. Solution structure of an oligodeoxynucleotide containing the human N-Ras codon 12 sequence refined from 1H NMR using molecular dynamics restrained by nuclear overhauser effects. Chem Res Toxicol. 1996;9:114–125.

Philadelphia: Lippincott Williams & Wilkins; 2007: Figure 57-19. Figure 37-11A: Data used to render the image were deposited in the RCSB Protein Data Bank (www .rcsb.org/pdb; PDB ID: 2BAT) by Varghese JN, McKimm-Breschkin JL, Caldwell JB, et al. The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins. 1992;14:327–332. Figure 37-11C: Adapted with permission from Lave WG, Bischofberger N, Webster RG. Disarming flu viruses. Sci Amer. 1999;280:78–87. Figure 38-8: Adapted with permission from Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer. 2003;3:155–168, Box 2. Figure 38-9: Adapted with permission from de Lange T. Shelterin: the protein complex that shapes and safeguards human telomerases. Genes Dev. 2005;19:2100–2110, Figure 2.

Figure 33-3: Adapted with permission from Dekker NH, Rybenkov VV, et al. The mechanism of type IA topoisomerases. Proc Natl Acad Sci USA. 2002;99:12126–12131, Figure 1.

Figure 38-11: Adapted with permission from Lodish H, Berk A, Zipursky SL, et al., eds. Molecular cell biology (4th ed.). New York: W.H. Freeman and Company/Worth Publishers; 2000:797, Figure 19-2.

Figure 33-4: Adapted with permission from Berger JM, Gamblin SJ, Harrison SC, Wang JC. Structure and mechanism of DNA topoisomerase II. Nature. 1996;379:225–232, Figure 5.

Figure 38-12: Adapted with permission from Lodish H, Berk A, Zipursky SL, et al., eds. Molecular cell biology (4th ed.). New York: W.H. Freeman and Company/Worth Publishers; 2000:806, Figure 19-15.

Figure 33-7: Adapted with permission from PharmAid. Copyright 2003, Jeffrey T. Joseph and David E. Golan.

Figure 38-21A: Data used to render the image were deposited in the RCSB Protein Data Bank (www .rcsb.org/pdb; PDB ID: 1AO1) by Caceres-Cortes J, Sugiyama H, Ikudome K, et al. Interactions of cobalt(III) pepleomycin (green form) with DNA based on NMR structural studies. Biochemistry. 1997;36:9995–10005.

Figure 33-12A: Adapted with permission from Schlunzen F, Zarivach R, Harms J, et al. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature. 2001;413:814–821, Figure 5. Figure 33-12B: Adapted with permission from Schlunzen F, Zarivach R, Harms J, et al. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature. 2001;413:814–821, Figure 4. Figure 36-1: Adapted with permission from Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria. Nature. 2002;415:674–679, Figure 2. Figure 36-4: Adapted from www.cdc.gov/ncidod/ emergplan/box23.htm. Figure 36-6: Adapted with permission from Huston CD, Haque R, Petri WA. Molecular-based diagnosis of Entamoeba histolytica infection. Expert Rev Mol Med. 1999:1–11, Figure 1. Figure 37-3: Adapted from an illustration kindly provided by Professor Stephen Harrison, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School. Figure 37-4: Adapted from Hay AJ. The action of adamantanamines against influenza A viruses: inhibition of the M2 ion channel protein. Sem Virol. 1992;3:21–30, Figure 3. With permission from Elsevier. Figure 37-8A: Adapted with permission from Knipe DM, Howley PM, eds. Fields virology (5th ed.). Philadelphia: Lippincott Williams & Wilkins; 2007: Figure 57-18. Figure 37-8B: Adapted with permission from Knipe DM, Howley PM, eds. Fields virology (5th ed.).

Figure 38-21B: Data used to render the image were deposited in the RCSB Protein Data Bank (www .rcsb.org/pdb; PDB ID: 1AIO) by Takahara PM, Rosenzweig AC, Frederick CA, Lippard SJ. Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin. Nature. 1995;377:649–652. Figure 38-21C: Data used to render the image were deposited in the RCSB Protein Data Bank (www.rcsb.org/pdb; PDB ID: 1D10) by Frederick CA, Williams LD, Ughetto G, et al. Structural comparison of anticancer drug/DNA complexes adriamycin and daunomycin. Biochemistry. 1990;29:2538–2549. Figure 38-22: Adapted with permission from Downing KH. Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu Rev Cell Dev Biol. 2000;16:89–111, Figure 9. Figure 39-4A: Adapted with permission from Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol. 2005;23: 4776–4789, Figure 1. Figure 41-1: Adapted with permission from Janeway CA, Travers P, Walport M, eds. Immunobiology: the immune system in health and disease (4th ed.). New York: Garland Publishing, Inc.; 1999:4, Figure 1.3. Figure 41-4: Adapted from Abbas AK, Lichtman AH, Pober JS. Cellular and molecular immunology (4th ed.). Philadelphia: WB Saunders; 2000:169, Figure 8-3. With permission from Elsevier.

Credit List 927

Figure 41-5: Adapted from Abbas AK, Lichtman AH, Pober JS. Cellular and molecular immunology (4th ed.). Philadelphia: WB Saunders; 2000:173, Figure 8-5. With permission from Elsevier. Figure 41-6: Adapted with permission from Janeway CA, Travers P, Walport M, eds. Immunobiology: the immune system in health and disease (4th ed.). New York: Garland Publishing, Inc.; 1999:378, Figure 10.11. Table 41-2: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Table 3-7. With permission from Elsevier. Figure 42-2: Adapted with permission from Serhan CS. Eicosanoids. In: Kooperman WJ, ed. Arthritis and allied conditions: a textbook of rheumatology (14th ed.). Philadelphia: Lippincott Williams & Wilkins; 1999:516, Figure 24.2. Figure 42-4: Adapted with permission from Serhan CS. Eicosanoids. In: Kooperman WJ, ed. Arthritis and allied conditions: a textbook of rheumatology (14th ed.). Philadelphia: Lippincott Williams & Wilkins; 1999:524, Figure 24.6. Figure 43-2: Adapted with permission from Janeway CA, Travers P, Walport M, eds. Immunobiology: the immune system in health and disease (4th ed.). New York: Garland Publishing, Inc.; 1999:474, Figure 12.12. Figure 43-3: Adapted with permission from Leurs R, Church MK, Taglialatela M. H1 antihistamines:

inverse agonism, anti-inflammatory actions and cardiac effects. Clin Exp All. 2002;32:489–498, Figure 1.

Figure 48-2: Adapted with permission from So A, Busso N. A magic bullet for gout? Ann Rheum Dis. 2009;68:1517–1519, Figure 2.

Figure 44-1: Adapted from Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease (6th ed.). Philadelphia: WB Saunders Company; 1999: Figure 14-1. With permission from Elsevier.

Figure 49-1: Data used to render the image were deposited in the RCSB Protein Data Bank (www .rcsb.org/pdb; PDB ID: 1HXW) by Kempf DJ, Marsh KC, Denissen JF, et al. ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. Proc Natl Acad Sci USA. 1995;92:2484–2488.

Figure 45-7: Adapted with permission from Fox DA. Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition of tumor necrosis factor. Arch Intern Med. 2000;160:437–444, Figure 1. Figure 45-8: Adapted with permission from Fox DA. Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition of tumor necrosis factor. Arch Intern Med. 2000;160:437–444, Figure 2. Figure 47-1: Adapted from Mason RJ, Broaddus VC, Murray JF, Nadel J, eds. Murray and Nadel’s textbook of respiratory medicine (4th ed.). Philadelphia: WB Saunders Company; 2005. With permission from Elsevier. Figure 47-3: Adapted from Mason RJ, Broaddus VC, Murray JF, Nadel J, eds. Murray and Nadel’s textbook of respiratory medicine (4th ed.). Philadelphia: WB Saunders Company; 2005. With permission from Elsevier. Figure 47-4: Adapted with permission from Drazen JM. Treatment of asthma with drugs modifying the leukotriene pathway. N Engl J Med. 1999;340: 197–206, Figure 1.

Figure 49-4: Adapted with permission from Schreiber SL. Target-oriented and diversityoriented organic synthesis in drug discovery. Science. 2000;287:1964–1969. Figure 50-2: Adapted from the CDER handbook by the U.S. Food and Drug Administration, available at http://www.fda.gov/. Figure 50-3: Adapted from the CDER handbook by the U.S. Food and Drug Administration, available at http://www.fda.gov/. Table 50-2: Adapted from http://www.fda.gov/fdac/ special/newdrug/testtabl.html. Figure 52-4: Adapted with permission from Luch A. Nature and nurture—lessons from chemical carcinogenesis. Nat Rev Cancer. 2005;5:113–125, Figure 3. Figure 52-5: Adapted with permission from Luch A. Nature and nurture—lessons from chemical carcinogenesis. Nat Rev Cancer. 2005;5:113–125, Figure 4.

Index Note: Page numbers followed by f denote figures; those followed by t denote tables; and those followed by b indicate boxes.

A AA. See Alcoholics Anonymous AADC. See aromatic amino acid decarboxylase abacavir, 77, 671t abatacept, 801, 806t, 907t Abbreviated New Drug Application (ANDA), 870 ABC. See ATP binding cassette abciximab platelets and, 396t protein therapeutics and, 909t thrombus formation and, 386 Abelcet, 624 A␤-fibers, 267 aBMD. See areal bone mineral density abraxane, 693, 698t absence seizures mechanism of, 230f T-type calcium channel and, 230, 230f absorption. See drug(s) absorption/distribution/metabolism/excretion. See ADME ABVD (Doxorubicin, bleomycin, vinblastine, dacarbazine), 726 AC. See adenylyl cyclase acamprosate addiction and, 305 drug dependence and, 308t acarbose, 538t ACAT. See acetyl-CoA-cholesterol acyltransferase accelerated approval, clinical drug development, 866 accessory electrical pathways, 408 bundle of Kent, 408, 408f ACE (Angiotensin converting enzyme), 13, 39f, 335, 371t ACE inhibitors (Angiotensin converting enzyme inhibitors), 343–344, 343f, 371t acute coronary syndromes and, 452f blood pressure and, 343f drug summary table, 349t heart failure and, 341, 460t, 461 indications and contraindications, 445t pharmacologic effects, 441f vasodilators and, 444 acebutolol, 141t, 142, 146t cardiac rhythm and, 413, 419t acetaminophen, 276, 276f, 282t, 756, 762t conjugation reactions and, 73t cytochrome P450 enzymes and, 52t drug binding and, 33f opioids vs., 275 oxidation/reduction enzyme and, 73t poisoning, mechanism, 65, 65f toxicity, echinacea and, 62 acetazolamide, 351t acetic acid derivatives, 756, 761t acetic acids, 761t acetohexamide, 538t acetylcholine (ACh) degradation, inhibitors of, 128t degradation of, 114–115 inhibitors, of synthesis, storage and release, 112f, 120, 128t

928

muscarinic receptors, effects of, 119f muscle contraction, quantal release and, 118f neuromuscular junction and, 115, 117f neuronal systems and, 101, 101f, 101t neurotransmitters and, 101–102, 101t, 102f, 103t peripheral nervous system and, 106 storage and release, 111–112, 112f synaptic cleft and, 90–91 synthesis, 110–111, 112f acetylcholinesterase (AChE), 13, 110, 114 acetylcholinesterase (AChE) inhibitors, 13, 92, 120–122, 121f structures and mechanisms, 886f acetyl-CoA-cholesterol acyltransferase (ACAT), 314 acetyltransferases, 593 ACh. See Acetylcholine; Muscarinic acetylcholine AChE. See acetylcholinesterase AChE inhibitors. See acetylcholinesterase inhibitors acid hypersecretion, peptic ulcer disease and, 811 acids, tissue damage and, 885 acid-sensitive ion channels (ASICs), 266 acquired genes, antibiotic resistance, 609 acquired immunodeficiency syndrome (AIDS), 649 marijuana and, 269 acquired tolerance, 287 acrivastine, 775t ACS. See acute coronary syndromes ACTH. See adrenocorticotropin hormone actin, cardiac myocyte contraction and, 425t action potentials (AP), 84f, 86–89, 87f, 88f, 89f cells and, 82 local anesthetics, 147 sodium channels and, 226, 226f voltage-gated sodium channel, 153 activated partial thromboplastin time (aPTT) assay, heparin and, 391 activated protein C (drotrecogin), 898 active metabolites, 60 active site, drug and, 4 active targeting, drug delivery and, 922 active transport, 28 activin, 473, 509 acute adrenal insufficiency, 497 acute coronary syndromes (ACS), 448–449 pathogenesis of, 449f pharmacologic management, 452f acute humoral rejection, 791 acute lithium intoxication, 218 acute mountain sickness, 345 acute myeloid leukemia (AML) cancer drugs and, 67 receptor tyrosine kinase FLT3 and, 708–709 acute rejection, solid organ transplantation and, 791–792 acute renal failure aminoglycoside use and, 591 loop diuretics and, 346 acute urticaria (Hives), IgE-mediated hypersensitivity reaction and, 768f, 769 acute withdrawal syndrome, 289 acyclovir (ACV), 568, 670t

mechanism of action, 657f viruses and, 655–658 ad hoc studies, drug study and, 876 ADA. See adenosine deaminase adalimumab, 758, 762t, 804t, 907t adaptive immunity, 732–736 addiction, 284–287 case study, 285 drug summary table, 307–309t future directions, 306 mechanisms, 291–295 opioids and, 274 pharmacology of, 284–309 treatments of, 302–306 Addison’s disease, 493, 500 additive drug interactions, quantification, 718–719, 719f additivity, 718 adducts, carcinogens and, 888 adefovir, 655, 671t adenine, 581, 675 adenosine, 759 cardiac rhythm and, 416, 421t DNA structure, 685f neurotransmitters and, 102, 103t adenosine 5⬘-monophosphate-activated protein kinase (AMPK), 526 adenosine deaminase (ADA), 675 adenosine deaminase, protein therapeutics and, 901t adenosine diphosphate (ADP) nucleotide synthesis, 675 receptor pathway inhibitors drug table summary, 395–396t thrombus formation and, 386 UA/NSTEMI, 453 adenosine triphosphate (ATP) neurotransmitters and, 102, 103t adenylyl cyclase (AC), 9–10, 10f, 427 A␦-fibers, 149, 149t ADHD. See attention-deficit hyperactivity disorder adhesins, fungus and, 620 adhesion, 13 adhesion receptors, 13 adjuvant drugs, anesthesia, 255–256 ADME (Absorption/distribution/metabolism/ excretion), 27, 28f, 856 ADP. See adenosine diphosphate adrenal androgens, 501–502 adrenal cortex case study, 490 hormone synthesis in, 491f pharmacology of, 489–504 regions of, 490f adrenal gland function, drugs testing, 479t pharmacology, drug summary tables for, 503–504t adrenal insufficiency, 493–494 adrenal medulla, 95f, 96 adrenal sex steroid, 504t adrenaline. See Epinephrine adrenergic agonists, 277–278

Index 929

adrenergic drugs pain relief and, 269 pharmacology, 132–142 table, 143–146t adrenergic neurons, drug abuse and, 301 adrenoceptor, 135–136, 136t adrenocortical hormone synthesis inhibitors, 498–499 adrenocorticotropin hormone (ACTH) adrenal cortex and, 489–490 aldosterone synthesis and, 500 anterior pituitary gland, 467, 472f Adriamycin, 691 adult-onset diabetes. See diabetes mellitus adverse effects, 56 AEDs. See antiepileptic drugs affective disorders clinical characteristics of, 211–212 pathophysiology of, 211–213 affective flattening, schizophrenia, 195, 196b afferent neurons, 147 affinity, drug-receptor interaction, 3 A-fibers, 149, 149t aflatoxins, 890 African-Americans, drug metabolism and, 53 afterdepolarization, 406 afterload, 354, 422–423, 456 contractile dysfunction, 461 2AG. See 2-arachidonylglycerol agalsidase B, protein therapeutics and, 900t age anesthesia induction and, 251–252, 252f drug metabolism and, 53 aging, 120 agonist dose-response relationship, 22f agonist dose-response relationship, competitive antagonist vs., 22, 22f agonists, 6, 20 action, 26t AIDS. See acquired immunodeficiency syndrome air pollution, toxicity and, 892 airway hyperresponsiveness, asthma and, 824f immune function in, 821–822 airway smooth muscle, immune function and physiology, 820–822 ALA-D. See delta-aminolevulinic acid dehydratase alanine aminotransferase (ALT), 66 alanine racemase, 603 albendazole, 642, 648t albuterol, 140, 145t, 828, 834t asthma, 821 bronchioles and, 96 alcohol coronary artery disease, 302 dependence, 289 drug user subtypes and, 295 drugs of abuse and, 286t metabolism, inhibitor, 307t peptic ulcer disease, 816 psychiatric disease and, 284 alcohol dehydrogenase, 47 alcoholics, 295. See also fetal alcohol syndrome Alcoholics Anonymous (AA), 285, 303 alcoholism type 1, 295 type 2, 295 aldosterone, 499 aldosterone hyperfunction, 500 aldosterone hypofunction (hypoaldosteronism), 500 aldosterone receptor antagonists acute coronary syndromes and, 452f heart failure and, 460–461 alefacept, 801, 806t, 908t alemtuzumab, 712, 715t, 806t, 907t alendronate, 559t alendronate, bone mineral homeostasis and, 552, 559t alfentanil, 275, 280t alglucosidase alfa, protein therapeutics and, 900t aliskiren, 342–343, 349t, 444 alkaline phosphatase, 66, 543 alkylamines, 774t, 775t

alkylating agents, 723t DNA structure and, 686–688, 696t immunosuppression and, 794, 796 allergic reaction, 768 allergic rhinitis, 769 allergy disease, 771 allodynia, peripheral sensitization and, 271 alloimmunity, 790 allopregnanolone, 168 allopurinol, 685 6-mercaptopurine, interaction, 841, 842f gout and, 841, 841f immune responses, immunotoxicity and, 64 uric acid synthesis and, 844t allostasis, addiction and, 291 allosteric antagonist, 21f all-trans retinoic acid (ATRA), 786 allylamines, 622, 626t allylglycine, GABAergic transmission and, 169t almotriptan, 217, 223t alogia, schizophrenia, 195 alosetron, irritable bowel syndrome and, 218, 224t ALOX5 gene, 76 alpha 2-antiplasmin, 380 alpha cells, glucagon from, 524 alpha helix, 4f, 9 alpha-1-proteinase inhibitor, 895 alpha-1-proteinase inhibitor, protein therapeutics and, 900t alpha-adrenergic agonists, 139–140, 144t alpha-adrenergic antagonists, 140–141, 145t blood pressure and, 443 drug summary table, 371t relative indications and contraindications, 445t vascular tone, 365–366 alpha-adrenoceptors, actions, 135–136, 136t alpha-galactosidase A, protein therapeutics and, 900t alpha-glucosidase inhibitors, 533, 538t alpha-linolenic acid, 741 alpha-methyldopa, 140 alpha-methyltyrosine, 137, 143t alpha-tubulin, 682 alphaxalone, 175 GABAA receptors and, 184t alprazolam, 51t clinical uses for, duration of action, 172t cytochrome P450 enzymes and, 51t GABAA receptors and, 183t alprenolol, cytochrome P450 enzymes and, 51t alprostadil, 763t ALT. See alanine aminotransferase alteplase, 393 Group Ib proteins, 898 STEMI, 454 alteplase, protein therapeutics and, 903t altretamine, 687, 696t aluminum hydroxide, 816, 818t drug summary table, 560t secondary hyperparathyroidism and, 556 alveolar partial pressure, 241 equilibration, anesthesia and, 246–247 of isoflurane, 243f ventilation and cardiac output vs., 251, 251f alveolar ventilation, anesthesia and, 247–248 alvimopan, 275, 282t Amanita phalloides, 885 amantadine, 125, 203t, 568, 670t NMDA receptor antagonists, 185t Parkinson’s disease, 195 viral uncoating and, 654f amatoxins, 885 ambenonium, 121, 128t AmBisome, 624 ambrisentan, 365, 370t amebiasis, 638, 638f amide-linked local anesthetics, 151–152, 151f, 156, 158–159, 161–162t amidophosphoribosyltransferase (amidoPRT), 837 amidoPRT. See amidophosphoribosyltransferase amikacin, 589, 596t, 720 amiloride, 347, 352t, 442t, 474

amino acid neurotransmitters, 102 amino penicillins, 610 “aminoacyl” site, 585 aminocandin, 625 aminocaproic acid, 393, 400t aminoglutethimide, 499, 499t, 504t aminoglycosides, 596t, 717t 30S ribosomal subunit, 589–591, 590f, 596t antimicrobial combination therapy, 720 beta-lactams and, 609 aminophylline, 829, 835t amiodarone cardiac rhythm and, 414–415, 415t, 421t cytochrome P450 enzymes and, 51–52t thyroid hormone homeostasis, 487 amitriptyline, 144t, 215, 221t, 277 cytochrome P450 enzymes and, 51–52t AML. See acute myeloid leukemia amlodipine, 363, 369t, 443 amobarbital, GABAA receptors and, 183t amodiaquine, 568 amoxicillin, 609, 610, 615t H. pylori, 817 AMPA receptors epilepsy and, 179–180 glutamate-gated ion channels, 176, 177t interaction, glutamate receptors and, 180f stroke and trauma, 179 amphetamine, 139, 143t drug abuse and, 299–302 schizophrenia and, 196 serotonin storage and, 213–214 Amphotec, 624 amphotericin B, 567, 624, 628t, 640, 720, 922 drug combinations and, 721 immune responses, immunotoxicity and, 64 Leishmania and, 640 renal toxicity, drug-induced, 66 ampicillin, 609, 610, 615t AMPK. See adenosine 5⬘-monophosphate-activated protein kinase amprenavir, 662, 672t amrinone, 362 amsacrine, 692, 697t amygdala, 98 amyl nitrate, 883 amylase, protein therapeutics and, 901t amylin, 529, 539t anakinra, 758, 762t, 800, 805t, 908t analgesia, 150–151 case presentation, 265 morphine and, 274 pharmacology of, 264–283 analgesic index, 241–242 analgesics, 150, 275–277 anandamide families, 269, 301 anaphylaxis, 62 beta-lactams and, 610 H1-antihistamines and, 771 histamine and, 769 anastrozole, 511, 520t, 723t ANDA. See Abbreviated New Drug Application androgen, 489–490 conjugation enzyme and, 73t hormone replacement, 523t hypogonadism, 519 synthesis, 505–506 androgen receptor antagonists, 515, 522t anesthesia, 150–151. See also General anesthesia; Local anesthesia; Topical anesthesia; specific e.g. Infiltration anesthesia abbreviations and symbols, 262 adjuvant drugs, 255–256 age and, 251–252, 252f drug summary table, 260–261t equations, 263 induction, children and, 251–252, 252f, 252t induction rate, physiologic and pathophysiologic variables in, 251–253, 252t inhibitory GABAA, 257, 258, 258f ion channels, 257–258, 258f

930 Index anesthesia (continued) muscle group and, 246, 246f stages of, 242f vessel-poor group and, 246f vessel-rich group and, 246, 246f V/Q mismatch, 252, 252t anesthesiologist, uptake model and, 245–250, 250–253 anesthetics, 8. See also Amide-linked local anesthetics; General anesthetics; Inhaled general anesthetics; Local anesthetics; Perfusion-limited anesthetics; Ventilation-limited anesthetics cys-loop superfamily, 259 future directions, 258–259 induction times, 250f intravenous, 255, 255f angina pectoris, 354, 447, 447f angiogenesis, 705–706 angiogenesis inhibitors, 710–711, 715t angiotensin converting enzyme. See ACE angiotensin converting enzyme inhibitors. See ACE inhibitors angiotensin I, 335 angiotensin II (AT II), 335 zona glomerulosa, 489 angiotensin II receptor antagonists, 344 cytochrome P450 enzymes and, 52t drug summary table, 350t subtype I, vasodilators and, 358, 444 angiotensinogen, 335 anhedonia, 291 anidulafungin, 625, 628t animal models, drug discovery and, 856 anisoylated plasminogen streptokinase activator complex (APSAC), 906t anistreplase, protein therapeutics and, 906t Ann Arbor staging system, Hodgkin’s disease and, 725 ANP. See A-type natriuretic peptide antacids, 816, 818–819t antagonism, 718 antagonists action, 21, 21f, 26t competitive receptor, 21–22 noncompetitive receptor, 22–23 nonreceptor, 23 drug interactions, quantification, 718–719, 719f anterior pituitary gland, 465, 467 cell types, hormonal targets of, 467t individual axes and, 468–474 anthracyclines, 690f, 691, 723t antianginal drugs, UA/NSTEMI, 453 antiarrhythmic drugs, 277 cardiac rhythm and, 408–409 class I, 409–410, 410f class IA, 410–411, 411f, 418t drug summary table, 418t class IB, 411f, 412 drug summary table, 419t class IC, 411f, 412–413 drug summary table, 419t class II, 413 drug summary table, 419t pacemaker cell action potential and, 413f class III, 413–415 drug summary table, 420–421t class IV, 415–416 drug summary table, 421t pacemaker cell action potentials and, 415f cytochrome P450 enzymes and, 51t antibacterial agents. See antibiotics antibacterial drug classes, sites of action, 567f antibiotics broad-spectrum, warfarin, 389t digoxin and, 430 future directions, 595 resistance, 609 translation, 588 antibodies, 733 anti-CD20 monoclonal antibody, 801 anti-CD25 monoclonal antibodies, 801 anti-CD52 monoclonal antibodies, 801

anticholinergic drugs asthma, 821, 827, 834t gastric acid and, 816, 819t geriatric, cognitively impaired patients, 126b anticoagulants drug summary table, 397t, 400t thrombus formation and, 387–392 anticoagulation inhibitors, 393, 400t anticonvulsants. See antiepileptic drugs (AEDs) antidepressants, 105, 277 cytochrome P450 enzymes and, 51–52t oxidation/reduction enzyme and, 73t sites of action, 215f antidigoxin antibodies, 430 antidiuretic hormone (ADH), 334 posterior pituitary gland, 474 signaling pathways, natriuretic peptide and, 336f, 337 antiepileptic drugs (AEDs), 277 antimuscarinic toxicity and, 126 cytochrome P450 enzymes and, 51t drug summary table for, 239t future directions, 235 immune responses, immunotoxicity and, 64 Na⫹ channels and, 231, 232t sites of action on pain pathways, 278f anti-factor VIII antibodies, 914 antifungal drugs cellular targets of, 621f cytochrome P450 enzymes and, 51–52t, 51t antigen-antibody complexes, 62 antigen-presenting cells (APCs), 730–732 anti-HBV nucleoside analogues, 659, 671t antihelminthic drugs, 647–648t onchocerciasis and, 641 pharmacology of, 641–642 antiherpesvirus nucleoside analogues, 655, 670–671t antihistamines, 769 anti-HIV agents cytochrome P450 enzymes and, 51t viral genome replication inhibition, 655 anti-HIV nucleoside analogues, 655, 659, 671t anti-HIV protease inhibitors, 664f antihypertensive agents classes of, 440t demographic factors, 445 homeostatic responses to, 441f indications, contraindications, 445t pharmacologic effects, 441f anti-IgE antibodies, 831, 836t anti-inflammatory agents, asthma and, 829–831 antimalarial agents, 644–645t P. falciparum, 633f pharmacology of, 632–636 antimalarial drug resistance, 636–637 antimetabolites, 575, 674, 723t, 794 antimicrobial combination therapy, 719–721 antimicrobial dihydropteroate synthase inhibitors, 579t, 596t drug targets, 575–576 antimicrobial drugs 30S ribosomal subunit and, 589–592 50S ribosomal subunit and, 592, 593f selective targeting, mechanism of, 564–566, 564t targeting 30S ribosomal subunit, 596–597t targeting 50S ribosomal subunit, 597–598t antimuscarinics, 124–126 antimycobacterial drugs, 612 drug summary table, 617t mycolic acid synthesis and, 606f antineoplastic combination chemotherapy, 722–725 examples of, 725–726 antineoplastic dihydrofolate reductase inhibitor, 580t antineoplastic drugs classes of, 570f pharmacology drug summary table, 579–580t principles of, 563–578 selective targeting, mechanism of, 564t structural proteins, 13 antiparkinsonian medications, drug summary table, 203t

antiplatelet agents drug summary table, 395t mechanism of action, 385f thrombus formation and, 381–386 antiprotozoal agents, 638–640, 646–646t antipsychotics adverse effects, 197–198 atypical, 199–200 chemical structure, 199f drug summary table, 204–205t schizophrenia and, 197 antiresorptive agents, bone homeostasis disorders and, 551–556 Anti-Rh IgG vaccine, 911t anti-Rhesus D antigen Ig, 912 antisense oligonucleotides, 851b antistaphylococcal penicillins, 610 antithrombin III, 379, 379f protein therapeutics and, 900t antithrombotic mechanisms, 373 antithymocyte globulin (ATG), 800, 805t, 908t anti-TNF agents, 799f anti-VEGF antibodies, 710–711 antivenins, 62 antiviral combination therapy, HIV and, 721–722 antiviral drugs classes of, viral life cycle and, 569f future directions, 668–669 immune system and, 673t mechanism of action, unknown, 665–668, 672–673t apcitide, 912, 913t APCs. See antigen-presenting cells apoB editing complex-1 (apobec-1), 313–314 apoB gene. See apolipoprotein B gene apobec-1. See apoB editing complex-1 apoCII deficiency, 322–323 apolipoprotein B gene (apoB gene), editing, 313–314, 313f apolipoprotein B-containing lipoproteins (apoB), 313 assembly and secretion, 313–315, 314f intravascular metabolism, 315–316, 315f LDL particles, formation and clearance, 317–318, 317f receptor mediated clearance, 316–317, 316f apomorphine, schizophrenia and, 196 apoptosis, 571 cell toxicity and, 62, 63t drug insensitivity to, 573–574 aprotinin, 393, 400t APSAC. See anisoylated plasminogen streptokinase activator complex aPTT assay. See activated partial thromboplastin time assay aquaporin water channel, 338 aquaretics, heart failure and, 460t araC. See cytarabine arachidonic acid, 373 generation, 741–742 metabolism, physiology of, 740, 740f 2-arachidonylglycerol (2AG), 269 arcitumomab, 913t arcuate nuclei, central dopamine pathways and, 190 area postrema, 191 area under the curve (AUC), 30f areal bone mineral density (aBMD), 548 arformoterol, 834t argatroban, 392, 398t arginine, 470 aripiprazole, 200, 206t aromatase inhibitors, 506, 513, 520t aromatic amino acid decarboxylase (AADC) dopamine synthesis and, 187 Parkinson’s disease and, 193 aromatic L-amino acid decarboxylase, 107 serotonin and, 208, 209f arsenic, toxicity and, 890–891 artemether, malaria and, 634, 644t artemisinin, 644t malarial plasmodia and, 634, 634f artesunate, malaria and, 634, 644t articaine, 159, 162t

Index 931

asbestos, toxicity of, 891–892 asbestosis, 891 ASICs. See acid-sensitive ion channels asoprisnil, 516, 522t aspartate aminotransferase (AST), 66 aspartate, neurotransmitters and, 102, 103t Aspergillus, 621 aspirin, 23, 275, 276, 395t, 755, 761t coronary artery disease and, 451 hydrolysis reactions and, 46t overdose, 37 prostaglandin synthesis pathway, 382, 384f UA/NSTEMI, 453 aspirin exacerbated respiratory disease, 831 aspirin-induced airway hyperreactivity, 756 aspirin-triggered lipoxins (ATLs), 756, 759 AST. See aspartate aminotransferase asthma, 753, 820 airway hyperresponsiveness, 824f allergic response of, 825f as bronchoconstrictive disease, 822–824 case study, 821 clinical management, 832, 832t conclusions and future directions, 832–833 drug delivery, 831–832 drug summary table, 834–836t H1-antihistamines and, 771 as inflammatory disease, 824–827 leukotriene pathway, 826f origin, TH2 cells and, 823f, 824 pathophysiology of, 822–827 astroglia, 106 AT II. See angiotensin II AT II receptor subtype I (AT1 receptor), 335, 371t AT1 receptor. See AT II receptor subtype I AT1 receptor antagonists drug summary table, 371t indications and contraindications, 445t atazanavir, 662, 672t atenolol, 141t, 142, 146t, 371t cardiac rhythm and, 413, 419t down-regulation of sympathetic tone, 442 ATG. See antithymocyte globulin ATLs. See aspirin-triggered lipoxins atomoxetine, 217, 222t atopy, 824 atorvastatin, 51t, 78, 325–326, 330t atovaquone, malarial plasmodia and, 635, 645t ATP. See adenosine triphosphate ATP binding cassette (ABC), biliary excretion and, 37 ATP/ADP ratio, 526 ATP-modulated K⫹ channels. See ATP-sensitive K⫹ channel (K⫹/ATP channel) ATP-sensitive K⫹ channel (K⫹/ATP channel), 365, 526–527 ATRA. See all-trans retinoic acid atrial fibrillation, 409b atrial flutter, 409b atropine, 96, 116, 124, 130t, 827 attention-deficit hyperactivity disorder (ADHD), 213–214 A-type natriuretic peptide (ANP), 335–336 atypical antidepressants, 217 drug summary table, 222–223t, 309t atypical antipsychotics, 199–200 drug summary table, 205–206t schizophrenia and, 197 atypical depression, 212 AUC. See area under the curve aura, 228 autoimmune disease, 792 tissue damage, characterization by, 793t autoimmune reactions, 62 autoimmunity, 64, 792. See also Hypersensitivity response autolysins, cell wall degradation, 606 automated databases, pharmacoepidemiology and, 875 autonomic ganglion neurotransmission, 115–116 synaptic signals, 118f

autonomic ganglionic blockade, tissues and, 119t autonomic nervous system, 94–95 cellular organization, 99 vascular tone and, 357–358 autoreceptors, 90 dopamine receptor proteins and, 189 Avandia. See rosiglitazone avolition, schizophrenia, 196 awake states, primary generalized seizures, 230, 230f axitinib, 711t AZA. See azathioprine azacytidine, 695t 5-Azacytidine, 686, 686f, 784, 787t azathioprine (AZA), 74, 684, 694t, 794, 795f, 803t conjugation enzyme and, 73t DNA structure, 684f azelastine, 775t azidothymidine (AZT), 659, 660b azithromycin, 592, 597t AZT. See azidothymidine; zidovudine aztreonam, 610, 611, 616t

B B cells, 732t, 733 bacitracin, 607, 614t, 717t baclofen, 175, 184t GABAA receptors and, 184t GABAergic transmission and, 169t bacteria pharmacologic intervention, targets for, 566–567 ribosomes, 588 bacterial cell wall architecture, 600f biosynthesis, pharmacologic agent inhibition and, 602f synthesis bacterial infections and, 599–617 biochemistry, 599–606 pharmacologic agents, classes of, 606–612 bacterial DNA, topoisomerases and, 582–583 bacterial growth kinetics, bacteriostatic vs. bactericidal drugs, 718f bacterial infections case study, 582 cell wall synthesis and, 599–613 drug summary table, 596–598t, 614–617t pharmacology of, 581–595 bacterial protein synthesis, 583–586 bactericidal antibiotics, 717t bactericidal drugs, 566, 589 bacteriostatic antibiotics, 717t Bacteroides, lincosamides and, 592, 594 bactoprenol phosphate, 603 balanced anesthesia, 256 barbiturates, 165, 170, 173–175, 238t clinical uses, duration of action and, 174t CNS and, 98 drugs of abuse and, 286t, 296–299 GABAA receptors, 183t GABAergic transmission and, 169t immune responses, immunotoxicity and, 64 intravenous anesthesia and, 255 seizures and, 234–235 basal ganglia, 97–98, 97f, 189 basal insulins, 533, 538t basal nucleus of Meynert, 101 base excision repair (BER), 676, 680f bases, tissue damage and, 885 basic multicellular units (BMU), 542 basiliximab, 801, 806t, 908t basophil/mast cell, 732t basophils, 731, 781 bazedoxifene, 515, 521t BBB. See blood-brain barrier B-cell non-Hodgkin’s lymphoma, 912 BCR-Abl tyrosine kinase, 5, 6, 713–714t imatinib and, interaction with, 5f intracellular signaling pathways and, 3 BCR-Abl/C-KIT/PDGFR inhibitors, 708, 724t BDNF. See brain-derived neurotrophic factor becaplermin, 904t

beclomethasone, 497, 498f, 503t, 830 asthma and, 835t befloxatone, 139, 144t serotonin degradation and, 214, 221t behavioral tolerance, 287 belatacept, 801, 806t benazepril, 349t bendroflumethiazide, 352t benign prostatic hyperplasia, 511 benperidol, 198f benzbromarone uric acid excretion and, 842 benzo[a]pyrene, metabolism of, 889f benzocaine, dentistry, 157b benzodiazepines, 165, 167f, 232t, 233f, 238t, 261t adverse effects, 180 CNS and, 98 cytochrome P450 enzymes and, 51t drugs of abuse and, 286t, 296–299 duration of action and, clinical uses and, 172t GABAA receptor activation, 171f GABAA receptors, 170–173, 170f GABAergic transmission and, 169t general anesthesia and, 255 pharmacokinetics and metabolism, 172 seizures and, 234 benzothiazepines, 363, 370t, 405 benztropine, Parkinson’s disease, 125, 195, 203t benzylamines, 622, 626t BER. See base excision repair beta adrenergic agonists asthma and, 834t heart failure and, 452f beta adrenergic antagonists, heart failure and, 452f beta barrel, 3 beta pleated sheet, 3 beta thalassemia, 779 beta-1 receptors, off-target effects and, 58 beta-(1,3)-D-glucan, 619–620 beta-(1,6)-D-glucan, 619 beta-2 receptors, off-target effects and, 58 beta-adrenergic receptor agonists, 140, 145t asthma and, 827–829, 834t cAMP and, 429 cardiac contractility, 431–432, 435t drug summary table, 435–436t beta-adrenergic receptor antagonists. See beta-blockers beta-adrenergic receptor kinase, cardiac contractile dysfunction and, 429 beta-adrenergic receptor(s) actions, 136–137, 136t epinephrine and, 14, 15f local anesthesia and, 155 off-target effects and, 60 regulation, 15f tissue localization and action, 10t beta-agonists. See Beta-adrenergic receptor agonists beta-antagonists. See beta-blockers (Beta-adrenergic receptor antagonists) beta-arrestin, 137 beta-blockers (Beta-adrenergic receptor antagonists), 23, 141–142, 141t, 146t, 413 acute coronary syndromes and, 452f class II antiarrhythmic drugs, 413, 419t coronary artery disease, 450 heart failure and, 459f, 460t, 461 hypertension, 442–443 hyperthyroidism and, 486–487 indications and contraindications, 445t off-target effects and, 60 oxidation/reduction enzyme and, 73t thyroid gland and, 488t vascular tone and, 366, 371t beta-cells, insulin and amylin from, 524 beta-endorphin, 269, 472 beta-glucocerebrosidase, 896 protein therapeutics and, 900t beta-lactam antibiotics, 718 bacterial cell wall and, 600 specific agents, 610–612 structural features, 608f toxicity, 610f

932 Index beta-lactam ring, 608, 608f beta-lactamase inhibitors, 609 drug combinations and, 721 beta-lactamases, 572 DNA plasmids and, 609 beta-tubulin, 682 betaxolol, 142, 146t bethanechol, 96, 122–123, 129t bevacizumab, 710–711, 715t, 724t, 907t bezafibrate, 328 B-fibers, 149, 149t “biased libraries,” 853 bicarbonate, gastric acid secretion and, 809 bicuculline, 170 GABAA receptors, 182t GABAergic transmission and, 169t Bier’s block, 157 biguanides, 532–533, 535, 539t bile acid absorption inhibitors, 326, 330t biliary excretion, drug reabsorption and, 37 biliary lipid secretion, 320–321, 321f bimatoprost, 763t binders, 858 binding site, 3 bioavailability drug absorption and, 29–30, 30f equation, 29 biochemical assays, 855 bioequivalence, ANDA and, 870 biogenic amines, 102, 103t, 104–106 biological membranes centran nervous system, 29 diffusion, 28–29, 29f traversing, 28 Biologics License Application (BLA), 867 biomarkers, clinical trials and, 866 biopharmaceutical review, IND and, 867 biosimilars, 915 biotransformation, drugs and, 34, 43 biperiden, 125 bipolar affective disorder (BPAD), 207, 211, 212b birth defects, off-target effects and, 60 bis-alkylate, 687 bisoprolol, cardiac rhythm and, 413, 419t bisphosphonates drug summary table, 559t hypercalcemia, 554b structures, 552–555, 553f bitolterol, 828, 834t bivalirudin, 392 drug summary table, 398t protein therapeutics and, 905t UA/NSTEMI, 453 BLA. See Biologics License Application “black box” warning, 869 black lung, 891 bleomycin, 690–691, 690f, 697t, 723t combination chemotherapy, 726, 726f blood flow abnormal, 381 drug distribution and, total and weight-normalized, 32t blood pressure (BP) ACE inhibitors, 343f determination, 439, 439f essential hypertension, 439 blood-brain barrier (BBB), 29, 106–107, 107f, 911 BMU. See basic multicellular units BNP. See B-type natriuretic peptide bone mineral balance, 542 structure of, 541, 543f bone anabolic agents, 555–556, 560t bone mass age and, 546, 550f PTH and, 546 bone mineral homeostasis case study, 542 diseases, 548–551, 549–550t drug summary table, 559–561t

future directions, 557–558 physiology of, 541–548 bone remodeling osteoblasts vs. osteoclasts, 542 regulation of, 542–543 bortezomib, 710, 714t, 724t bosentan, 365, 370t botulinum toxin, 120, 128t protein therapeutics and, 905t BP. See blood pressure BPAD. See bipolar affective disorder brachial plexus blocks, 152–153 bradykinin asthma, 821 nociceptors and, 148 receptors, 267 brain blood-brain barrier and, compounds and, 107f catecholamine pathways in, 186 dopamine receptor proteins and, 189, 190f seizures and, 225–226 brain capillary, peripheral capillary vs., 107f brain reward pathways opioids and, 296f psychological dependence and, 289 brain-derived neurotrophic factor (BDNF), neurotransmission and, 267 brainstem, 97, 97f, 99 brand name, drugs, 869 breast carcinoma, 511 bretylium, cardiac rhythm and, 414, 420t British anti-Lewisite, 884 brofaromine, 139, 144t serotonin degradation and, 214, 221t bromocriptine, 194, 470, 471, 478t, 511 brompheniramine, 774t bronchial smooth muscle, local anesthetics, 158 bronchodilators, asthma, 827–829 B-type natriuretic peptide (BNP), 335–336, 344 drug summary table, 350t BuChE. See butyrylcholinesterase bucindolol, 78 budesonide, 498f, 503t, 835t buffering capacity, tissue damage and, 885 bufuralol, cytochrome P450 enzymes and, 51t bumetanide, 346, 351t, 442t bundle of Kent, accessory electrical pathways, 408, 408f bupivacaine, 159, 161–162t central nerve blockade, 157 infiltration anesthesia, 156 on-target adverse effect, 58 buprenorphine, 275, 281t addiction and, 297f, 305 drug dependence and, 308t bupropion, 217, 222t addiction and, 305 atypical depression and, 212 drug dependence and, 309t serum sickness and, 62 burn patients, ketamine and, 265 buspirone, 217, 223t butaclamol, 198f butenafine, 622, 626t butoconazole, 623, 627t butorphanol, 275, 281t butyl trimethyl ammonium (TMA), partial agonists, 23f butyrates, 784 butyrophenones, schizophrenia and, 197, 199f, 204t butyrylcholinesterase (BuChE), 114

C C fiber nociceptors, 149, 149t, 267 C1 inhibitor, protein therapeutics and, 900t CA. See Cocaine Anonymous Ca2⫹. See calcium cabergoline, 470, 471, 478t, 511 CAD. See coronary artery disease

Caenorhabditis elegans, 642 caffeine asthma and, 829 drugs of abuse and, 286t, 301 CAH. See congenital adrenal hyperplasia CAII. See carbonic anhydrase II CAIV. See carbonic anhydrase IV calcifediol, 557, 560t calcimimetics drug summary table, 561t secondary hyperparathyroidism, 556–557 calcineurin, 797 calcitonin, 555 calcium homeostasis and, 547–548 drug summary table, 559t calcitonin gene-related peptide (CGRP), 267, 480 asthma, 821 calcitonin-salmon, protein therapeutics and, 903t calcitriol, 560t calcium absorption and, 546–547 secondary hyperparathyroidism, 556 vitamin D, 557 calcium (Ca2⫹), 542. See also calcium channel blockers; calcium channels; calcium gluconate bone mineral homeostasis, disease of, 561t carbonate, hypocalcemic states, 557 chloride, hypocalcemic states, 557 citrate, hypocalcemic states, 557 cycling, regulation of, 426–427 homeostasis cardiac contractile dysfunction and, 427–429 hormonal control of, 543–548, 545t hormonal control of, 543–548 hypocalcemic states, 557 lactate, hypocalcemic states, 557 oral, hypocalcemic states, 557 phosphate, hypocalcemic states, 557 release, 425–426 storage, 425–426 calcium acetate, bone mineral homeostasis and, 556, 560t calcium carbonate, 557, 560t, 561t, 816, 819t bone mineral homeostasis and, 556 calcium channel blockers chemical classes, 363–364 class IV antiarrhythmic agents, 415, 421t coronary artery disease and, 450–451 cytochrome P450 enzymes and, 51t drug summary table, 369–370t epilepsy and, 233–234 grapefruit juice effect and, 53 mechanism of action, 363 oxidation/reduction enzyme and, 73t pharmacokinetics, 364 relative indications and contraindications, 445t selectivity of, 364t sites of action, 364f toxicities and contraindications, 364–365 vasodilators and, 358f, 359, 443 calcium channel inhibitors, 237t calcium channels, 89 local anesthetics and, 158 calcium citrate-malate, 561t calcium gluconate, 557, 561t calcium pump, 424, 424f calcium-sensitizing agents, 433 drug summary table, 436t cAMP (cyclic AMP), 9, 10f dopamine receptors and, 189 phosphodiesterase inhibitors and, 362 physical dependence, 289 SERCA and, 426 campath-1 (CD52), 801 camptothecins, 691, 723t Canadian Food and Drugs Act, 869 canakinumab, 800, 805t, 908t cancer, chemotherapeutic agents. See also chemotherapeutic agents; tumor(s); specific e.g. prostate cancer carcinogen and, 67

Index 933

cells, 568–571 chemotherapeutic agents classes, 723–724t, 723t drug metabolism and, 74 drug summary table, 694–698t environmental exposures and, 887t NSAID therapy and, 754 pharmacologic treatment, 674–693 pharmacology of, signal transduction and, 699–715 receptor tyrosine kinases and, 701t candesartan, volume regulation and, 350t cannabinoid receptors, 269, 299, 300f, 301 cannabinoids, 286t, 301 capecitabine, 684, 694t capillary fluid filtration, 333–334, 334f capreomycin, 720 capromab pendetide, 912, 913t CAPS. See cryopyrin-associated periodic fever syndromes capsid, 650 captopril, 349t, 371t, 444 carbachol, 122, 123f, 123t, 129t carbamazepine, 232t, 236t, 277 BPAD and, 218 cytochrome P450 enzymes and, 51t immune responses, immunotoxicity and, 64 P450 enzymes and, 39, 50 sodium channels and, 231–232, 232t, 236t trigeminal neuralgia, 272 carbapenems, 608, 611–612, 616t carbenicillin, 609, 611, 615t carbidopa, 107 L-DOPA metabolism, 194f Parkinson’s disease, 193 carbinoxamine, 774t carbon monoxide (CO) poisoning, 881–882 mechanism of, 882f carbonic anhydrase II (CAII), 338 carbonic anhydrase inhibitors drug summary table, 351t renal Na⫹ reabsorption and, 345 carbonic anhydrase IV (CAIV), 338 carboplatin, 689f, 690, 696t carboprost, 763t carboxy penicillins, 610 carcinogen, 67 carcinogenesis, due to drug therapy, 67 cell proliferation and, 568–570 carcinogenicity, chronic toxicology and, 887–892, 888f cardiac action potentials, 402–405 phase 0, 404–405 phase 1, 405 phase 2, 405 phase 3, 402, 405 phase 4, 402, 405 cardiac compensation, 457 cardiac remodeling, hypertrophy, 457–458 Frank-Starling mechanism and, 457 neurohumoral activation, 458 cardiac contractility case study, 423 pharmacology of, 422–433 cardiac contraction, physiology of, 422–427 cardiac death, 449 cardiac disease, drug metabolism and, 54 cardiac function, blood pressure elevation and, 439 cardiac glycosides, 429–431 drug summary table, 434t heart failure and, 462 cardiac ion currents, molecular identity of, 416, 416t cardiac myocytes, contraction, functional anatomy, 425 cardiac output (CO) anesthesia induction and, 251, 251f determinants, 457f distribution, volume capacity and, general anesthesia and, 246f, 247 cardiac remodeling, 457–458

cardiac rhythm case study, 402 drug summary table, 418–421t pharmacology of, 401–417 cardiovascular disease, 754 lipoproteins and, 311 cardiovascular toxicity, drug-induced, 67 carmustine, 687, 687f, 696t, 921 carteolol, 142, 146t carvedilol, 141t, 142, 146t cardiac rhythm and, 413, 419t cytochrome P450 enzymes and, 51t case-control studies data analysis from, 876, 876f schematic design, 876, 876f caspofungin, 567, 625, 628t catatonic behavior, schizophrenia, 195 catecholamines conjugation enzyme and, 73t metabolism, 189f metabolism, inhibitors, 139 receptors, 135–137 reuptake inhibitors, 134f, 139, 144t reuptake, metabolism and, 133–135, 134f, 138f storage, inhibitors, 138–139, 138f, 143t synthesis, 105f, 132–133, 134f, 186, 188f inhibitors, 134f, 137, 143t catechol-O-methyltransferase (COMT), 135 catecholamine metabolism and, 187–189 monoamines and, 210 Caucasians, drug metabolism and, 52, 73 caudate, 98 CBER. See Center for Biologics Evaluation and Research CBG. See corticosteroid-binding globulin CCD. See cortical collecting duct CCR5 antagonist, NNRTIs and, 662b CD. See collecting duct CD4 cell count, 44 CD40 ligand (CD40L), 735–736, 735f CD52. See campath-1 CDER. See Center for Drug Evaluation and Research cefaclor, serum sickness and, 62 cefazolin, 611, 616t cefepime, 611, 616t cefoperazone, 616t cefotaxime, 616t cefotetan, 616t cefoxitin, 616t ceftaroline, 616t ceftazidime, 611, 616t ceftizoxime, 616t ceftriaxone, 616t cefuroxime, 611, 616t Celebrex. See celecoxib celecoxib (Celebrex), 757, 762t drug study and, 877 immune responses, immunotoxicity and, 64 peripheral sensitization, 271, 276 celiac ganglion, 96 celiprolol, 141t cell adhesion blockade, 802, 806t cell membrane electric circuit model of, 84f ion channels and, 84, 84f stability, inhibitors of, 612, 617t cell recruitment, inflammatory response and, 737 cell surface adhesion receptors, 13 cell wall bacterial, 599 biosynthesis, 601 enzymes of, 603b pharmacologic agent inhibition and, 602f degradation, autolysins and, 606 synthesis, cross-linking, 604 cell-cycle nonspecific drugs, 570–571 cell-cycle specific drugs, 570, 571f cell(s) drug binding and, 14–15 drugs and, 28–29 of immune system, 730–732, 731f, 732t

cellular assays, 855 cellular communication, 82 cellular excitability, 82–89 cellular immunity, 733–734 cellular senescence, 681 Center for Biologics Evaluation and Research (CBER), 862 Center for Drug Evaluation and Research (CDER), 862 center-surround signaling, 100 central adrenergic neurotransmission, pharmacology of, 207–224 central diabetes insipidus, vasopressin, 347 central dopamine pathways, 189–191, 191f central monoamine neurotransmission, future directions, 219 central nerve blockade, 157 central nervous system (CNS) acetylcholine and, 100f, 116–119 adenosine, caffeine and, 301 amino acid neurotransmitters, 102–103, 104f anatomy, 96–99, 97f capillaries in, peripheral vasculature vs., 107f cellular organization, 99, 100f depression, barbiturates and, 165 drugs and, 29, 124–126, 125b, 126b electrical neurotransmission in case study, 226 pharmacology of, 225–239 GABA and, 164–165 local anesthetics and, 157 neurotransmitters and, 100, 103t, 180 partial pressure, 241 small molecule neurotransmitters, 102, 103t, 104f central sensitization, 271–272, 272f central sympatholytics, 443 central tolerance, autoimmunity and, 792 cephalexin, 611, 616t cephalic phase, gastric acid secretion, 809 cephalosporins, 608, 611, 616t cerebellar hemispheres, 98, 98f cerebellar vermis, 98, 98f cerebellum, 98, 98f cerebral cortex, 97, 97f cerebral hemispheres, 96, 97f cerebral white matter. See white matter cerebrum, 97–98, 97f certolizumab, 762t, 799, 804t, 907t cervical spinal nerves, 95f, 96 cestodes (Tapeworms), 640 cetirizine, 771, 775t CETP. See cholesterol ester transfer protein cetrorelix, 474, 479t, 520t, 909t cetuximab, 707, 713t, 724t, 906, 907t, 915 cevimeline, 123, 129t CFR. See coronary flow reserve cGMP. See cyclic guanosine monophosphate cGMP phosphodiesterase type V (PDE5), 362 cGMP-dependent protein kinase, 355 CGRP. See calcitonin gene-related peptide channel selectivity, 84–86, 85f, 85t, 86f ChAT. See choline acetyltransferase chelators, heavy metal, 884f chemical antagonist, 21, 23 chemical burns, 885 chemical dependence. See addiction chemical reaction-based systems, drug delivery and, 920–921 chemical transmission, cells and, 83 chemical-synapse, neuromuscular junction and, 90 chemistry review, IND and, 863, 867 chemokines, inflammatory response and, 737 chemosensitive transduction receptors, nociceptor neurons, 266, 266t chemotaxis, inflammatory response and, 737 chemotherapeutic agents. See also antineoplastic drugs; chemotherapy; combination chemotherapy cancer, 66 classes of, 723–724t, 723t selective targeting mechanisms, 564–566, 564t tumor resistance, mechanisms of, 725t

934 Index chemotherapy, 570–571 case study, 700 cytotoxic agents and, 67 children, anesthesia induction in, 251–252, 252f chitin, 619 Chlamydia trachomatis, 599 chloral hydrate, GABA physiology and, 176 chlorambucil, 696t cancer and, 67 conjugation reactions and, 46t DNA structure and, 687 chloramphenicol, 107f, 231, 592–594, 593f, 594f, 597t, 719 drug metabolism and, 53 oxidation/reduction reactions, 45t chlordiazepoxide, 231 clinical uses for, duration of action, 172t GABAA receptors and, 183t chlorinated hydrocarbons, toxicity and, 892 2-chloroprocaine (Nesacaine), 158, 161t chloroquine, 567, 644t antimalarial drug-resistance and, 636 drug-resistance and, geographic, 633, 633f malarial plasmodia and, 632–633 Vd of, 33 chlorothiazide, 442t chlorpheniramine, 770, 774t chlorpromazine, 197, 198f, 204t chlorpropamide, 538t chlorprothixene, 204t chlorpyrifos, 885 chlortetracycline, 591, 597t chlorthalidone, 346, 352t, 442t cholecalciferol, 546, 560t cholesterol absorption, 314f, 326–327, 330t balance, 321 metabolism case study, 312 pharmacology, 311–329 cholesterol ester transfer protein (CETP), 320 cholesterol synthesis inhibitors, 324–326, 330t cholestyramine, 326, 330t, 485 choline, 111 choline acetyltransferase (ChAT), 110–111, 112f cholinergic neurotransmission, 110–119 cholinergic pharmacology, 110–127, 128–130t cholinergic receptors, 113–119 cholinergic toxicity, 125b cholinergic transmission, physiological effects, 115–119 cholinesterase inhibitor poisoning, 125b cholinesterases, 52, 114 choreoathetosis-with-salivation syndrome (CS), 887 chromosomal genes, antibiotic resistance, 609 chromosome maintenance, 681f DNA repair, 676–679 chronic bronchitis, 822b chronic coronary artery disease (CAD) clinical management, 450–452 pathophysiology of, 446–448 chronic inflammation, 738 chronic kidney disease, 550–551 agents for, 551–556 secondary hyperparathyroidism, 556–557 chronic myeloid leukemia (CML), 708 chronic obstructive pulmonary disease (COPD), 822b chronic rejection, 792 chylomicron, 314f chylomicron remnant, 316, 316f ciclesonide, 830, 835t cidofovir, 658, 671t cigarette smoke, toxicity of, 888–889 cilastatin, 617t pharmacodynamic drug-drug interactions, 61 pharmacokinetic drug-drug interactions, 61 cilomilast, 833 cimetidine, 39, 231, 772, 775t, 818t cytochrome 450 enzymes and, 51t development of, 851

oxidation/reduction reactions and, 45t peptic ulcer disease and, 812–813 cinacalcet drug summary table, 561t secondary hyperparathyroidism, 556 cingulate gyrus, 97f, 98 ciprofloxacin, 2, 39, 587, 596t cytochrome P450 enzymes and, 52t immune responses, immunotoxicity and, 64 off-target effects and, 60 ciproxifan, 772 circulatory failure, contractile dysfunction and, 455, 455t circumventricular organs, 191 cirrhosis, 341–342 drug metabolism and, 54 Na⫹ retention and, mechanisms, 341–342, 342f cisapride, 217 drug metabolism and, 54 cisatracurium, 261t cisplatin, 689, 689f, 696t, 723t citalopram, 216, 221t, 277 c-kit inhibitors, 713–714t c-kit ligand, 779 cladribine, 685f, 686, 695t, 867 clarithromycin, 51t, 597t H. pylori, 817 class effects, 58 clavulanic acid, 609, 615t, 721 clearance, of drugs, 34f, 37–38 clear-core synaptic vesicles, 91 clemastine, 774t clevidipine, 363, 369t clindamycin, 593f, 594f, 597t, 721 malarial plasmodia and, 635–636, 645t clinical drug development, 863–867 evaluation, regulatory approval and, 860–871 testing, in humans, 864–866, 865t clinical hold, 863 clinical pain syndromes, 270 clinical pharmacology, 866 clinical trials authorizations for, 863 development of, 864–865, 865t in humans, 864–866, 865t planning and execution, 868t clobenpropit, 772 clomiphene, 514–515, 521t clomipramine, 215, 221t cytochrome P450 enzymes and, 51–52t clonazepam, 172, 238t clinical uses for, duration of action, 172t GABAA receptors and, 183t seizures and, 234 clonidine, 105, 139, 144t, 277–278, 443, 470 detoxification and, 303 hypothalamus and, 98 pain and, 269 clopidogrel, 375, 383–384, 396t cytochrome P450 enzymes and, 52t, 53 UA/NSTEMI, 453 clorazepate clinical uses for, duration of action, 172t GABAA receptors and, 183t clorgyline, 139, 144t closed state, CNS and, 226 Clostridium difficile, 594, 607 clotrimazole, 623, 627t cloxacillin, 609, 610, 615t clozapine, 198f, 205t cytochrome P450 enzymes and, 52t schizophrenia and, 200 CML. See chronic myeloid leukemia CMP. See cytidylate CMV. See cytomegalovirus CNP. See C-type natriuretic peptide CNS. See central nervous system CNS partial pressure, 241 CO. See carbon monoxide; cardiac output coagulation cascade, 372, 375–379, 378f

coal worker’s pneumoconiosis (CWP), 891 coating agents, 816, 819t cocaethylene, 302 cocaine, 144t, 147, 156, 158, 161t, 299–302 mechanism of action, 134f norepinephrine transporter and, 139 physical dependence, 293 route of administration, drugs and, 294f schizophrenia and, 196 single-source divergent neuronal systems, 101 Cocaine Anonymous (CA), 303 codeine (Methylmorphine), 74, 274, 280t cytochrome P450 enzymes and, 51t oxidation/reduction enzyme and, 73t cognitive function, cholinergic link, 117 cohort studies data analysis from, 877f schematic design, 876, 876f colchicine, gout and, 840–841, 841f, 844t colesevelam, 326, 330t colestipol, 326, 330t collagenase, 898, 905t collecting duct (CD), nephron and, 339–340, 340f collecting duct (CD) diuretics, 347 drug summary table, 352t colloid, thyroglobulin and, 481 colloidal bismuth, 816, 819t combination chemotherapy. See also antineoplastic combination chemotherapy case study, 717 drug resistance and, 574 future directions, 726 HIV and, 721–722 principles, 716–726 combination therapy diabetes mellitus and, 536 heart failure and, 462 combinatorial chemistry, 848, 852–853, 852f Common Technical Document (CTD), 867 compassionate use protocols, 869 competitive antagonist, 7, 21, 21f agonist dose-response relationship vs., 21–22, 22f complement, inflammatory response and, 736–737 complement inhibition activation, 802, 806t compound-centered drug design, 848 COMT. See catechol O-methyltransferase concentration (of gas), partial pressure vs., 242b concentration effect, anesthesia, 254 concentration-dependent bactericidal agents, 718, 718f concentric hypertrophy, 457–458 conditioned opponent response, 287 conditioned tolerance, 287 conduction block, impulse and, 408 “confounding by indication,” 877 congenital adrenal hyperplasia (CAH), 501, 501f congenital nephrogenic diabetes insipidus, 344 conivaptan, 350t, 479t conjugation, 572 conjugation/hydrolysis reactions, 34–35, 47, 49f consensus interferon, 902t contraception. See also male contraception; oral contraceptives; specific e.g. combination contraceptive formulations emergency, 517 hormones and analogues, 516–517 contractile dysfunction cellular pathophysiology of, 427–429, 428f etiologies, 455–457 contractile state, 422 contractility, regulation of, 424–427, 456 contrast media, 66 controllers, asthma treatment, 827 convergent signaling, 100 convergent synthesis, 857, 857f COPD. See chronic obstructive pulmonary disease coronary artery disease (CAD), alcohol and, 302 coronary artery occlusion, 448, 448f coronary flow reduction, 447–448 coronary flow reserve (CFR), 447 coronary steal phenomenon, 359

Index 935

corpus callosum, 97, 228 corpus luteum, menstrual cycle and, 509 cortical collecting duct principal cell, 337, 339–340, 340f cortical spreading depression, migraine headaches and, 272 corticosteroid-binding globulin (CBG), 490–491 corticosteroids asthma and, 829–830, 835t thyroid hormone homeostasis, 487 corticotropin-releasing hormone (CRH), 106, 472 cortical secretion, 492–493 cortisol analogues, 494, 495f backbone, synthetic modifications, 494–495, 494f hypothalamic-pituitary unit and, 492–493 immune responses and, 492, 493f cortisone, 495f costimulation immune system and, 731 T cell activation pathway and, 734, 735f costimulation inhibition, 806t cosyntropin, 479t, 913t cough variant asthma, 824 “counter-regulatory” hormones, 525 coupling, thyroglobulin and, 481 covalent bonding, 3 COX. See cyclooxygenase COX-1, 742t COX-2, 742t COX-2 selective inhibitors, 757–758, 757f, 762t adverse effects, 742t COX-3, 742–744 cranial nerves, 99 craniosacral system, 95f, 96 cravings (for drug), addiction and, 289 Cremophor, immune responses, immunotoxicity and, 64 CRH. See corticotropin-releasing hormone cromakalim, 365, 370t cromolyns, 769, 836t asthma and, 830 cross-dependence, 296 crossover design, clinical trials and, 864 cross-tolerance, 303 crotalidae polyvalent immune Fab (Ovine), 909t cryopyrin-associated periodic fever syndromes (CAPS), 800 CS. See choreoathetosis-with-salivation syndrome CTD. See Common Technical Document C-type natriuretic peptide (CNP), 336 13 C-urea breath test, 810–811 Cushing’s syndrome, 494, 811 CWP. See coal worker’s pneumoconiosis cyanide poisoning and treatment, 883 cyclases, activation of, 9, 11f cyclic AMP. See cAMP cyclic guanosine monophosphate (cGMP), 355 cyclizine, 775t cyclooxygenase enzymes, NSAIDs and, 271, 275 cyclooxygenase inhibitors, 395t, 754–758 thrombus formation and, 382–383, 384f cyclooxygenase pathway, 742–745 cyclophosphamide, 687, 687f, 696t, 723t, 796, 803t activation and metabolism, 689f DNA damage and, 67 oxidation/reduction enzyme and, 73t structure and, 687f cycloserine, 607, 607f, 614t cyclosporine, 51t, 796–797, 797f, 803t, 849 immune responses, immunotoxicity and, 64 immunosuppression, 922 oxidation/reduction enzyme and, 73t cyfluthrin, 887 CYP2D6. See cytochrome P450 2D6 CYP3A4 enzymes, 61 cypermethrin, 887 cyproheptadine, 774t cys-loop superfamily, anesthetics and, 259 cytarabine (araC), 686, 686f, 695t

cytidine, DNA structure of, 686, 686f cytidylate (CMP), 676 cytochrome P450 2D6 (CYP2D6), 72–76 drug metabolism, 74 pharmacogenetics, 74f cytochrome P450 enzymes, 72 drugs and, 49–50, 51–52t oxidation/reduction reactions, 34, 45t, 47, 48f cytochromes, adrenal cortex and, 490 cytokine antagonists, 762t cytokine inhibitors, 758 immune function and, 798–801, 805t cytokine receptor antagonists, 800, 805t cytokine release, glucocorticoids and, 496 cytokine release syndrome, 800 cytokines, 737 cytomegalovirus (CMV), 655 cytoprotective roles, 744 cytosine, 581 cytotoxic agents, 794–796, 803t cytotoxic T cells, 732t

D D1 receptors, 189, 190f D2 receptors, schizophrenia and, 189, 190f DA. See dopamine dabigatran, 392, 399t dacarbazine, 687, 696t, 726 daclizumab, 801, 806t, 909t dalfopristin, 572, 593f, 594, 598t dalteparin, 391, 398t dantrolene, 254 DAP. See diaminopimelic acid dapsone, 73t, 576, 579t daptomycin, 572, 612, 617t darbepoetin (NESP), 783, 787t darbepoetin alfa, protein therapeutics and, 901t darbepoetin-alpha, 895 darifenacin, 125, 130t darunavir, 662, 672t dasatinib, 708, 714t DAT. See dopamine transporter daunorubicin, 697t, 922 dazoxiben, 758, 763t dCMP. See deoxycytidylate DCT. See distal convoluted tubule de novo synthesis, 837 dead-end complex, 608 debrisoquine, 73, 74f oxidation/reduction enzyme and, 73t DEC. See diethylcarbamazine decitabine, 695t, 784, 787t Declaration of Helsinki, 862 deep vein thrombosis, contraceptives, 517 deferasirox, 884 deferoxamine, 884 degarellx (GnRH receptor antagonist), 907t degenerin/ENaC, 266–267 dehydroepiandrosterone (DHEA), 168, 501, 504t delavirdine, 661, 671t cytochrome P450 enzymes and, 51t delayed afterdepolarization, 407, 407f delayed rectifier K⫹ channels, 88 delayed-type hypersensitivity. See hypersensitivity response type IV delta-aminolevulinic acid dehydratase (ALA-D), 884 deltamethrin, 887 delusions, schizophrenia, 195 demeclocycline, 344, 591, 597t dendritic cells, 732, 732t, 781 denileukin diftitox, 712, 715t, 723t, 910, 910t denosumab, 555, 559t dense-core synaptic vesicles, 91 dentistry, local anesthetics and, 157b deoxycytidylate (dCMP), 676 deoxyribonuclease I (DNAse1), 898 deoxythymidylate (dTMP), 676 dependence mechanisms, 288–291, 288f, 290f medical complications, 302

pharmacologic treatment of, 304–306 treatment, 302–303 dependence syndrome, 289 dephosphorylase, 604 depolarized membrane, 83 depolarizing blockade, 123 depression. See specific e.g. melancholic depression dermatomal distribution, 96 desensitization, drug-receptor interactions, 14, 15f desflurane, 249, 255, 260t desipramine, 74, 215, 221t, 277 addiction and, 305 cytochrome P450 enzymes and, 51t drug dependence and, 309t desirudin, 398t, 905t desloratadine, 771, 775t desmethyldiazepam, 172 desmopressin, 347 desogestrel, 516, 516f, 523t desvenlafaxine, 216, 222t detergents, tissue damage and, 885 detoxification, dependence and, 303 dexamethasone, 494, 495, 495f, 503t, 754, 762t, 803t dexfenfluramine, serotonin storage and, 214, 220t dexlansoprazole, 813, 818t dexmedetomidine, 140, 144t dexrazoxane, 691 dextroamphetamine, serotonin storage and, 213–214, 220t dextromethorphan, 74, 272, 277, 283t cytochrome P450 enzymes and, 51t oxidation/reduction enzyme and, 73t DGAT. See diacylglycerol acyltransferase DHA. See docosahexaenoic acid DHDOC. See 5␣-dihydrodeoxycorticosterone DHEA. See dehydroepiandrosterone DHF. See dihydrofolate DHFR. See dihydrofolate reductase DHOD. See dihydroorotate dehydrogenase DHT. See dihydrotestosterone diabetes insipidus, 474, 475f. See also central diabetes insipidus diabetes mellitus morbidity and mortality, 532 pathophysiology, 530–532, 530t therapy for, pharmacologic classes and agents in, 532–536 type 1, 530–531, 530t protein therapeutics, 896 type 2, 531–532 protein therapeutics, 896 diabetic ketoacidosis (DKA), 531 diacylglycerol acyltransferase (DGAT), 314 Diagnostic and Statistical Manual of Mental Disorders bipolar disorder and, 212b MDD and, 211b schizophrenia and, 196b substance dependence and, 287b diaminopimelic acid (DAP), 601 diastolic heart failure, 427, 456 diazepam, 171–172, 238t, 261t clinical uses for, duration of action, 172t GABAA receptors and, 183t general anesthesia and, 255 seizures and, 234 withdrawal and, 303 diazinon, 885 diazoxide, 540t diclofenac, 276, 756, 761t dicloxacillin, 609, 610, 615t dicyclomine, 816, 819t didanosine, 671t diencephalon, 96, 97f, 98 dopamine receptor proteins and, 189 diet drug metabolism and, 53–54 peptic ulcer disease, 816 dietary supplement. See supplements Dietary Supplement Health and Education Act of 1994, 870

936 Index diethyl ether, 240, 249, 260t diethylcarbamazine (DEC), filarial infections, 642, 648t differential functional blockade, 153 differentiating agents, 723t diffuse neuronal systems, 101f diffuse system of organization, 100 diffusion, drug delivery, 919–920 diffusion hypoxia, 254 diffusion rate, equation, 245 digitoxin, 429, 431, 434t oxidation/reduction reactions and, 45t digoxin, 33, 49, 50, 424, 429–431, 434t conjugation reactions and, 46t heart failure and, 460t pharmacokinetics of, 430t positive inotropic mechanism, 429f digoxin immune Fab, 434t, 910t dihydroartemisinin, 644t malarial plasmodia and, 634 5␣-dihydrodeoxycorticosterone (DHDOC), 168 dihydrofolate (DHF), 576, 676 dihydrofolate reductase (DHFR), 575, 576–577, 639, 676 dihydrofolate reductase (DHFR) inhibitors, 565 future of, 565b sulfonamides and, synergy of, 577–578 values for, 577t dihydroorotate dehydrogenase (DHOD), 632 dihydropyridines, 363, 369t, 405 cardiac rhythm and, 415 dihydrotestosterone (DHT), 506, 508f dihydroxyphenylalanine (DOPA), 132 diiodotyrosine (DIT), 481 diisopropyl fluorophosphate, 120, 129t diltiazem, 39, 51t, 363, 364t, 370t, 405, 443 cardiac rhythm and, 415, 421t dimenhydrinate, 771, 774t dimercaprol, 884 dipeptidyl peptidase-4 (DPP-4), 530 dipeptidyl peptidase-4 (DPP-4) inhibitors, 536, 539t diphenhydramine, 58, 126b, 770, 771, 774t dipyridamole, 359, 395t direct thrombin inhibitors, 391–392 drug summary table, 398t UA/NSTEMI, 453 Directly Observed Therapy Short Course. See DOTS discovery biology, 855–856 diseases, affecting drug metabolism, 54 disodium cromoglycate asthma and, 830 disopyramide, 411, 418t disorganized speech, schizophrenia, 195 distal convoluted tubule (DCT), 337, 339f distribution systems, drug absorption, 27, 28f disulfiram addiction treatment and, 304 alcohol metabolism, inhibition, 307t cytochrome P450 enzymes and, 52t drug dependence and, 307t DIT. See diiodotyrosine diuretics. See also specific type e.g. collecting duct diuretics heart failure and, 459–460 hypertension and, 441–442, 442t intravascular volume, reduction of, 441–442 nonreceptor mediated mechanisms in, 15 relative indications and contraindications, 445t divergent signaling, 100 DKA. See diabetic ketoacidosis DNA alkylating agent, 50 damage and repair mechanisms, 679f gyrase, 583, 587 plasmid drug resistance and, 572 purine analogues, 686 pyrimidine analogues, 686 repair, chromosome maintenance and, 676–679 replication, 581–595 strands, hydrogen bonding, 583f

structure drugs modifying, 582f, 686–691, 687t supercoiling, type II topoisomerases and, regulation, 584f transcription, 581–595 translation, 581–595 DNA polymerase, 655 DNAse1. See deoxyribonuclease I dobutamine, 140, 145t, 432, 435t heart failure and, 460t, 462 docetaxel, 692–693, 698t docosahexaenoic acid (DHA), 331t docosanol, 667, 673t dofetilide cardiac rhythm and, 414, 420t domoate, 225–226 donepezil, 106, 121, 122t, 129t DOPA (dihydroxyphenylalanine), 132 decarboxylase, 107 dopamine (DA), 137, 186–189, 188f, 431–432, 435t, 470 conjugation enzyme and, 73t future directions, 200–201 hypothalamic-releasing factors, 471, 471f metabolism inhibitors, 195 drug summary table, 202–203t neurotransmitters and, 102, 103t Parkinson’s disease, 191–195, 192f precursors, 193–194 drug summary table, 202t receptor agonists, 194–195 drug summary table, 202t receptors, 189, 190f thought disorders and, 195–200 dopamine beta hydroxylase, 104, 132, 187 dopamine dysregulation syndrome, 195 dopamine hypothesis, 196 dopamine neurons, Parkinson’s disease and, 191 dopamine transporter (DAT), 187, 210, 300 dopaminergic neurotransmission, 186 biochemistry and cell biology, 186–191 case study, 187 drug summary table, 202–206t pharmacology, 193–195 dopaminergic pathways, movement regulation and, 191f doripenem, 611, 617t dornase alfa, protein therapeutics and, 905t dorsal columns, 96 dorsal horn neurons, central sensitization, 271, 272f dorsal root ganglia, sensory neurons and, 96 dorsal roots, somatic nervous system and, 96 dose, 39–42, 40f tolerance, 285 toxicity, 61, 63f dose-response curves, 241, 243f dose-response relationships, 18–20 DOTS (Directly Observed Therapy Short Course), 720 double-blind study, clinical trials, 864, 866 double-strand break repair, 679–681, 680f down-regulation, 14–15 doxazosin, 141, 145t, 371t, 443 doxecalciferol, 560t doxepin, 221t, 771, 775t doxercalciferol, 560t, secondary hyperparathyroidism, 556 doxorubicin, 697t, 723t, 922 combination chemotherapy, 726 heart failure and, 691 doxycycline, 242, 591, 592, 597t malarial plasmodia and, 635–636, 645t doxylamine, 771 DPP-4. See dipeptidyl peptidase-4 DPPD (recombinant purified protein derivative), 913t dronedarone, cardiac rhythm and, 415, 421t droperidol, 198f, 204t general anesthesia and, 256 droperidol and fentanyl combination therapy, 256 drospirenone, 515, 516, 523t drug abuse, 274, 284–309 medical complications, 286t, 302

drug addiction. See addiction drug approval drug review and, 896f life cycle of, 862f process, 867–870, 869f drug delivery systems asthma, 831–832 case study, 918 conclusion and future directions, 922–923 intelligent, 921–922 liposome-based, 922 modalities, 917–923 oral, 917–918 polymer-based, 919–922 pulmonary, 918–919 transdermal, 919 drug design compound-centered, 849–851 structure-based, 853 target-centered, 851–853 drug development, 857–858 case study, 848 rare diseases and, 866–867 drug discovery efficacy models in, 856t phases of, 849f preclinical development and, 847–859 process, 848–853 drug dose. See dose drug formulation, 858–859, 858t drug naming, 869–870 Drug Price Competition and Patent Term Restoration Act of 1984, 870 drug production, regulatory aspects, 870 drug resistance drug therapies and, 572–574 genetic causes, 572–574 mechanisms of, 572, 573t drug study design and interpretation, 877–878 duration and post approval, 874 size and generalization, 872–873 strategies, 876–877, 876f, 877f surrogate, 872–873 drug targeting passive, 922 pharmacology, drug resistance mechanisms, 572–574 similar, selective inhibition of, 565–566 unique, 565 drug toxicity, 56–57 contexts of, 60–69 future directions of, 69–70 mechanisms of, 57–60, 58f, 59f online resources, 70t treatment, principles of, 69 drug transport, 47–49 drug user, subtypes, alcohol and, 293 drug-drug interactions, 61–62, 718–719, 719f H2 receptor antagonists, 812 plasma protein binding and, 33–34 drug-eluting stent, 454 drug-herb interactions, 61–62 drug-receptor(s) affinity bond strength of, 3–4, 5t interactions, 2–16, 7t binding, 17–18, 19f binding curves, 18, 19f interactions, 15f, 20 cellular regulation of, 14–15 membrane effects on, 5–6 signal processing, 13–14, 14f types of, 6–13, 7f, 7t drug(s). See also antineoplastic drugs; chemotherapeutic agents; chemotherapy; clinical drug; oral drug; specific drug e.g. penicillin absorption bioavailability, 29–30, 30f local factors affecting, 31–32 mechanism, 27, 28f

Index 937

administration routes and, 30–31, 30t, 31f adrenergic table, 143–146t adverse effects case study, 873 future directions, 879 health care system, 878–879 bacterial cell wall biosynthesis and, 602f binding cells and, 14–15 receptor and, impact of, 4–5 cancer, 674–693 signal transduction and, 699–715 CNS and, 124–126, 125b, 126b combinations, unfavorable, 721 conformation and chemistry, 2–6 dependence, pharmacology of, 284–309 distribution to body compartments, 32–33, 32t four-compartment model, 34, 36f kinetics and thermodynamics of, 34, 34f, 35f, 36f plasma protein binding, 33f schematic model, 35f volume of, 33–34 distribution phase, 34, 34f electrical signaling, 82 elimination, 34, 34f, 35f, 36, 36f erythrocyte production, 779–781 excretion, 36 generic, 870 interactions, 718–719, 719f intravenous, bioavailability, 30f ion channels and, 84 labeling, 869 metabolism, 34–36, 43–54 conjugation/hydrolysis reactions, 34, 46t diseases affecting, 54 factors affecting, 50–54 future directions, 54 oxidation/reduction reactions, 34, 45t pathways, 44–50, 45t, 46t sites of, 43, 44f mucosal defense, gastric acid and, 816 overdose, 61 oxidation, cytochrome P450 system and, 48f pH trapping, 29 physiologic barriers to, 27–29 poisoning by, 881–893 in pregnancy, 68–69, 68b reabsorption, biliary excretion and, 37 route of administration, plasma cocaine concentrations and, 294f safety challenges, 872–874 selectivity, molecular and cellular determinants, 6 trapping, plasma protein binding and, 33f tumors and, 707–712 viral life cycle and, 651f viruses and, 651–668 drug-seeking behavior, 274, 285, 293f dTMP. See deoxythymidylate d-tubocurarine, 126 duloxetine, 216, 222t, 269, 277 Durham-Humphrey Amendment, 870 dusts, toxicity of, 891–892 dutasteride, 511, 520t dynamic instability, of microtubules, 682, 683f dynorphins, 106, 269 dyskinesias, 193 dyslipidemia, 324 dysphoria, addiction and, 301

E early afterdepolarization, 406–407, 407f early-acting growth factors, 778 ebastine, 775t EC50. See potency of drug ECG. See electrocardiogram echinacea, hepatic glutathione stores and, 61–62 echinocandins, 620, 625, 628t ECL cells. See enterochromaffin-like cells econazole, 623, 627t

eculizumab, 802, 806t, 908t ED. See erectile dysfunction ED50. See median effective dose edema loop diuretics and, 345–346 pathophysiology, 340–342 edetate disodium, heavy metal chelators and, 884 Edinger-Westphal nucleus, 96 EDRF (Endothelial-derived relaxing factor), 356 edrophonium, 96, 120–121, 128t EEG. See electroencephalogram efalizumab, 65, 908t efavirenz, 51t, 568, 661, 662, 671t effective refractory period, 409b efficacy (Emax), 19 efflux pumps, tetracycline-resistant microbes and, 592 eflornithine, African trypanosomiasis and, 640, 647t EGFR. See epidermal growth factor receptor eicosanoids case study, 741 drug summary table, 761–764t future directions, 759–760 inflammation and, roles of, 752t inflammatory response and, 737 metabolic inactivation, 748 pathophysiology of, 752–754 pharmacologic classes and agents, 754–759 pharmacology, 740–760 eicosapentaenoic acid (EPA), 331t, 741 EKG. See electrocardiogram elderly anticholinergic drugs, adverse effects and, 126b antimuscarinics and, 125 drug metabolism and, 53 plasma drug concentration and, 39b renal excretion, drugs and, 36 electrical dysfunction, pathophysiology of, 406–408 electrocardiogram (ECG or EKG), 405b, 405f electrochemical gradient, 84 electrochemical transmission, 88–92 electroencephalogram (EEG) absence seizures and, 230f focal seizures and, 227 electrogenic transport, 85 electron transport chain inhibitors, 644–645t malarial plasmodia and, 634–635 electron transport chain, malarial plasmodia and, 631–632, 632f electroporation, 919 electrostatic force, 84, 85f eletriptan, 217, 223t elimination half-life, drugs and, 38 elongation, 585 eltrombopag, 788t Emax. See efficacy EMEA. See European Medicines Agency emergency (morning-after) contraception, 517 emesis, 191 EMLA (eutectic mixture of local anesthetic), 156, 162t emphysema, 822b emtricitabine (FTC), 659, 671t enalapril, 349t, 371t, 444 encainide cardiac rhythm and, 412, 419t drug study and, 874 endocrine axis, 468 endocrine disorders protein therapeutics and, 903–904t, 909t endocrine pancreas case study, 525 drug summary table, 538–540t pharmacology of, 524–537 endocytosis, 28 endogenous catecholamines, 137 endogenous pathway modifiers, 723t endometriosis, 511–512 endoperoxide, 373 endoplasmic reticulum (ER), 47 endorphins, 106 endothelial cell contraction, 766

endothelial dysfunction, 448 endothelial injury, 381 endothelial isoform of nitric oxide synthase. See eNOS endothelial-derived relaxing factor. See EDRF endothelin, 357, 357f endothelin receptor antagonists, 365, 370t endothelin-converting enzyme, 357 end-plate potential (EPP), 91, 115, 118f end-plate region, 117f end-systolic pressure-volume relationship (ESPVR), 456, 457f energy homeostasis, 525–526, 526t enflurane, 249, 255, 260t cytochrome P450 enzymes and, 52t enfuvirtide (T-20), 652–653, 653f, 670t, 906, 909t enkephalins, 106, 269 eNOS (endothelial isoform of nitric oxide synthase), 356 enoxacin, cytochrome P450 enzymes and, 52t enoxaparin, 391, 398t entacapone, 195, 203t Entamoeba histolytica, 629, 637 entecavir, 655, 671t enteral drug administration, 30–31, 30t enteral formulations, 858 enteric coating, 858 enterochromaffin-like (ECL) cells, histamine and, 808 enterohepatic circulation, 37, 321 envelope, capsid and, 650 environment, drug metabolism and, 53–54 environmental toxicology, 881 acute and subchronic toxicology, 881–887 carcinogenicity and chronic toxicology, 887–892 case study, 882 conclusion and future directions, 893 enzyme activity assays, 855 enzyme inhibition, 50 structural basis of, 5f enzyme variation, drug metabolism and, 72–75, 73t enzymes, 13 of cell wall biosynthesis, 603b eosinophil, 732t eosinophils, 731, 781, 826–827 EPA. See eicosapentaenoic acid ephedra, FDA and, 871 ephedrine, 139, 827 EPI. See epinephrine epidermal growth factor receptor (EGFR) inhibitors, 699, 707, 713t epilepsy agents for, 233 lamotrigine and, 180 epileptic seizures, classification of, 228t epinephrine (EPI), 10, 132, 137, 156, 186, 432, 435t, 821, 827, 834t beta adrenergic receptors and, 14 conjugation enzyme and, 73t neurotransmitters and, 102, 103t epipodophyllotoxins, 691–692, 723t epirubicin, 697t epitope, 732 eplerenone, 347, 352t, 442t, 500, 504t epoetin alfa, drug development and, 867 epoetin alfa, protein therapeutics and, 901t epoprostenol, 763t epoxygenase pathway, 748 EPP. See end-plate potential EPSPs. See excitatory postsynaptic potentials eptifibatide, 386, 396t equilibration of alveolar, with inspired partial pressure, 246–247, 247f equilibration of tissue, with alveolar partial pressure, 247–248, 248t ER. See endoplasmic reticulum; estrogen receptor erectile dysfunction (ED), 362 EREs. See estrogen response elements ergocalciferol, 546, 560t vitamin D, 557 ergosterol synthesis inhibitors, 622–624 pathway, 619, 619f

938 Index ergotamine, 217, 278 ergots, 217 eribulin, 692, 698t erlotinib, 707, 713t ertapenem, 611, 617t erythrocyte production, 779–781 agents stimulating, 783, 787t erythrocytes, 776 erythromycin, 51t, 566, 592, 594f, 597t, 719 metabolic drug interactions, 54 erythromycin A, 593f erythropoiesis, 779–781, 781t erythropoietin, 779–781, 787t, 895, 898, 901t synthesis, regulation of, 780f erythropoietin, protein therapeutics and, 901t ESBLs. See extended-spectrum ␤-lactamases Escherichia coli, 585, 604, 609 escitalopram, 216 esmolol, 141t, 142, 146t esomeprazole, 813, 818t off-target effects, 60 ESPVR. See end-systolic pressure-volume relationship essential hypertension, blood pressure and, 439 estazolam, 171 clinical uses for, duration of action, 172t GABAA receptors and, 183t ester-linked local anesthetics, 151, 151f, 156, 158, 161t estradiol, 922 estramustine, 688, 696t estrogen receptor (ER), 506 antagonists, drug summary table for, 521t estrogen response elements (EREs), 506 estrogen-progestin contraception, 516–517, 523t estrogen(s). See also ethinyl estradiol contraception, 516f decrease of, 512 hormone replacement, 518 oxidation/reduction enzyme and, 73t synthesis, 505–506 ET-1, 357 ET-2, 357 ET-3, 357 etanercept, 758, 762t, 798–799, 804t, 906, 907t ethacrynic acid, 346, 351t, 442t conjugation reactions, 46t ethambutol, 612, 617t, 719 ethanol cytochrome P450 enzymes and, 52t GABA physiology and, 176 metabolic drug interactions, 54 oxidation/reduction enzyme and, 73t toxicity, 889–890 ethanolamines, 774t ethinyl estradiol, 516, 523t ethionamide, 612, 617t, 720 ethnicity, drug metabolism and, 53 ethosuximide, 232t, 237t absence seizures and, 234 ethyl ether, 255 ethylenediamines, 774t ethynodiol, 516, 523t etodolac, 756, 761t etomidate, 175, 261t GABAA receptors and, 184t intravenous anesthesia and, 255 etonogestrel, 517, 523t etoposide, 697t, 723t oxidation/reduction enzyme and, 73t etoposide (VP-16), 691 etravirine, 661, 671t European Medicines Agency (EMEA), 70, 869 European Union, 869 eutectic mixture of local anesthetic. See EMLA everolimus, 12, 710, 714t, 804t, 922 STEMI, 454 everolimus-eluting stents, 797–798 excitability. See cellular excitability excitatory neurotransmitters, 164–165, 165f excitatory nicotinic acetylcholine receptors, 257 excitatory postsynaptic potentials (EPSPs), 90, 116

excitotoxicity, 178 ischemic stroke, 179, 179f excretion, drug, 27, 28f exemestane, 511, 520t exenatide, 535–536, 539t protein therapeutics and, 904t exogenous glucagon, 540t exogenous insulin, 533–534, 538t extended release formulations, drug delivery and, 917 extended-spectrum ␤-lactamases (ESBLs), 609 external advisory committees, FDA and, 867–868 extracellular enzymes, 13 extraction ratio, 38 extrapyramidal effects antipsychotics and, 199 schizophrenia and, 197 extrinsic pathway, coagulation cascade and, 377, 378f ezetimibe, 326–327, 330t conjugation reactions and, 46t

F facilitated diffusion, 28, 107 factor IX, protein therapeutics and, 898, 899t factor V Leiden, 381 factor VII, coagulation cascade and, 377 factor VIII, protein therapeutics and, 898, 899t factor Vlla, protein therapeutics and, 903t factor X, coagulation cascade and, 377 factor XII, coagulation cascade and, 377 famciclovir, 658, 670t familial combined hyperlipidemia (FCH), 323 familial hypercholesterolemia (FH), 322 familial hypertriglyceridemia, 322–323 familial lipoprotein lipase deficiency, 322–323 famotidine, 775t peptic ulcer disease and, 812, 818t FAS. See fetal alcohol syndrome FAS1. See fatty acid synthetase 1 FAS2. See fatty acid synthetase 2 fasting state, 525–526 fat group (FG), anesthesia and, 246 fatty acid synthetase 1 (FAS1), 605 fatty acid synthetase 2 (FAS2), 605 FCH. See familial combined hyperlipidemia FDA (U.S. Food and Drug Administration), 847 approval, 868–869 approved drugs, pharmacokinetics, 37, 860 major legislation and, 861–862 review process, 867–868, 869f role of, 879 FDA Amendments Act (FDAAA), 862, 879 FDAAA. See FDA Amendments Act febuxostat, 842, 844t fed state, 525 felbamate, 232t, 235, 239t NMDA receptor antagonists, 185t NMDA receptors and, 103 felodipine, 51t, 369t cytochrome P450 enzymes and, 51t FemA enzyme, 603 FemB enzyme, 603 femoral blocks, 157 FemX enzyme, 603 fenamate derivatives, 756 fenamates, 761t fenestrae, 106 fenfluramine off-target effects and, 60 serotonin storage and, 214 fenofibrate, 326, 328 lipoprotein metabolism and, 331t fentanyl, 256, 261t, 275, 280t fenthion, toxicity, 885 fermentation enzymes anaerobic organisms and, 638, 639f luminal parasites and, 637–638 fertility protein therapeutics and, 902t

fesoterodine, 125, 130t fetal alcohol spectrum disorder, 302 fetal alcohol syndrome (FAS), 890 fetal hemoglobin (HbF), agents inducing, 783–784, 787–788t fetus, teratogenesis and, 67–68, 68b fexofenadine, 775t off-target effects, 60 FG. See fat group FGF-23. See fibroblast growth factor 23 FH. See familial hypercholesterolemia fibrates, 326, 331t lipid metabolism and, 327–328, 327f fibrin sealant protein therapeutics and, 903t fibrinogen, 375 protein therapeutics and, 900t fibrinoid arteriolar necrosis, 446 fibrinolysis inhibitors, 393, 400t fibroblast growth factor 23 (FGF-23), 547 Fick’s law, 245, 254, 263 filgrastim, 784–785 drug-induced toxicity, 69 immunotoxicity and, 65 filgrastim (rhG-CSF), 788t protein therapeutics and, 901t finasteride, 511, 520t first pain, A␦-fibers and, 149, 150f first-order kinetics, 37 first-pass effect, 43 first-pass liver metabolism, enteral drug administration, 31 FK-binding proteins (FKBP), 797 FKBP. See FK-binding proteins FLAP inhibition. See 5-lipoxygenase activating protein inhibition flecainide cardiac rhythm and, 412, 419t cytochrome P450 enzymes and, 51t drug study and, 874 flocculonodular lobe, 98, 98f flow rate, global equilibration and, 245 FLT3 inhibitors, 708–709 fluconazole, 567, 623–624, 627t cytochrome P450 enzymes and, 52t flucytosine, 620–621, 621f, 626t, 720 fludarabine, DNA structure, 685f fludarabine phosphate, 695t DNA structure of, 686, 686f fludrocortisone, 494, 495, 495f, 500, 504t flukes. See trematodes flumazenil, 172 benzodiazepine overdose and, 69 GABAA receptors and, 183t general anesthesia and, 255 flunisolide, 497, 503t, 835t flunitrazepam, GABA physiology and, 176 fluoride, bone and, 555, 560t 5-fluorocytosine, 567 fluoroquinolones, 587 off-target effects and, 60 5-fluorouracil (5-FU), 566, 694t combination chemotherapy, 725 DNA structure, 684f fluoxetine, 39, 92, 216, 221t, 277 addiction and, 305 cytochrome P450 enzymes and, 51–52t drug dependence and, 309t flupentixol, 198f fluphenazine, 198f, 204t schizophrenia and, 197, 199 flurazepam, 171 clinical uses for, duration of action, 172t GABAA receptors and, 183t flurbiprofen, 756, 761t flutamide, 511, 515, 522t, 723t fluticasone, 497, 498f, 503t, 830, 835t fluticasone propionate, 498f fluvastatin, 325–326, 330t fluvoxamine, 216, 221t cytochrome P450 enzymes and, 52t

Index 939

fMet. See formylated methionine foam cell, 318 focal seizures, 227–228, 228t focal seizures, pathophysiology of, 227–228 focus, 227 folate analogues, structures of, 575–577, 575f metabolism inhibitors malarial plasmodia and, 636, 645t selective targeting, synergistic drug interactions and, 574–578 structures of, 575–577, 575f synthesis and functions, 576f folic acid. See Folate follicle-stimulating hormone (FSH) anterior pituitary gland, 467, 474 Group Ib proteins, 898 follicular phase, menstrual cycle and, 509 follitropin (rFSH), 474, 479t fomivirsen, 665, 672t fondaparinux, 391, 398t Food and Drug Administration. See FDA Food and Drug Law, history, 861–862 food contaminants, 884–885 Food, Drug, and Cosmetic Act, 862 food illnesses, 884 formestane, 511, 520t formoterol, 828, 834t formylated methionine (fMet), 585 fosamprenavir, 662, 672t foscarnet, 661, 671t fosfomycin, 606–607, 614t fosinopril, 349t fosmidomycin, 606–607, 614t four-compartment model, drug distribution, 36f Frank-Starling law, 424, 456, 457, 458f FRC. See functional residual capacity free ligand, concentration (L), 17 frontal cortex, dopamine receptor proteins and, 189 frontal lobe, 97, 97f frovatriptan, 217, 223t FSH. See follicle-stimulating hormone; human follicle-stimulating hormone; urofollitropin FTC. See emtricitabine 5-FU. See 5-fluorouracil 5-FU/folinic acid, 684 full agonist, 6–7 full agonist dose-response curves, 23f fulvestrant, 511, 521t functional residual capacity (FRC), anesthesia and, 247 fungal cell wall, biochemistry, 618–620 fungal infections case study, 619 drug summary table, 626–628t pathophysiology of, 620 pharmacology of, 618–625 fungal membrane biochemistry of, 618–620 stability, inhibitors, 628t stability, polyenes and, 624–625, 628t fungal mitosis inhibitor, 622, 626t fungal nucleic acid synthesis inhibitor, 620–621, 621f, 626t fungal wall synthesis inhibitors, 625, 628t fungi, 885 drugs and, 567–568 toxic, 885 furosemide, 346, 351t, 441, 442t

G G protein receptor kinase (GRK), 137 G protein-coupled receptors (GPCRs), 9–10, 9f, 113, 427 G proteins, 9, 10t, 14, 113 adenylyl cyclase and, 10f dopamine receptors and, 189 peripheral sensitization and, 271 second messengers and, 9, 10f G1-S cell cycle transition, 703f

G6PD deficiency (Glucose-6 phosphate dehydrogenase deficiency), primaquine and, 635 GABA (␥-aminobutyric acid), 8–9, 268 agonists, drug table for, 308t binding site, 177 channel potentiators, drug summary table for, 238t ivermectin and, 642 metabolism, inhibitors, 166, 166f, 182t neurotransmission, 164–165, 165f alcohol and, 289 drug summary table, 183–185t opioids and, 295 pharmacologic classes and agents affecting, 168–176, 169t physiology, 165–168 neurotransmitters and, 102, 103t pathways, benzodiazepines and, 234 pharmacologic classes and agents affecting, 177–178, 178t physiology, 176 receptors, 166–168 helminths and, 641 structure, 167f GABA transporter (GAT), 166 GABAA. See also Inhibitory GABAA channels, alcohol and, 299 receptor, 166–168, 167f agonists and antagonists, 182t barbiturates, 183–184t benzodiazepines, 183t, 298, 298f receptor modulators, 183–184t GABAA-mediated chloride conductance, structure, 167f GABAB receptor agonists, 175, 184t receptor antagonists, 175 receptors, 166, 168, 168f GABAC, receptor, 166–168, 167f GABA-mediated surround inhibition, 229, 229f drugs enhancing, 234 gabapentin, 232t, 234, 237t, 277 GABA-transaminase (GABA-T), 166, 166f gabazine, 182t GABAergic transmission and, 169t gaboxadol, 170 GABAA receptors and, 182t GABAergic transmission and, 169t GAD. See glutamic acid decarboxylase galantamine, 121, 122t, 129t gallbladder disease, 517 galsulfase, protein therapeutics and, 900t gamma (␥)-hydroxybutyric, GABA physiology and, 176 gamma-vinyl GABA (Vigabatrin), 182t, 234 GABA metabolism and, 169–170 GABAergic transmission and, 169t seizures and, 235 ganciclovir, 658, 670t, 922 ganglionic blockade, 116 ganirelix, 474, 479t, 909t gastric acid, secretion, 807–810 gastric phase, gastric acid secretion, 809 gastrins, 106, 807 gastrointestinal stromal tumor (GIST), 708 gatifloxacin, 596t GATs. See GABA transporter G-CSF. See granulocyte colony-stimulating factor gefitinib, 76, 707, 713t gemcitabine, 686, 695t gemfibrozil, 326, 328 lipoprotein metabolism and, 331t gemtuzumab ozogamicin, 712, 715t, 723t, 910, 910t gender, drug metabolism and, 53 gene expression inhibitors, 793–794, 803t general anesthesia cardiac output distribution and, volume capacity and, 246f pharmacology, 254–256 recovery, 253–254, 254f

general anesthetics CNS and, 98 cytochrome P450 enzymes and, 52t mechanism of action, 256–258 pharmacology, 240–263 structures, 257f general growth factors, 778 generalized seizures, 227, 228t generic drugs, 870 generic name, drugs, 869 genes alcohol dependence and, 294 drug metabolism and, 53 genetic polymorphisms drug metabolism and, 73t examples of, drug targets and, 76t genome(s) biochemistry, 675–683 replication, viral life cycle and, 650 synthesis, 674–693 gentamicin, 39f, 589, 596t renal toxicity, drug-induced, 66 geranylgeranylation, 59 gestodene, 516, 516f, 523t GFR. See glomerular filtration rate GH. See growth hormone GHRH. See growth hormone-releasing hormone Giardia, 638 ginkgo biloba, platelet aggregation and, 61 GIST. See gastrointestinal stromal tumor glibenclamide (Glyburide), 538t gliclazide, 538t glimepiride, 538t glipizide, 538t gliquidone, 538t global equilibration, anesthesia and, 245 globus pallidus, 98, 191 glomerular dysfunction, 342 glomerular filtration rate (GFR) digoxin and, 430 natriuretic peptides and, 336 renal excretion, drugs and, 36 glomerulonephritis, 753–754 glossopharyngeal nerve, 96 GLP-1. See glucagon-like peptide-1 glucagon, 470, 529, 537, 540t, 913t glucagon-like peptide-1 (GLP-1), 530, 535–536 glucocorticoid receptor agonists, drug table, 503t glucocorticoid receptor antagonists, drug table, 503t glucocorticoid response elements (GREs), 492 glucocorticoid(s), 489–490, 754, 762t analogues, 494, 495f potencies and durations of action, 495–496, 496t bone mineral metabolism and, 547 dosing, 496–497 gene expression and, 793–794 gout and, 841 pathophysiology, 493–494 physiology, 490–493 routes of administration, 497–498 structure and potency, 494–495 synthesis, inhibitors of, 503–504t withdrawal, 497 glucose homeostasis, regulation of, 525–526, 527f glucose-6 phosphate dehydrogenase deficiency. See G6PD deficiency glutamate, 155, 164, 239t metabolism and, 176 neurotransmitters and, 102–103, 103t glutamate receptors, 176–178 classes of, interaction of, 179f drugs inhibiting, 235, 239t glutamate synthesis, 166f, 176 glutamate-gated chloride channels, ivermectin and, 642 glutamatergic neurotransmission pathophysiology and pharmacology, 178–180 pharmacology, 182–185t physiology of, 176–178

940 Index glutamic acid decarboxylase (GAD), 166, 166f glutaminase, 166f, 176 glutamine synthetase, 166f, 176 Glyburide. See glibenclamide glycine neurotransmitters and, 102, 103t pain and, 268 glycine, neurotransmitters and, 103t glycohemoglobin (HbA1c), 532 glycopeptides, 607–608 glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists drug summary table, 396t thrombus formation and, 386 UA/NSTEMI, 453 glycopyrrolate, 124, 130t glycylcyclines, 572, 591–592, 592, 597t GM-CSF. See granulocyte-macrophage colonystimulating factor; granulocyte-monocyte colony-stimulating factor GMP. See Good Manufacturing Practice GnRH. See gonadotropin-releasing hormone Goldman equation, 86 Goldman-Hodgkin-Katz equation, 85f, 86 golimumab, 758, 762t, 799, 804t, 907t gonadal hormone action, pharmacologic modulation of, 512f gonadal hormone inhibitors, 512–516 gonadorelin, 479t gonadotropin expression, drugs altering, 479t gonadotropin-releasing hormone (GnRH), 106, 479t, 508, 508f, 904t, 920 gonadotropin-releasing hormone (GnRH) agonists, 512–516, 520t gonadotropin-releasing hormone (GnRH) antagonists, 512–516, 520t, 909t gonadotropins, 473 Good Manufacturing Practice (GMP), 870 goserelin, 479t, 904t gout, 837–839 case study, 838 conclusion and future directions, 843 drug summary table, 844–845t natural history, 849t pathophysiology of, 839–840 pharmacologic agent, and classes, 840–842 GPCRs. See G protein-coupled receptors GPIIb-IIIa, 396t. See glycoprotein IIb-IIIa antagonists graded dose-response relationships, 19, 19f graft-versus-host disease (GVHD), 790, 792 graft-versus-leukemia effect (GVL), 792 gram-negative bacteria, 600 gram-positive bacteria, 600 grandiosity, 212 granulocyte colony-stimulating factor (G-CSF), 781 granulocyte colony-stimulating factor (G-CSF), protein therapeutics and, 901t granulocyte-macrophage colony-stimulating factor (GM-CSF) protein therapeutics and, 902t granulocyte-monocyte colony-stimulating factor (GM-CSF), 779, 781, 784–785 granulocytes, 730, 776 granulocyte-stimulating factors, 781–782 granulosa cells, gonadal hormone action and, 508–509 granzymes, 733 grapefruit juice, cytochrome P450 enzymes and, 51t grapefruit juice effect, 53 Graves’ disease, 484 gray baby syndrome, 53, 593 grepafloxacin, 60 GREs. See glucocorticoid response elements GRH. See growth hormone-releasing hormone griseofulvin, 622, 626t GRK. See G protein receptor kinase growth factor receptor antagonists, 707–710 growth factor(s) growth factor receptors, 699–700, 700f RAS-MAP kinase pathway, 702–703f structure and function, 700f

growth hormone (GH) agents decreasing, 477t anterior pituitary gland, 467 deficiency, pathophysiology and pharmacology of, 469–470 excess, pathophysiology and pharmacology of, 470–471 protein therapeutics and, 899t replacement, 477t growth hormone-releasing hormone (GHRH), 106, 912, 913t anterior pituitary gland, 467–468 growth regulation, protein therapeutics and, 904t guanabenz, 140, 144t, 443 guanadrel, 138, 143t guanethidine, 138, 143t, 443 guanfacine, 140, 144t guanine, 581, 675 guanine alkylation, DNA and, 688f guanylyl cyclase, 355 GVHD. See graft-versus-host disease GVL. See graft-versus-leukemia effect

H H1-antihistamines. See histamine 1 (H1) -antihistamines H1N1 swine flu, 665 H2-receptor antagonists. See histamine 2 (H2) -receptor antagonists H3 receptors. See histamine 3 receptors H4 receptors. See histamine 4 receptors H5N1 avian influenza, 665 Haemophilus influenzae, 592–594 half-life, of drugs factors altering, 38–39, 38t local anesthetics and, 158 hallucinations, schizophrenia and, 195 haloperidol, 58, 74, 198f, 204t cytochrome P450 enzymes and, 51t parenteral drug administration and, 31t schizophrenia and, 197, 199 halothane, 73t, 242, 248f, 249, 249f, 251f, 254, 260t cytochrome P450 enzymes and, 52t oxidation/reduction reactions and, 73t haptens, 62 Hashimoto’s thyroiditis, 483f, 484 Hatch-Waxman Act, 870 HbA. See hemoglobin A HbA1c. See glycohemoglobin HbF. See fetal hemoglobin; hemoglobin F HbS. See hemoglobin S HBsAg vaccine, 911t HBV. See hepatitis B virus HC1. See hydrochloric acid hCG. See human chorionic gonadotropin HDL. See high-density lipoprotein Health Canada, 869 “healthy user” effect, 877–878 heart, 401 electrical physiology of, 401–406 heart failure (HF), 422 afterload reduction, 461 case study, 45 clinical management, 458–462 doxorubicin, 691 Frank-Starling relationship in, 458f neurohumoral effects of, 458, 459f pharmacologic modulation of, 459f pathophysiology of, 455–458 pharmacologic agents, 460t preload reduction, 459–461 sodium retention, mechanisms, 340–341, 341f heavy metal chelators, 884f Helicobacter pylori, 638 peptic ulcer, 810–811, 810f treatment, 816–817 helminths (worms), 640–641 case study, 640 physiology of, 641 helper-T cells (TH1 cells), 732t, 734f, 821, 824

hemagglutinin, viral uncoating and, 653–655 hematopoiesis case study, 777 drug summary table, 788–789t future directions, 786 pharmacology, 776–786 physiology, 776–782 hematopoiesis, protein therapeutics and, 901–902t hematopoietic cells, 777t hematopoietic growth factors, 776, 778–779 hematopoietic system, cells of, 778t heme metabolism inhibitors, 644t malarial plasmodia and, 631–632, 631f heme protein mono-oxygenases, 47 hemicholinium-3, 112f, 120, 128t hemochromatosis, drug metabolism and, 54 hemoglobin, 779 hemoglobin A (HbA), 779 hemoglobin F (HbF), 779 hemoglobin S (HbS), 779 hemolysis, primaquine and, 635 hemophilia A, 898 hemophilia B, 898 hemorrhagic disorders, 381, 383b hemostasis, 372, 374f pharmacology of, 372–394 physiology of, 372–380 protein therapeutics and, 903t, 909t regulation of, 379–380, 395–400t Henderson-Hasselbalch equation, drugs and, 29 heparin, 23, 389 clinical uses, 391 mechanism of action, 389–391 UA/NSTEMI, 453 heparin-induced thrombocytopenia (HIT), 391 heparin-induced thrombocytopenia syndrome, 381 hepatic enzymes, drugs and, 54 hepatic metabolism, routes, 156 hepatitis B immune globulin, 909t hepatitis B virus (HBV), 651, 898 vaccine, 912 hepatitis C antigens, 898, 914t hepato-renal reflex, cirrhosis and, Na⫹ retention and, 341–342 hepatotoxicity, thioamines and, 65–66 HER2/neu inhibitors, 713t hERG channels, 59 heroin, 286t case study, 285 methadone vs., pharmacodynamics of, 304–305, 304f morphine vs., 296 herpesvirus, 655 heterologous desensitization, 14 HETEs. See hydroxyeicosatetraenoic acids hexamethonium, 115, 133, 443 tetanic fade, 118f hexose transporter, 106 HF. See heart failure HGPRT. See hypoxanthine-guanine phosphoribosyltransferase Hibernian fever, 800 HIF-1alpha. See hypoxia-inducible factor-1alpha high innate tolerance, 294 high-density lipoprotein (HDL), 311 drug abuse and, 302 formation, 318–320 intravascular maturation, 320 liver and, 319f, 320 mediated cholesterol efflux, 320 metabolism disorders, 323 reverse cholesterol transport and, 318–320, 319f high-throughout screening, 851–852 high-voltage-activated (HVA) calcium channel, 232t, 233 hippocampal formation, 98 hippocampus, 98 dopamine receptor proteins and, 189 histamine 1 (H1)-antihistamines adverse effects, 771–772

Index 941

first generation, 770f, 774–775t structure, 770, 770f pharmacologic effects, clinical uses and, 771 receptor, model, 770f second generation, 770–771 histamine 2 (H2)-receptor antagonists, 772, 775t peptic ulcer disease and, 812–813, 813f, 818t structure, 772f histamine 3 receptors (H3 receptors), 768, 772 histamine 4 receptors (H4 receptors), 768, 772–773 histamine(s) anaphylaxis and, 769 case study and, 766 conjugation enzyme and, 73t drug summary table, 774–775t gastric acid and, 807–808 inflammation response and, 736 neuronal systems and, 102 neurotransmitters and, 102, 103t, 104, 106 oxidation/reduction reactions and, 45t pathophysiology, 768–769 clinical manifestations of, 769 pharmacology, 765–769 agents and classes, 769–773 strategies, 769t physiology of, 765 actions in, 765–767, 767t receptors, 767–768 subtypes, 767t histidine decarboxylase enzyme, histamine and, 765 histrelin, 479t, 904t HIT. See heparin-induced thrombocytopenia hit-to-lead development, 852 HIV (Human immunodeficiency virus), 649 case study, 44 combination chemotherapy, 660b enfuvirtide, 652–653 gp41-mediated fusion, 653f integrase inhibitors, 661–662, 663f life cycle, 652f protease inhibitor, 662 HIV antigens, 914t HIV DNA, 663f HIV pol gene product, ritonavir and, 666f HIV protease inhibitor, 662 hives. See acute urticaria HLA. See human leukocytic antigen HMG-CoA (hydroxymethylglutaryl-coenzyme A) on-target effects and, 58–59 reductase, 22 Hodgkin’s disease Ann Arbor staging system for, 725t antineoplastic combination chemotherapy and, 725 homologous desensitization, 14 homologous recombination, 676–678, 681 homovanillic acid (HVA), 189, 189f horizontal transmission, 572 hormone release inhibitors, 485–486 hormone replacement therapy (HRT), 559t. See also reproduction hormones bone homeostasis disorders and, 552 hormone deficiency and, 518 hormone synthesis, in adrenal cortex, 490–491, 491f hormone-dependent tissues, inappropriate growth of, 511–512 hormone(s). See also specific e.g. steroid hormones action and metabolism, 506–508 bone mineral metabolism and, 547–548 islets of Langerhans, 524 modulators, 723t HPA axis. See hypothalamic-pituitary-adrenal axis HPETEs. See hydroperoxyeicosatetraenoic acids hPTH 1-34, 560t hPTH 1-84, 560t HPV vaccine, 911t HSV. See herpesvirus 5-HT. See serotonin (5HT) human albumin, protein therapeutics and, 901t human chorionic gonadotropin (hCG), 510 protein therapeutics and, 902t

human deoxyribonuclease I, protein therapeutics and, 905t human follicle-stimulating hormone (FSH), protein therapeutics and, 902t human immunodeficiency virus. See HIV human leukocytic antigen (HLA), 531 human parathyroid hormone residues 1-34 protein therapeutics and, 903t human solute linked carrier (SLC), 28 humoral immunity, 733–734 HVA. See homovanillic acid HVA calcium channel. See high-voltage-activated calcium channel hyaluronidase, protein therapeutics and, 905t hydralazine, 371t, 443 conjugation enzyme and, 73t drug metabolism and, 53, 72 immune responses, immunotoxicity and, 64 vascular tone and, 365 hydrochloric acid (HC1), tissue damage and, 885 hydrochlorothiazide, 346, 352t, 441, 442t, 474 hydrocodone, 274, 280t hydrocortisone, 503t hydroflumethiazide, 352t hydrofluoric acid, tissue damage and, 885 hydrogen bonds, 3 hydromorphone, 274 hydroperoxyeicosatetraenoic acids (HPETEs), 745 hydrophilic protein segments, 3 hydrophobic effect, 3–4 hydrophobic protein segments, 3 hydroxyapatite, 541 hydroxyeicosatetraenoic acids (HETEs), 745 hydroxy-methylglutaryl-coenzyme A. See HMG-CoA 11␤-hydroxysteroid dehydrogenase, 491, 492f 4-hydroxytamoxifen, 50 5-hydroxytryptamine. See Serotonin 5-hydroxytryptophan, 208 hydroxyurea, 685, 695t, 723t, 784, 787t hydroxyzine, 770, 771, 775t hyperacute rejection, 790–791 hyperalgesia, 271 hyperbaric pressure, anesthesia and, 256 hypercalcemia loop diuretics, 346 treatment, 554b hypercalcemia of malignancy, 553 hypercholesterolemia, 322, 342 hypercoagulability, 381, 382t hyperinsulinemia, therapy for, 536–537 hyperkalemia digoxin and, 430 diuretics and, 346, 347 loop diuretics, 346 hyperphosphatemia, 551 hyperpolarized membrane, 83 hyperreactivity, asthmatic airways and, 824 hyperresponsiveness, asthmatic airways and, 822–824, 824f hypersensitivity asthmatic airways and, 824 pain, pathophysiology and, 269 hypersensitivity reactions, 62 mechanisms of, 59f types of, 64t hypersensitivity response. See also IgE-mediated type I hypersensitivity type I, 62 asthma and, 824 type II, 62 hypersensitivity response type III (immune complex mediated hypersensitivity), 62 hypersensitivity response type IV (delayed-type hypersensitivity), 62, 824 hypertension adults and, classification of, 439, 439t case study, 438 clinical management, 440–446 diuretics for, 441–442, 442t pathophysiology, 438–440

hypertensive crisis, 445–446 tyramine toxicity and, 215 hyperthyroidism drug metabolism and, 54 treatment of, 485–487 hypertriglyceridemia, 322–323 hypocalcemia, treatment, 554b hypoglycemia, 470, 532 hypogonadism, 512 hypokalemia digoxin and, 430 potassium and, 442 hypomanic episode, 212 hypoparathyroidism, 557 hypotension ACE inhibitors and, 344 nesiritide and, 344 hypothalamic physiology, 465–468 hypothalamic-pituitary portal vascular system, 465, 466f hypothalamic-pituitary-adrenal axis, 472–473, 472f, 494 hypothalamic-pituitary-gonadal axis, 473–474, 473f hypothalamic-pituitary-growth axis, 468–469, 469f hypothalamic-pituitary-prolactin axis, 471–472, 471f hypothalamic-pituitary-reproduction axis, 508–509, 508f disruption of, 511 hypothalamic-pituitary-target organ feedback, 468 hypothalamic-pituitary-thyroid axis, 472 in health and disease, 483–484, 483f hypothalamus, 98 dopamine cell bodies in, 190–191 dopamine receptor proteins and, 189 pituitary gland vs., 465–468 hypothyroidism, treatment, 484–485 hypoventilation, anesthetic and, 251 hypoxanthine-guanine phosphoribosyltransferase (HGPRT), 685, 837–838 hypoxia, 354 hypoxia-inducible factor-1alpha (HIF-1alpha), 706, 706f

I IB. See Investigator’s Brochure ibandronate, 552, 559t ibritumomab tiuxetan, 712, 715t, 910, 910t IBS. See irritable bowel syndrome ibuprofen (Motrin), 276, 756, 761t conjugation enzyme and, 73t cytochrome P450 enzymes and, 52t drug study and, 877 gout and, 844t immune responses, immunotoxicity and, 64 ibutilide, cardiac rhythm and, 414, 420t ICH. See International Conference on Harmonization idiopathic hypereosinophilic syndrome, 708 idiosyncratic drug reactions, 77 idiosyncratic toxicity, 60 IDL. See intermediate-density lipoprotein idoxuridine, 671t idursulfase, protein therapeutics and, 900t IECs. See Independent Ethics Committees ifosfamide, 696t IgE-mediated type I hypersensitivity, 768, 768f asthma, 825–826 IGF-1. See insulin-like growth factor 1 IHD. See ischemic heart disease IL-1 inhibitors. See interleukin-1 inhibitors IL-2. See interleukin-2 IL-3. See interleukin-3 IL-5. See interleukin-5 IL-11. See interleukin-11 IL-12 inhibitors. See interleukin-12 inhibitors IL-23p40 inhibitors. See interleukin-23p40 inhibitors iloperidone, 206t imatinib, 708, 713t BCR-Abl kinase and, interaction with, 2, 3f receptor affinity, 4, 5f

942 Index imatinib mesylate, 699 imciromab pentetate, 913t IMiD. See immunomodulatory drug imidazole, 622–624, 627t unfavorable drug combinations, 721 imiglucerase, drug development and, 867 imipenem, 611, 617t cilastatin and, 61 imipramine, 52t, 74, 144t, 213, 215, 221t, 277 cytochrome P450 enzymes and, 51–52t imiquimod, 668, 673t immediate hypersensitivity, 62 immune cells, depletion of, 800–801, 805–806t immune complex mediated hypersensitivity. See hypersensitivity response type III immune diversity, 733 immune rejection, modes of, 791t immune responses activation of, 732 cortisol and, 492, 493f harmful, 62–63 immune system, 730 adaptive branch, cells of, 732–736 antiviral drugs and, 673t cells of, 731f, 732t drugs modulating, 668 innate branch, cells of, 730–732 immunoglobulin, 733 immunomodulatory drug (IMiD), 711, 723t with antineoplastic applications, 785–786 drug summary table, 789t immunoregulation, protein therapeutics and, 902–903t immunosuppressants, cytochrome P450 enzymes and, 51t immunosuppression case study, 791 cytotoxic agents and, 794–796 drug summary table, 803–806t future directions, 802 pharmacology, 790–802, 794f immunotoxicity, 64–65 IMP. See inosinate impulse conduction, defects in, 407–408 impulse formation, defects in, 406–407 inactivated state, CNS and, 226 inactivation, 8, 14 inactive state, 20 inamrinone, 436t heart failure and, 460t, 462 increased intracranial pressure, 345 incretins, 539t IND application. See Investigational New Drug application indapamide, 352t, 442t Independent Ethics Committees (IECs), 862 indinavir, 51t, 662, 672t indium-111-octreotide, 913t indomethacin, 276, 756, 761t induced fit, 4 inducible nitric oxide synthase (iNOS), 429 induction times, anesthetics, 250f inferior cervical ganglion, 96 inferior mesenteric ganglion, 96 infiltration anesthesia, 156 inflammation chemical mediators, 736–737, 736t eicosanoids and, roles of, 752t histamine and, 736 immune system and case study, 730 principles of, 729–739 inflammatory bowel disease, 753 TNF-␣ and, 906 inflammatory response, 737–738, 738f infliximab, 758, 762t, 799, 799f, 804t, 906, 908t influenza virus, viral uncoating, 653–654, 654f informed consent, 862 INH. See isoniazid inhalants, drugs abuse and, 286t, 301–302 inhaled anesthetics alveolar partial pressure of, determinants of, 247, 247f

pharmacodynamics of, 240–244, 242f pharmacokinetics of, 244–254 properties of, 243t recovery rate from, 254f inhaled general anesthetics, 260t inhaled nitric oxide gas, 360, 369t inhibin A protein, 509 inhibin B protein, 509 inhibin, hypothalamic-pituitary-reproductive axis and, 473 inhibitory G protein, 429 inhibitory GABAA, anesthesia and, 257–258, 258f inhibitory neurotransmitters, 164, 165f, 268 inhibitory postsynaptic currents (IPSCs), 167 inhibitory postsynaptic potentials (IPSPs), 90, 116, 167 initiation, translation system and, 585 initiators, cancer and, 67 injury, pain and, 270 innate immunity, 730–732 innate responses, 730 innate tolerance, 287 inorganic phosphate, 557, 561t iNOS. See inducible nitric oxide synthase inosinate (IMP), 675 inotropic agents, heart failure and, 462 inotropic receptors, 92 INR. See international normalized ratio insomnia, H1-antihistamines and, 771 inspired partial pressure, 241 alveolar with, equilibration of, 246–247, 247f rate of approach to, anesthetic agents and, 249f tissue groups with, equilibration of, 247–249, 249f Institutional Review Boards (IRBs), 862 insulin, 106 analogues, 5 beta-cells, 524 biochemistry, 526 cytochrome P450 enzymes and, 52t development of, 851 human, processing of, 528f protein-based therapies and, 898, 899t release, pancreatic beta cells, 528f replacement, 533–535, 534t exogenous insulin as, 533–534 resistance, 531 secretagogues, 535, 538t secretion, 526–529 sensitizers, 539t theory, 511 insulin aspart, 534, 538t, 899t insulin detemir, 534, 538t, 899t insulin glargine, 534, 538t, 899t insulin glulisine, 534, 538t insulin lispro, 534, 538t insulin receptor substrate (IRS) proteins, 11 insulin receptors, 11 activation, downstream effects, 529, 529f target tissues, 529 insulin-like growth factor 1 (IGF-1), 468 replacement, 477t integrase, viral enzyme, 659 integrated inflammation pharmacology, 807–819 asthma, 820–836 gout, 837–845 schema, 752 integration, viral proteins and, 651 intelligent drug delivery, 921–922 intercellular communication, cellular excitability and, 82, 92 intercellular signal transduction, biochemistry, 699–706 interferon alfa-2b, 902t interferon alfacon-1, 902t interferon alfa-n3, 902t interferon-alpha, 673t interferon alpha-2a, 902t interferon beta-1a, 903t interferon beta-1b, 903t interferon gamma-1b, 903t interferon-␣2b, 920

interferon-alpha, 668, 673t, 723t interferon-beta, 668 interferons, 785, 789t interferon-␥, 668, 821 type I, 668 type II, 668 interictal spikes, 227 interleukin-1 (IL-1) inhibitors, 800, 805t interleukin-2 (IL-2), 723t, 785, 903t interleukin-3 (IL-3), 779 interleukin-5 (IL-5), 781 interleukin-11 (rhIL-11), 785, 898, 902t, 903t interleukin-12 (IL-12) inhibitors, 799, 805t interleukin-23p40 (IL-23p40) inhibitors, 799, 805t interleukins, 737, 779, 782 intermediate-density lipoprotein (IDL), 317 intermediolateral columns, 95 International Conference on Harmonization (ICH), 854b, 862 international normalized ratio (INR), 389 intestinal phase, gastric acid secretion, 809 intra-arterial injection (IT injection), 31 intra-articular administration, 498 intracellular bacteria, 609 intracellular communication, cellular excitability and, 82 intracellular drug concentration, reduced, 572–573 intracellular receptors, 12–13, 13f intracellular signal transduction biochemistry, 699–706 pathways, 700–701 intravascular volume determinants of, 332–333 reduction of, 441–442 intravenous anesthetic agents, 255, 255f, 260–261t intravenous injection (IV injection), drugs and, 31 intravenous regional anesthesia, 157 intrinsic pathway, coagulation cascade and, 377, 378f inverse agonists, 7, 24 inverse tolerance, 285 Investigational New Drug (IND) application, 863 Investigator’s Brochure (IB), 863 iodide, 480 thyroid hormone and, 488t iodide uptake inhibitors, 478t, 485, 488t iodoquinol, malarial plasmodia and, 639 iodothyronine, 482 ion channels, 7, 8t cell membranes and, 84, 84f effects on, anesthesia and, 257–258, 258f inactivated period, 8 pharmacology of, 89 refractory period, 8 ionic interactions, 3 ionization state (pKa), 4 ionotropic GABA receptors (GABAA and GABAC), 166–168, 167f ionotropic glutamate receptors, 173f, 176, 176–177, 177t ionotropic receptors, 90 ions, Nernst potential for, 91, 113 iontophoresis, 919 ipodate hyperthyroidism and, 487 thyroid gland and, 488t ipratropium, 96, 124, 130t, 827, 834t iproniazid, 139, 144t, 213 serotonin degradation and, 214, 220t IPSCs. See inhibitory postsynaptic currents IPSPs. See inhibitory postsynaptic potentials irbesartan, 350t cytochrome P450 enzymes and, 52t IRBs. See Institutional Review Boards irinotecan, 691, 697t, 915 genetic polymorphisms, drug metabolism and, 73t irreversible antagonist, 21, 21f irritable bowel syndrome (IBS), 218 IRS proteins. See insulin receptor substrate proteins ischemia, 354 ischemic heart disease (IHD) classification of, 447f clinical management, 449–454

Index 943

pathophysiology of, 446–449 islets of Langerhans, 524 isocarboxazid, serotonin degradation and, 214, 220t isoetharine, 828, 834t isoflurane, 241, 245f, 249, 249f, 255, 257f, 260t alveolar partial pressure, 243f cytochrome P450 enzymes and, 52t isoflurophate, 121f isolated systolic hypertension, 438 isoniazid (INH), 231, 612, 617t conjugation reactions and, 46t cytochrome P450 enzymes and, 52t drug metabolism and, 52–53, 72 GABAergic transmission and, 169t immune responses, immunotoxicity and, 64 rifampin and, 588 tuberculosis and, 44 isoprostanes, 748 isoproterenol, 140, 145t, 432, 436t, 827–828, 834t isosorbide 5-mononitrate, 360, 360f, 361, 368t isosorbide dinitrate, 360, 360f, 361, 368t drug metabolism and, 53 isotretinoin, pregnancy and, 68 IT injection. See intra-arterial injection itch, 766, 771 itraconazole, 51t, 567, 623, 627t IV injection. See intravenous injection ivermectin, helminths and, 641–642, 647t

J Jacksonian march, 228 JAK2 inhibitors, 709 JAK-STAT pathway, 701 jimson weed, toxicity and, 885 Joint National Commission (JNC), 441 juxtaglomerular apparatus, 334 juxtaglomerular cell renin release, mechanisms, 334–335, 335f

K K⫹. See potassium kainate receptors, glutamate-gated ion channels, 176–177, 177t, 179 kanamycin, 589, 596t, 720 K⫹ATP channels, 365 Kayexalate. See sodium polystyrene sulfonate Kefauver-Harris Amendments, 862 keratinocyte growth factor (KGF), 904t kernicterus, 47, 576 ketamine, 255, 261t, 277, 283t ketanserin, 218, 223t ketoconazole, 473, 499, 499t, 504t, 623, 627t cytochrome P450 enzymes and, 50, 51t, 61 ketolides, 592, 597t ketones, 756, 761t ketoprofen, 756, 761t ketorolac, 276, 756, 761t KGF. See keratinocyte growth factor kidney drugs and, 36, 36f, 39f local anesthetics and, 156 toxicity, tetracyclines and, 592 kidney disease, chronic, drug use in, 39b kininase II, 335 kinins, 267 Kirsten ras gene, 700–701 Klebsiella pneumoniae, 609 Krebs cycle, glutamate synthesis, 166, 166f

L LABAs. See long-acting beta-agonists labetalol, 141t, 142, 146t, 442 cardiac rhythm and, 413, 419t laclase, protein therapeutics and, 900t lacosamide, sodium channels and, 232, 232t, 237t lactic acidosis, 535 lactotrophs, 189 lamellae, 543 lamivudine, 574, 659–661, 671t lamotrigine, 232t, 236t, 277 mood disorders and, 218

NMDA receptor antagonists, 185t seizures and, 180 sodium channels and, 232, 236t Langerhans cells, 781 lanreotide, 470 protein therapeutics and, 904t lansoprazole, 813, 818t cytochrome P450 enzymes and, 52t lapatinib, 707, 713t Laplace’s law, 457 laronidase, protein therapeutics and, 900t L-asparaginase, 898, 905t latanoprost, 763t late perimenopause, 548 late-acting growth factors, 778 latency, herpesviruses, 655 latent pacemaker, 406 laterodorsal tegmental area, nicotinic receptors and, 299 LCAT. See lecithin-cholesterol acyltransferase LD50. See median lethal dose LDL. See low-density lipoprotein LDL-receptor-related protein (LRP), 317 particles, formation and clearance, 317–318, 317f L-DOPA. See Levodopa lead drug discovery and, 848 poisoning by, 883–884 lead optimization, 853 leak channels, 87 learned tolerance, 287 lecithin-cholesterol acyltransferase (LCAT), 320 leflunomide, 794, 795–796, 796f, 803t left ventricular pressure-volume loop, 456f Legionella, 592 Leishmania, 640 lenalidomide, 711, 715t lepirudin, 392, 398t, 898, 905t leptin, 525 Lesch-Nyhan syndrome, 839 letrozole, 511, 520t leukocyte(s) production, 781–782 agents stimulating, 784–785, 788t suppressors of, acute gout and, 840–841, 844t leukotriene B4, 822b leukotriene inhibitors, 758–759, 759f leukotriene pathway-modifying agents, 830–831, 836t leukotriene receptor antagonists, 764t leukotriene synthesis inhibitors, 759 leukotriene(s), 745–748, 764t asthma, 825–826, 826f biosynthetic pathways, 746f leuprolide, 473, 479t, 723t, 904t levalbuterol, 834t levamisole, 785, 789t levetiracetam, 232t, 239t levobunolol, 142, 146t levobupivacaine, 157, 159 levocabastine, 775t levocetirizine, 775t levodopa (L-DOPA), 98, 104, 105f, 186–187, 193, 202t, 849 blood-brain barrier and, 106–107, 107f genetic polymorphisms, drug metabolism and, 73t Parkinson’s disease and, 193 levofloxacin, 60, 587, 596t, 720 levonorgestrel, 523t, 919 contraception, 516, 516f morning after contraception, 517 levorphanol, 281t levosimeridan, 436t levothyroxine hypothyroidism and, 485 thyroid gland and, 488t Leydig cells, gonadal hormone action and, 508 LFA-3, 801 LH. See luteinizing hormone LH hypothesis, 511

lidocaine, 156, 158–159, 161t, 277 cardiac rhythm and, 412, 419t dentistry and, 157b genetic polymorphisms and drug metabolism, 73t hydrolysis reactions and, 46t infiltration anesthesia, 156 loading dose, 41 on-target adverse effect, 58 oxidation/reduction reactions and, 45t patch, 156 toxicity, 158 life-threatening infections, combination antimicrobial drugs and, 721 ligand. See drug(s) ligand-binding domain, 8 ligand-gated channels, 7, 8t ligand-gated nicotinic acetylcholine receptor, 8f ligand-receptor complex concentration (LR), 17 limbic system, 98 dopamine receptor proteins and, 189 lincosamides, 594, 597t lineage-dominant growth factors, 778 lineage-specific growth factors, 779 linear synthesis, 857f linezolid, 572, 593f, 594, 598t linoleic acid, 741 liothyronine, thyroid gland and, 488t lipase, protein therapeutics and, 901t lipid-lowering agents, coronary artery disease and, 451 lipid(s), 311 solubility hypothesis, anesthesia and, 256–257 theories, 256 lipopeptides, 572 lipopolysaccharides (LPS), 601 lipoprotein lipase (LPL), 315–316 lipoprotein(s) ApoB-containing, metabolism of, 313–318 characteristics, 311–313, 312t metabolism case study, 312 pharmacology, 311–329 particles, structure of, 313, 313f liposome-based delivery systems, 922 liposomes, oral drug delivery and, 917 lipoteichoic acids, 601 lipotropin, 472 lipoxins, 748, 759 biosynthesis, 747f lipoxin-stable analogues, 759 5-lipoxygenase activating protein (FLAP) inhibition, 759 lipoxygenase inhibition, 758–759, 764t lipoxygenase pathway, 745–748, 745t liraglutide, 539t lisdexamfetamine, serotonin storage and, 213–214, 220t lisinopril, 349t, 371t, 444 lithium, 207 drug summary table, 224t mood disorders and, 218–219 thyroid hormone homeostasis, 487 liver drug metabolism and, 43, 44f, 54 failure, toxicity and, 65–66 HDL cholesterol and, 319f, 320 local anesthetics and, 156 oxidation/reduction reactions, 34 phenylalanine and, 186 liver cytochrome P450 oxidases, 47 LMW heparin. See low molecular weight heparin loading dose, 40f, 41 local anesthesia phasic inhibition, 153–155, 155f sodium channels, 151, 152f tonic inhibition, 154, 155f local anesthetic hydrophobicity, 151, 152f local anesthetics, 8, 147. See also amide-linked local anesthetics; ester-linked local anesthetics administration of, 156–157 amine group of, 151–152

944 Index local anesthetics (continued) calcium channels, 158 chemistry, 151–152, 151f in dentistry, 157f drug summary table, 161–162t future directions, 159–160 hydrophobicity, diffusion, binding, 152f individual agents, 158–159 major toxicities, 157–158 mechanism of action, 152–155, 153f nociception, 147–151, 148f pharmacokinetics of, 155–156 pharmacology of, 147–160 toxicities, 157–158 voltage-gated ion channels, 8 local circuit neurons, 99, 100, 100f local control mechanisms, vascular tone and, 358 local equilibration, anesthesia and, 245 locus ceruleus, 99, 101, 101f, 208, 300 lofexidine, withdrawal and, 303 log cell kill model, 571, 572f lonafarnib, 709 long tract neuronal systems, 99–100, 100f long-acting beta-agonists (LABAs), 828 loop diuretics, 345–346, 351t, 440t, 441–442, 442t lopinavir, 50, 574, 662, 672t loratadine, 51t, 771, 775t lorazepam, 183t, 238t, 261t clinical uses for, duration of action, 172t general anesthesia and, 255 seizures and, 234 losartan, 50, 51t, 350t, 371t, 845t cytochrome P450 enzymes and, 52t uric acid excretion and, 842, 845t lovastatin, 51t, 325–326, 330t oxidation/reduction enzyme and, 73t low molecular weight heparin (LMW heparin), 389, 391 coagulation factor inactivation and, 390t drug summary table, 398t low-density lipoprotein (LDL), 311 low-threshold mechanoreceptors, 148 loxapine, 204t LPL. See lipoprotein lipase LPS. See lipopolysaccharides LR. See ligand-receptor complex concentration LRP. See LDL-receptor-related protein luminal protozoa, physiology, 637–638 luteal phase, menstrual cycle, 509 luteinizing hormone (LH) anterior pituitary gland, 467 reproduction, 509 lutropin alfa, protein therapeutics and, 902t Lyme disease, 912 lymphocytes, 776 lymphocyte-signaling inhibitors, 796–798, 803–804t lymphocyte-stimulating factors, 782 lymphoid stem cells, 781 lymphopoiesis, 781–782 lyophilized powder, 859 lysine analogues, 393, 400t

M M2 proton channel, viral uncoating and, 654 MAC. See minimum alveolar concentration mAChR. See muscarinic cholinergic receptors macrolide antibiotics cytochrome P450 enzymes and, 51t grapefruit juice effect and, 53 macrolides, 592 oxidation/reduction enzyme and, 73t macromolecular therapies, 852t macrophages, 731–732, 732t, 776 macula densa cells, 335 magnesium hydroxide, 816, 818t, 835t magnesium sulfate, 835t, hypocalcemia, 554b maintenance dose, 41–42 major depressive disorder (MDD), 207 case study, 208

clinical characteristics, 211–212 criteria, 211b future directions, 219 major histocompatibility complex (MHC), 531, 732–733, 733f major mood disorders, 207 malarial plasmodia electron transport chain, 631–632, 632f heme metabolism, 631, 631f life cycle, 629–631, 631f malathion, toxicity, 885 male contraception, 518 malignant hyperthermia, 254 mammalian target of rapamycin inhibitors. See mTOR inhibitors mania, 207 manic episode, 212 mannitol, 15, 345, 351t mannoproteins, 619 MAO. See monoamine oxidase MAO-A inhibitors. See monoamine oxidase A inhibitors MAO-B inhibitors. See monoamine oxidase B inhibitors MAOIs. See monoamine oxidase inhibitors maprotiline, 215, 277 maraviroc, 651–652, 670t maresins, 759 biosynthesis, 748, 749–751f margin of safety, 56 marijuana, AIDS and, 269 mast cells asthma and, 825–826 degranulation, tissue damage, 730–731 matrix metalloproteinases, 822b matrix protein, viral uncoating and, 654 MBC. See minimum bactericidal concentration M-CSF. See monocyte colony-stimulating factor MDD. See major depressive disorder MDMA (methylenedioxymethamphetamine), 214, 286t, 301 MDR. See multidrug resistance MDR transporters. See multiple drug resistance transporters MDR1. See multidrug resistance protein 1 MDR-TB. See multidrug-resistant tuberculosis mebendazole, 642, 648t mecamylamine, 126, 131t, 133 mecasermin, 470, 477t mecasermin rinfabate, protein therapeutics and, 899t mechanisms of desensitization, types of, 14 mechlorethamine, 687, 696t combination chemotherapy, 726 meclizine, 771, 775t meclofenamate, 756, 761t medial forebrain bundle, 289 median effective dose (ED50), 20 median lethal dose (LD50), 241 median toxic dose (TD50), 20 medical review, IND and, 863 Medication Guides, drugs and, 869 medications. See drug(s) medroxyprogesterone acetate, 517, 523t medulla, 96, 97t mefenamate, 756, 761t mefloquine, 568, 644t malarial plasmodia and, 633–634 meglitinides, 535, 539t meglumine antimonate, leishmaniasis and, 640, 647t melancholic depression, 211 melanocyte-stimulating hormone (MSH), 472 melarsoprol, African trypanosomiasis, 639–640, 646t meloxicam, 757 melphalan, 696t DNA damage and, 67 DNA structure and, 687, 696t memantine Alzheimer’s disease and, 178 NMDA receptor antagonists, 185t membrane attack complex, 736, 802

membrane excitability, 88 membrane hyperpolarization, 165 membrane resistance, 165, 165f memory, cholinergic link, 117 menopause, 512 menstrual cycle, 509–510, 510f meperidine, 261t, 275, 281t mepivacaine, dentistry, 157b mepolizumab, 833 MEPP. See miniature end-plate potential mercaptopurine, 723t DNA structure, 684f formation of, from azathioprine, 795f genetic polymorphisms, drug metabolism and, 73t 6-mercaptopurine, 684–685, 694t, 841, 842f meropenem, 611, 612, 617t mescasermin, protein therapeutics and, 899t mesna, 688 mesocortical system, schizophrenia and, 197 mesolimbic system, schizophrenia and, 196 mesoridazine, 204t mestranol, 516, 523t metabolic acidosis, diuretics and, 347 metabolic drug interactions, 54 metabolic syndrome, 323 metabolism, drug absorption, 27, 28f metabotropic GABA receptors (GABAB), 166 metabotropic glutamate receptors (mGluR), 177–178, 177f, 178t metabotropic receptors, 90, 92 metal, toxicity of, 891–892 metal-ligand complexes, chelator and, 884 metaproterenol, 140, 145t, 828, 834t metastasis, tumor and, 570 metformin, 535, 539t methacholine, 120, 122, 129t methacycline, 591, 597t methadone, 275, 280t addiction, pharmacologic treatment of, 304–305, 304f detoxification and, 298f, 303 drug dependence and, 307t opioids and, 303 methamphetamine, 139 oxidation/reduction reactions and, 45t methanol, 47 metabolic drug interations, 54 methemoglobinemia, 576 methicillin, 609, 610, 615t methimazole, 486 thyroid hormone and, 488t methohexital, 173, 174 clinical uses for, duration of action, 174t GABAA receptors and, 183t methotrexate, 39f, 565, 573, 576, 580t, 723t, 803t combination chemotherapy, 725 methotrexate (MTX), 794–795 DHFR and, 577 methoxamine, 139, 144t methoxy polyethylene glycol-epoetin beta, protein therapeutics and, 901t methoxyflurane, 254f cytochrome P450 enzymes and, 52t methscopolamine, 124, 130t methyldopa, 144t, 443 genetic polymorphisms, drug metabolism and, 73t immune responses, immunotoxicity and, 64 methylenedioxymethamphetamine. See MDMA methylenetetrahydrofolate (MTHF), 676 methylmorphine. See codeine methylnaltrexone, 275, 282t methylphenidate, 139, 143t, 921 atypical depression and, 212 serotonin storage and, 213–214 methylprednisolone, 495, 503t, 762t methylxanthines, 829, 835t metolazone, 352t, 442t metoprolol, 73–74, 141t, 142, 146t, 371t, 442 cardiac rhythm and, 413, 419t cytochrome P450 enzymes and, 51t metronidazole, 638–639, 646t, 721

Index 945

anaerobic organisms and, 639f H. pylori, 817 metyrapone, 473, 499, 499t, 504t mevalonate pathway, 552 mexiletine, 277 cardiac rhythm and, 412, 419t cytochrome P450 enzymes and, 51t Meyer-Overton Rule, 242–244, 245f, 256–257 mezlocillin, 609, 611, 615t MG. See muscle group mGluR. See metabotropic glutamate receptors MGMT. See O6-methylguanine-DNA methyltransferase MHC. See major histocompatibility complex MIC. See minimum inhibitory concentration micafungin, 567, 625, 628t Michaelis-Menten kinetics clearance of drug and, 37, 38f drug toxicity and, 42f miconazole, 567, 623, 627t cytochrome P450 enzymes and, 52t microbiology review, IND and, 867 microsatellite instability, 678 microsomal triglyceride transfer protein (MTP), 313 microspheres, drug delivery and, 917 microtubule depolymerization inhibitors, 692–693, 698t microtubule polymerization inhibitors, 692, 692f, 697t microtubule(s) dynamic instability, 683f mitosis and, 682–683 structure, 682f midazolam, 51t, 171, 238t, 261t clinical uses for, duration of action, 172t GABAA receptors and, 183t general anesthesia and, 255 oxidation/reduction enzyme and, 73t seizures and, 234 midbrain, 96, 97f middle cervical ganglion, 96 mifepristone (RU-486), 499, 503t, 515, 522t cytochrome P450 enzymes and, 51t miglitol, 538t migraine headache, 217, 272–273 therapy, 278 milnacipran, 216, 222t milrinone, 362, 436t heart failure and, 460t, 462 miltefosine, 640, 647t mineralocorticoid receptor, 499 mineralocorticoid receptor agonists, 500, 504t mineralocorticoid receptor antagonists, 500–501, 504t mineralocorticoids, 489–490 pathophysiology, 500 pharmacologic classes, agents, 500–501 physiology, 499–500 miniature end-plate potential (MEPP), 115 minimum alveolar concentration (MAC), 241 minimum bactericidal concentration (MBC), 716–718 minimum inhibitory concentration (MIC), 716–718 Ministry of Health and Welfare, Japan, 869 minocycline, 591, 597t minoxidil, 365, 370t, 443 mirtazapine, 217, 223t mismatch repair pathway, 676–678, 679f misoprostol, 515, 763t peptic ulcer disease, 816 MIT. See monoiodotyrosine mitomycin C, 50 tumor cells and, 687, 696t mitosis, microtubules and, 682–683 mitotane, 473, 499, 499t, 503t mivacurium, 126, 131t, 261t mixed agonists, 275, 281t mixed episode, 212 mixed hyperlipidemia, 323 mizolastine, 775t MMF. See mycophenolate mofetil moclobemide, 139, 144t serotonin degradation and, 214, 221t

modafinil, serotonin storage and, 213–214, 220t moderation management, addiction and, 303 modulated receptor hypothesis, local anesthesia and, 153, 154f, 155t moexipril, 349t molds, 618 molecular mimicry, autoimmunity and, 792 molindone, 204t mometasone, 830, 835t monoamine hypothesis, 211 monoamine neurotransmission, monoamine theory of depression and, 213 monoamine oxidase (MAO), 47, 133f, 187–189, 189f, 210, 431 monoamine oxidase A inhibitors (MAO-A inhibitors), 215 monoamine oxidase B inhibitors (MAO-B inhibitors), L-DOPA metabolism and, 194f monoamine oxidase inhibitors (MAOIs), 105, 139, 144t atypical depression and, 212 serotonin degradation and, 214–215, 215f monoamine theory of depression, 212–213 monobactams, 608, 611–612 monoclonal antibodies, 800–801, 851b monocyte colony-stimulating factor (M-CSF), 781 monocyte/macrophages, 781 monoiodotyrosine (MIT), 481 monotherapy, hypertension and, 444–445 montelukast, 759, 764t, 831, 836t mood stabilizers, 218–219, 224t moperone, 198f MOPP regimen, 726 morbidity, major depressive disorder and, 207 moricizine, cardiac rhythm and, 412, 419t morphine, 261t, 269, 273–274, 280t, 849 drugs of abuse, 286t general anesthesia and, 256 genetic polymorphisms, drug metabolism and, 73t parenteral drug administration and, 31t motion sickness, H1-antihistamines and, 771 Motrin. See ibuprofen moxifloxacin, 596t, 720 6-MP. See 6-Mercaptopurine MPA. See mycophenolic acid MPTP, Parkinson’s disease, 193 MSH. See melanocyte-stimulating hormone MTHF. See methylenetetrahydrofolate mTOR inhibitors, 709–710, 714t, 797–798 MTP. See microsomal triglyceride transfer protein MTX. See methotrexate mu-opioid receptor agonists, 280t Muckle-Wells syndrome, 800 mucosal defense, agents promoting, gastric acid and, 816 mucous membrane, drug administration, 30t, 31 multidrug resistance (MDR), biliary excretion and, 37 multidrug resistance protein 1 (MDR1), 49 multidrug-resistant tuberculosis (MDR-TB), 720 multilineage growth factors, 778–779 multiple drug resistance transporters (MDR transporters), 107 MurA enzyme, 601 MurB enzyme, 601 MurC enzyme, 601 MurD enzyme, 601 MurE enzyme, 601 murein, bacterial cell wall and, 599 murein monomers, 612 synthesis inhibitors, 614t synthesis of, 601–603 murein polymer synthesis inhibitors, 607–608, 614t MurF enzyme, 603 muromonab-CD3, 909t muscarine, 123 muscarinic acetylcholine (ACh) receptors, partial agonists, 23, 23f muscarinic cholinergic receptors (mAChR), 110, 113, 114t, 127

agonists, 122–123, 123f, 123t, 129t antagonists, 119t, 124–126, 130t asthma, 821 muscarinic cholinergic toxicity, 125b muscimol, 170, 182t GABAergic transmission and, 169t muscle contraction, ACh and, 115, 118f muscle group (MG), anesthesia and, 246 muscles, myotomal distribution, 96 myasthenia gravis, treatment, 111f Mycobacterium leprae, 604 Mycobacterium tuberculosis, 604, 612 mycophenolate mofetil (MMF), 794, 795f, 803t mycophenolate sodium, 803t mycophenolic acid (MPA), 795, 795f, 803t Mycoplasma pneumoniae, 599 myeloid stem cells, 781 myelopoiesis, 781–782 myelosuppression, 692, 784 myocardial ischemia, 354 myocyte anatomy, 423 contraction, 423–424, 424f myofibrils, 425t myosin, cardiac myocyte contraction and, 425t myosin light chain kinase, 355 myosin light chain phosphatase, 356 myotomal distribution, 96

N NA. See Narcotics Anonymous Na⫹ channel-mediated inhibition, 231–232 nabumetone, 756, 761t NAc. See nucleus accumbens N-acetylcysteine (NAC), 362 acetaminophen overdose and, 69 N-acetyl-p-benzoquinoneimine (NAPQI) hepatotoxicity and, 65–66 N-acetyltransferase, 52 nAChR. See nicotinic cholinergic receptors; nicotinic receptors NAD. See nicotinamide adenine dinucleotide nadolol, 141t, 142, 146t NADPH. See nicotinamide adenine dinucleotide phosphate nafarelin, 479t, 904t nafcillin, 609, 610, 615t naftifine, 622, 626t NAG-arabinogalactan, 604 Na⫹/K⫹ATPase, 500 nalbuphine, 281t nalidixic acid, 587 naloxone, 275, 282t drug dependence and, 307t opioid overdose and, 69 naltrexone, 275, 282t addiction treatment and, 304 drug dependence and, 307t general anesthesia and, 256 NANC fibers, 820 NAPQI. See N-acetyl-p-benzoquinoneimine naproxen, 276, 755, 756, 761t genetic polymorphisms, drug metabolism and, 73t naratriptan, 278 Narcotics Anonymous (NA), 303 natalizumab, 65, 802, 806t, 908t nateglinide, 539t National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines, 324, 324t native pacemaker, 406 natriuresis, 334 natriuretic peptide uroguanylin (UGN), 336 natriuretic peptides, 335–336, 336f natural ligands, analogues of, 849–851 natural products, as drugs, 723t, 849, 850t nausea, H1-antihistamines and, 771 NBCe1, 338 NBM. See nucleus basalis of Meynert

946 Index NDA. See New Drug Application NE. See norepinephrine nebivolol, 142, 146t, 442 cardiac rhythm and, 413, 419t necrosis, cell toxicity and, 62, 63t nedocromil, 769, 836t asthma and, 830 nefazodone, 217, 223t cytochrome P450 enzymes and, 51t negative membrane potential, 84 negative reinforcement, drug addiction and, 291 negative symptoms, schizophrenia, 195 Neisseria meningitidis, 592 nelfinavir, 51t, 662, 672t nematodes (Roundworms), 640 neomycin, 589, 596t neonatal jaundice, phenobarbital and, 47 neostigmine, 96, 120, 121, 128t, 256 nephrogenic diabetes insipidus, 219, 347 nephrolithiasis, thiazides, 346 nephron anatomy, 337–338, 337f nephrotic syndrome, 342 NER. See nucleotide excision repair Nernst equation, 84 Nernst equilibrium potential, 402 Nernst potential, 84, 85f, 85t nerve cell membranes, local anesthetics and, 151 nerve injury, pain in, 272, 273f nervous system, physiology and pharmacology, principles, 93–108 Nesacaine, 158 nesiritide, 12, 344, 350t, 904t NESP. See darbepoetin NET. See norepinephrine transporter netilmicin, 589, 596t neural networks, CNS and, 226–227 neuraminidase inhibitors, 664, 667f neuroactive peptides, 102 neuroactive steroid, 180 neuroanatomy, 93–107 neurodegenerative diseases, 178 neuroendocrine system, blood pressure and, 440 neurogenic diabetes insipidus, 474, 475f neurohormonal mediators, vascular tone and, 358 neurohumoral activation, heart failure and, 458, 459f neuroleptic malignant syndrome (NMS), 198 neuroleptics, schizophrenia and, 197 neuromuscular blockers, 261t neuromuscular junction (NMJ) ACh and, 115, 117f chemical synapse, 90, 117f neuronal excitability, anesthesia and, 257 neurons, 89–90 surround inhibition and, 227, 227f neuropathic pain, 272, 273f neuropeptides, 106, 267 neuroprotective therapies, 193 neurorestorative therapies, 193 neurosteroids, 167f, 168 neurotoxicity, drug-induced, 66 neurotransmission central adrenergic, 207–219 central monoamine, 219 cholinergic, 110–119 CNS, electrical neurotransmission and, 225–235 dopaminergic, 186–201, 188f GABA, 164–165 alcohol and, 294 drug summary table, 182–184t pharmacologic classes and agents affecting, 168–169 physiology of, 165–168 GABAergic, 227 glutamatergic, 178–180, 182–184t monoamine, 213 serotonergic, 207–219 spinal cord dorsal horn, 268f neurotransmitter release, mechanism, 91f neurotransmitters, 89, 102, 102f, 103t, 104f amino acid, 102–103 biogenic amines, 104–106

CNS, 100, 102f excitatory, 164–165, 165f, 180 inhibitory, 165, 165f neuropeptides, 106 peripheral nervous system, 102f small molecule, 102, 104f, 106 neutropenia, leukocyte production and, 784 neutrophil, 732t neutrophils, 731, 781 nevirapine, 661, 671t cytochrome P450 enzymes and, 51t New Drug Application (NDA), 867 NFAT (nuclear factor of activated T cells), 797 NHE3 Na⫹/H⫹ exchanger, 338 niacin, 331t lipid metabolism and, 328–329, 328f nicorandil, 365, 370t nicotinamide adenine dinucleotide (NAD), 328 nicotinamide adenine dinucleotide phosphate (NADPH), 47, 48f, 328 nicotine, 73t, 292f, 299 drugs of abuse and, 286t oxidation/reduction enzyme and, 73t nicotine replacement therapy, 305 nicotinic acetylcholine receptor channel opening, kinetics, 116f structure of, 8, 115f nicotinic cholinergic receptors (nAChR), 110, 113–114, 114t agonists, 123–124, 129t antagonists, 124–126, 131t nicotinic cholinergic toxicity, 125b Niemann-Pick C1-like 1 protein (NPC1L1), 314 nifedipine, 51t, 363, 364t, 369t, 405, 443, 921 cardiac rhythm and, 415 nifurtimox, Chagas’ disease and, 640, 647t nigrostriatal pathways, physiology of, 191–192 nigrostriatal system, 189 nigrostriatal tract, 98 nilotinib, 708, 714t nitazoxanide, 639, 646t nitrates contraindications, 362 coronary artery disease and, 451 effects of, vasodilation and, 362 heart failure and, 461 nitric oxide, 106, 356–357, 357f asthma, 821 neurotransmitters and, 102, 103t nitrofurantoin, oxidation/reduction reactions, 45t nitrogen mustards, 674 DNA damage and, 67 nitroglycerin (NTG), 354, 356, 368t heart failure and, 461 pharmacodynamic drug-drug interactions, 61 nitrosoureas, 687 DNA damage and, 67 nitrous oxide, 255, 260t MAC, 241 partition coefficients, 244b, 248t properties of, 243t rate-limiting step, 249, 249f recovery from, 254, 254f nizatidine, 775t peptic ulcer disease and, 812, 818t NKCC2, 339 NMDA receptor antagonists, 277 drug summary table, 185t, 283t hyperalgesia and, 179 NMDA receptor(s) alcohol and, 299 central sensitization, 271–272 dorsal horn neurons and, 267 glutamate-gated ion channels, 176–177, 177t neurodegenerative disease and, 178 NMJ. See neuromuscular junction NMR. See nuclear magnetic resonance NMS. See neuroleptic malignant syndrome NNRTIs. See nonnucleoside reverse transcriptase inhibitors NO. See nitric oxide

nociception, 147–151, 148f, 269 nociceptive circuit, 265f nociceptive pain, 269 nociceptor cell bodies, 147 nociceptor neurons chemical stimuli and, 266, 266t peripheral sensitization and, 271 nociceptors, 265, 265f nofetumomab, 913t nonanesthetics, 257 nonarteritic ischemic optic neuropathy, 362 nonblinded trials, phase I studies and, 865 noncompetitive antagonists, 7, 21f, 22–23, 22f nondepolarizing (competitive) neuromuscular blockade, 126 nonhomologous end-joining, 678, 681 nonimmobilizers, 257 noninsulin-dependent diabetes mellitus. See diabetes mellitus nonnucleoside DNA polymerase inhibitors, 661, 661f, 671t nonnucleoside reverse transcriptase inhibitors (NNRTIs), 661, 661f, 671t CCR5 antagonist and, 662b nonopioid analgesics, 275–277 nonpacemaker cells, 401–402 nonprescription drugs, 870–871 nonreceptor antagonist, 21, 23 nonreceptor tyrosine kinases, 11f, 12 nonreceptor-mediated mechanisms, 15 nonselective COX inhibitors, adverse effects, 742t non-ST elevation myocardial infarction (NSTEMI), 448–449 clinical management, 452–453 nonsteroidal analgesics, 282t nonsteroidal anti-inflammatory drugs. See NSAIDs norelgestromin, 517 norepinephrine (NE), 10, 137, 186, 207, 432, 435t metabolic cycle, 208–209, 209f metabolism, 132, 134f neurotransmission, presynaptic regulation, 210f neurotransmitters and, 102, 102f, 103t pain and, 268 synthesis of, 209f norepinephrine transporter (NET), 135, 210, 300 norepinephrine-selective reuptake inhibitors. See NRIs norethindrone, 516, 516f, 523t cytochrome P450 enzymes and, 52t norethindrone acetate, 516, 516f, 523t norfloxacin, 596t cytochrome P450 enzymes and, 51–52t norgestimate, 516, 516f, 523t norgestrel, 516, 523t nortriptyline, 215, 221t, 277 cytochrome P450 enzymes and, 51t Novocain. See procaine NPC1L1. See Niemann-Pick C1-like 1 protein NPH (neutral protamine Hagedorn) insulin, 534, 538t NPR-A, 336 NPR-B, 336 NPR-C, 336 NRIs (Norepinephrine-selective reuptake inhibitors), 217, 222t NSAID-induced gastropathy, 755 NSAIDs (nonsteroidal anti-inflammatory drugs), 73t, 754–755, 761t chronic kidney disease, 39b classes of, 276–277 cytochrome P450 enzymes and, 52t general features, 275–276, 276f ginkgo and, 61 gout and, 840 immune responses, immunotoxicity and, 64 niacin and, 328 oxidation/reduction enzyme and, 73t pain, 270 peptic ulcer disease and, 811, 811f selection, 756–757 structural classes, 755f NSTEMI. See non-ST elevation myocardial infarction NTG. See nitroglycerin

Index 947

N-type voltage-gated calcium channels, 267 nuclear factor of activated T cells. See NFAT nuclear factor-kappa B (NF␬B) pathways, 704, 704f nuclear magnetic resonance (NMR), 853 nucleic acid synthesis, 676 nucleosides, 675 analogues, 655, 656f nucleotide excision repair (NER), 679–682 nucleotide(s), 675 synthesis, 675–676, 676f, 677f nucleus accumbens (NAc), 189, 289–291 nucleus basalis of Meynert (NBM), 117 nutrition, drug metabolism and, 53 nutritional rickets, vitamin D and, 557 nystatin, 567, 623–624, 628t

O O6-methylguanine-DNA methyltransferase (MGMT), 687 OAT. See organic anion transporter OATP. See organic anion transporting polypeptide OATP1. See organic anion transporting polypeptide 1 observer bias, 864 occipital lobe, 97, 97f OCT. See organic cation transporter octopamine, 138 octreotide, 470, 477t, 537, 540t protein therapeutics and, 904t oculomotor nerve, 95f, 96 odds ratio, drug study and, 876 ofatumumab, 806t “off-label” use, 870 off-target adverse effects, 56, 57, 58f, 59–60 ofloxacin, 587, 596t cytochrome P450 enzymes and, 52t Ohm’s law, cells and, 83, 83f oil/gas partition coefficient, 242 OKT3, 800–801, 805t olanzapine, 198f, 200, 205t olfactory tubercle, dopamine receptor proteins and, 189 olopatadine, 775t omalizumab, 831, 836t, 909t omega-3 fatty acids, 329, 331t generation, 741–742 omeprazole, 73t, 813, 814f, 818t cytochrome P450 enzymes and, 52t off-target effects, 60 oxidation/reduction enzyme and, 73t Onchocerca volvulus, 629, 640 life cycle, 641, 641f ondansetron, 218, 223t one-pot synthesis, 858 on-target adverse effects, 56, 57–59, 58f open state, sodium channel and, 226 opioid agonists drug summary table, 280t long-acting, drug table and, 307t pain relief and, 273–275 spinal cord and, mechanism of action, 274f, 275 opioid antagonists, 275, 282t long-acting, drug table, 307t opioid hyperalgesia, 295 opioid partial agonists, drug table for, 308t opioid peptide family, 106, 268–269 opioids, 106, 261t cytochrome P450 enzymes and, 51t drugs of abuse and, 286t, 292f, 295–296, 296f, 297f general anesthesia and, 256 methadone and, 304 pain and, 268 pathways and, 296f prescription, 296 sites of action, 278f withdrawal, 296 oprelvekin, 785, 789t protein therapeutics and, 902t opsonins, inflammatory response and, 738 optic tract, 100 oral contraceptives, 231

oral drug administration enteral, 30–31, 30t delivery, 917–918 oral drug, administration, enteral, 30–31, 30t oral hydrocortisone, 496 oral phosphate, 560t binders drug summary table, 560t secondary hyperparathyroidism, 556 hypercalcemia, 554b organ toxicity, 62–69 organic anion transporter (OAT), 28, 49 organic anion transporting polypeptide (OATP), 49 organic anion transporting polypeptide 1 (OATP1), 61 organic cation transporter (OCT), 28, 49 organic nitrates biotransformation, 359f drug summary table, 368t heart failure and, 460t mechanism of action, 359–361 sites of action, 360f vasodilators and, 359 organification, 481, 488t organification inhibitors, 485–486 organophosphate insecticides, 885 ornithine decarboxylase, 640 orphan diseases, drug development and, 867 Orphan Drug Act, 867 oseltamivir, 568, 664–665, 672t osmotic diuresis, 345 OspA vaccine, 911t ospemifene, 515, 521t osteitis fibrosa cystica, 550 osteoblasts, bone remodeling, 542, 544f osteoclasts, bone remodeling, 542, 544f osteomalacia, 550 osteoporosis, 548 pathophysiologic basis for, 551f vitamin D and, 557 osteoprotegerin (OPG), 542 ovarian hyperstimulation syndrome, 474 ovarian hypothesis, 511 overdrive suppression, 406 overflow model, cirrhosis and, Na⫹ retention and, 341, 342f oxacillin, 609, 610, 615t oxaliplatin, 690, 696t oxazolidinones, 572, 594, 598t oxcarbazepine, 277 cytochrome P450 enzymes and, 51t trigeminal neuralgia, 272 oxicam derivatives, 756 oxicams, 761t oxiconazole, 623, 627t oxidation reactions, 44, 45t oxidation/reduction reactions, 34, 45t, 47 oxybutynin, 125, 130t oxycodone, 274, 280t, 292f oxymetazoline, 139, 144t oxypurinol, 841, 844t oxytetracycline, 591, 597t oxytocin, 475

P P wave, 405b, 405f P2X ligand-gated channels, 267 P2Y G protein-coupled purinergic receptors, 267 P2Y1 receptors, 375 P2Y(ADP) receptors, 375 PABA. See Para-aminobenzoic acid pacemaker cells, 401–402 paclitaxel, 73t, 692–693, 698t, 723t, 849, 922 immune responses, immunotoxicity and, 64 oxidation/reduction enzyme and, 73t STEMI, drug-eluting stent and, 454 PAI. See plasminogen activator inhibitor pain. See also clinical pain syndromes; first pain; second pain adrenergic drugs, 277–278

nerve injury, 264 nociceptors and, 265, 265f pathophysiology, 269–273 pathways, 150f perception, 149–150 physiology, 264–269 sensation, transmission of, 149 treatment, future directions, 278–279 pain management drug classes, sites of action for, 278f drug summary table, 280–283f palifermin, 904t paliperidone, 206t palivizumab, 668, 906, 909t pamidronate, 552, 559t pancreas, 524 pancreatic anatomy, biochemistry and physiology, 524–530 pancreatic enzymes, 895 pancreatic enzymes, protein therapeutics and, 901t pancreatic polypeptide, PP cells from, 524 pancuronium, 96, 126, 131t, 261t general anesthesia and, 256 pancytopenia, 779 panitumumab, protein therapeutics and, 907t pantoprazole, 813, 818t cytochrome P450 enzymes and, 52t papain, 898, 905t para-aminobenzoic acid (PABA), 158, 575 parafollicular C cells, 480 parasites, 629 drugs and, 567–568 parasitic infections drug summary table, 644–648t pharmacology of, 629–643 parasympathetic nervous system, anatomy of, 94, 95f, 96 parathion, 885 parathyroid hormone (PTH) action of, 545f bone and, 555–556 calcium homeostasis, 544–546 parathyroid hormone-related peptide (PTHrP), 553 paraventricular nuclei, 190 parenteral drug administration, 30t, 31, 31t parenteral formulations, 858 paricalcitol, 560t secondary hyperparathyroidism, 556 parietal cell acid secretion, 809f parietal lobe, 97, 97f Park nucleotide, 603 Parkinson’s disease, 186 nigrostriatal pathways and, 191–192, 192f nondopaminergic pharmacology, 195 pathophysiology, 192–193 pharmacology and, 193–195 treatment, 195 paromomycin, 589, 596t, 639 paroxetine, 216, 221t, 277 cytochrome P450 enzymes and, 51t paroxysmal depolarizing shift (PDS), 227 paroxysmal nocturnal hemoglobinuria (PNH), 802 paroxysmal supraventricular tachycardia (PSVT), 409b PARP1 inhibitors, 693 pars compacta, 189 partial agonists, 7, 23–24, 23f, 275, 281t partial nicotine agonists, 309t partial pressure (of gas) concentration vs., 242b partition coefficients, 244b pasireotide, 470 passive targeting, drug delivery and, 922 patient registries, pharmacoepidemiology and, 875 pattern recognition, innate immune response and, 732 pazopanib, 711t, 715t PBPs. See penicillin-binding proteins PCP. See phencyclidine PCSK9. See proprotein convertase subtilisin-like kexin type 9 PDE inhibitors. See phosphodiesterase inhibitors PDE5. See cGMP phosphodiesterase type V PDGF. See platelet-derived growth factor

948 Index PDGFR inhibitors, 713–714t PDS. See paroxysmal depolarizing shift pedunculopontine nucleus, 101, 101f peg-asparaginase, 905t PEG-filgrastim, 784–785, 788t protein therapeutics and, 902t Peg-G-CSF, protein therapeutics and, 902t PEG-interferon, 914 peginterferon alfa-2a, 902t pegloticase, uric acid metabolism and, 842, 845t PEG-rHuMGDF. See pegylated recombinant human megakaryocyte growth and development factor pegvisomant, 470–471, 477t, 909t pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), 785, 788t pemetrexed, 684, 694t penbutolol, 142, 146t cytochrome P450 enzymes and, 51t penciclovir, 658, 670t penicillamine, 884 penicillin G, 609, 610, 615t penicillin V, 610, 615t penicillin-binding proteins (PBPs), 604 penicillin(s), 599, 604, 608, 720 discovery, 849 drug combinations, 721 hypersensitivity reactions and, 62 pharmacokinetic drug-drug interactions, 61 seizure and, 225–226 transpeptidase action and, 605f unfavorable drug combinations, 721 pentamidine, African sleeping sickness and, 639, 646t pentobarbital, 173 clinical uses for, duration of action, 174t GABAA receptors and, 183t phenobarbital vs., 174t pentostatin, 685, 685f, 694t PEP. See phosphoenolpyruvate pepsin, 811 peptic ulcer disease case studies, 808 drug summary table, 818–819t pathophysiology of, 810–811 pharmacologic classes, and agents, 812–817, 812f risk factor modification, agents for, 816–817 peptides, 851b peptidoglycan, 599 biosynthesis, 601–604 peptidoglycan glycosyltransferases (PGTs), 604 peptidoglycan polymerization, 607 “peptidyl” site, 585 peptidyl transferase, 585–586 perceived volume depletion, heart failure and, 341, 341f perchlorate, 485, 488t perforins, 733 perfusion-limited anesthetics, 249, 249f pergolide, 194 periaqueductal gray, 99 perindopril, 349t peripheral nerve blockade, 156–157 peripheral nerve fibers local anesthetics and, 153f types of, 149, 149t peripheral nervous system, anatomy of, 93–96 peripheral neuropathy, 692 peripheral sensitization, 270–271, 270f peripheral testosterone conversion to DHT, inhibitors, drug summary table for, 520t peripheral thyroid hormone metabolism inhibitors, 486, 488t peripheral tolerance, autoimmunity and, 792 peripheral transduction, 266f permanent plug, 373 permethrin, 887 peroxisome proliferator-activated receptor-␥ (PPAR␥), 525

peroxynitrite, 362 perphenazine, 204t cytochrome P450 enzymes and, 51t pertechnetate, 485, 488t pesticides, 885–887, 886f petit mal seizures, 230 PGG2. See prostaglandin G2 P-glycoprotein, 49, 691 pharmacokinetic drug-drug interactions, 61 PGTs. See peptidoglycan glycosyltransferases pH trapping, 29, 29f phagocytosis, inflammatory response and, 737–738 pharmacodynamic interactions, drug-drug, 61 pharmacodynamic tolerance, 287 pharmacodynamics, 6, 17–26 pharmacoepidemiology, 874–875 data sources, 875–876 pharmacology vs., 874, 874t study design and interpretation, 877–878 study strategies, 876, 876f, 877f pharmacogenetics-pharmacogenomics, pathway-based, 76–77, 77f pharmacogenomics, 42, 71 future directions, 79 pharmacology and, 71–78 physiology, 71 pharmacokinetic tolerance, 287 pharmacokinetics, 27–42 clinical applications, 37–42 drug-drug interactions, 61 future directions, 42 relationships, 42t pharmacology and pharmacogenomics, 71–72 drug targets, variation in, 75–76, 76t idiosyncratic drug reactions, 77 pathway-based pharmacogeneticspharmacogenomics, 76–77, 77f pharmacogenetics-pharmacogenomics, 77–78 pharmacogenomics, regulatory science and, 78 variation in enzymes, of drug metabolism, 72–75, 73t, 74f, 75f pharmacology review, IND and, 867 pharmacology/toxicology review, IND and, 863 phase I reactions. See biotransformation phase I studies, clinical trials and, 865–866 phase II reactions. See biotransformation phase III studies, clinical trials and, 866 phase IV studies, 874 phasic inhibition, local anesthesia and, 153–155, 155f phencyclidine (PCP), 301 drugs of abuse and, 286t phenelzine, 139, 144t serotonin degradation and, 214, 220t phenindamine, 774t phenobarbital, 173, 232t, 238t cytochrome P450 enzymes and, 51–52t GABAA receptors and, 183t pentobarbital vs., 174t seizures and, 234–235 phenothiazines, 204t, 774t schizophrenia and, 197 phenoxybenzamine, 140, 145t, 443 phentolamine, 141, 145t, 443 phenylalanine, 186 phenylalkylamines, 363, 370t, 405 phenylbutazone, 755 cytochrome P450 enzymes and, 52t phenylephrine, 139, 144t phenylethanolamine N-methyltransferase (PNMT), 132, 187 phenylethylamines, drugs of abuse and, 286t phenylpropanolamine, 139 phenytoin, 108, 232t, 236t, 485 cardiac rhythm and, 412, 419t cytochrome P450 enzymes and, 39, 45t, 51t immune responses, immunotoxicity and, 64 oxidation/reduction enzyme and, 73t sodium channels and, 236t phosphate homeostasis, hormonal control of, 543–548, 545t phosphatidylinositol 3-kinase (PI3-kinase), 529

phosphatidylinositol 3-kinase (PI3K)-AKT pathway, 700, 702–703f phosphodiesterase inhibitors (PDE inhibitors), 362–363, 432–433, 436t drug summary table, 369–370t, 395t heart failure and, 462 thrombus formation and, 383 phosphoenolpyruvate (PEP), 601 phospholamban, 426 phospholipase, 741 phospholipase inhibitors, inflammation and, 754 phospholipid transfer protein (PLTP), 320 phosphomycin, 606–607 phosphoribosyl pyrophosphate (PRPP), 837–839 phosphorylation, 11 phthalazinones, 775t physical dependence, 285 mechanisms, 289–291 opioids and, 274 psychostimulants and, 300–301 physiologic antagonist, 21, 23 physostigmine, 111f, 120–121, 128t PI3-kinase. See phosphatidylinositol 3-kinase picrotoxin, 170, 182t GABAergic transmission and, 169t pilocarpine, 106, 123, 129t pimozide, 198f, 204t pinacidil, 365, 370t pindolol, 141t, 142, 146t cardiac rhythm and, 413, 419t pioglitazone, 536, 540t piomelanocortin, 493 piperacillin, 609, 611, 615t piperazines, 642, 648t, 775t piperidines, 774t, 775t pirbuterol, 828, 834t pirenzepine, 120, 124, 130t pirmagrel, 758, 763t piroxicam, 276, 755, 756, 761t pitavastatin, 325–326, 330t pitrakinra, 833 pituitary gland, hypothalamus vs., 465–468 pituitary physiology, 465–468 pivotal trials, 865 pKa. See ionization state PKR. See protein kinase R plant sterols, 326–327 plasma lipoproteins, characteristics of, 312t plasma protein binding, drug trapping and, 33–34, 33f plasmin, 380 plasminogen activator inhibitor (PAI), 380 Plasmodium falciparum, 629–631 platelet granule release reaction, 373–374 platelet-derived growth factor (PDGF), 904t platelet(s), 373, 776 activation, 373, 375f adhesion, 373, 375f aggregation, 375–376, 375f production, 782, 782f drugs stimulating, 785, 788t platinum compounds, 689, 690f, 696t, 723t pleiotropic growth factors, 778 pleuromutilins, 572, 594–595, 598t PLTP. See phospholipid transfer protein pluripotent hematopoietic stem cell, 776 PNH. See paroxysmal nocturnal hemoglobinuria PNMT. See phenylethanolamine N-methyltransferase podagra, 839 Podophyllum, 691 polyanhydride polymers, surface erosion and, 921f polyclonal antibodies, 800 polycystic ovarian syndrome (PCOS), 511 polydipsia, 531 polyenes, 624–625, 628t polymer cross-linking inhibitors, 608–612, 615–617t polymer release mechanisms, 920f polymerase chain reaction (PCR) analysis, 44f polymer-based drug delivery system, 919–922, 920f polymerization, cell wall biosynthesis and, 603–604 polymicrobial infections, combination antimicrobial drugs and, 721

Index 949

polyphagia, 531 polythiazide, 352t polyuria, 531 POMC. See proopiomelanocortin pons, 96, 97f pooled immunoglobulins, protein therapeutics and, 901t porins, outer membrane, 601 posaconazole, 623, 627t positive chronotropic effect, 426 positive inotropic effect, 426 positive lusitropic effect, 427 positive reinforcement, drug addiction and, 291 positive symptoms, schizophrenia, 195 posterior horn, 96 posterior pituitary gland, 465, 474–475 postganglionic neuron, 94–95 postmyocardial infarction management, 454 postsynaptic receptors, 92 potassium (K⫹), cardiac rhythm and, 416 potassium channel openers, 365, 370t drug summary table, 370–371t vasodilators, 443–444 potassium hydroxide, 885 potassium loading, aldosterone synthesis and, 500 potassium phosphate, 561t potassium-sparing diuretics, 440t, 442, 442t potency of drug (EC50), 19 PP cells, 524 PPAR␥. See peroxisome proliferator-activated receptor-␥ PPD test. See purified protein derivative test PR interval, 405b, 405f pralidoxime, 887 pramipexole, 194, 202t pramlintide, 535, 539t protein therapeutics and, 899t prandial bolus insulins, 533, 538t prasugrel, 375, 386, 396t UA/NSTEMI, 453 pravastatin, 22, 325–326, 330t praziquantel, adult cestode and, 642, 648t prazosin, 141, 145t, 365, 371t, 443 pRB. See retinoblastoma protein preclinical research, 863 prednisolone, 494–495, 495f, 503t, 754, 762t prednisone, 39, 494, 503t, 723t, 754, 762t combination chemotherapy, 726 cytochrome P450 enzymes and, 52t pregabalin, 232t, 234, 237t, 277 preganglionic neuron, 94 pregnancy ACE inhibitors, 69 drugs, 68b glucocorticoids and, 498 pregnane X receptor (PXR), 49 preload, 354, 422, 456 preventers, asthma treatment, 827 prevertebral ganglia, 96 prilocaine, 159, 161t primaquine, 635, 644t primary generalized seizures, pathophysiology of, 229–230, 229f primary hemostasis, 373–376 primary hemostatic plug, 372, 375 primary hyperaldosteronism, 500 primary percutaneous intervention, STEMI, 454 primary prevention, statins and, 325 primary structure, amino acid and, protein structure of, 2 primase, 583 probenecid pharmacokinetic drug-drug interactions, 61 uric acid excretion and, 842, 845t procainamide, 418t conjugation enzyme and, 73t immune responses, immunotoxicity and, 64 procaine (Novocain), 147, 156, 158, 161t procarbazine, 687, 696t combination chemotherapy, 726 prochlorperazine, 198f

procyclidine, 125 prodrugs, 34, 50 progesterone receptor antagonists, 522t progestin-only contraception, 517, 523t progestins contraconception, 516–517, 516f hormone replacement, 518 synthesis, 505–506 proguanil, malarial plasmodia and, 636, 645t prolactin agents decreasing, 478t anterior pituitary gland, 467 schizophrenia and, 198 prolactinomas, 511 proliferation, tumor and, 569 proliferative phase, menstrual cycle and, 509 promethazine, 770, 771, 774t promoters, cancer and, 67 proopiomelanocortin (POMC), 472 propafenone cardiac rhythm and, 412, 419t cytochrome P450 enzymes and, 51t propantheline, 125, 130t prophylactic chemotherapeutics, drug resistance and, 574 propionic acid derivatives, 756 propionic acids, 761t propofol, 175, 260t GABAA receptors and, 184t intravenous anesthesia and, 255 propoxyphene, 281t propranolol, 73t, 141t, 142, 146t, 371t, 442 cardiac rhythm and, 413, 419t cytochrome P450 enzymes and, 51–52t oxidation/reduction enzyme and, 73t proprotein convertase subtilisin-like kexin type 9 (PCSK9), 318 propylthiouracil, 486 thyroid hormone and, 488t prostacyclin (PGI2), 379 cyclooxygenase pathway and, 744–745 prostaglandin G2 (PGG2), 382 prostaglandins biosynthesis, pharmacologic inhibition and, 743f cyclooxygenase pathway and, 744 gastric acid and, 808–809 peptic ulcer disease, 816 products/synthesis/receptors/function, 744t prostanoid structure, 744f prostanoid, 763t cyclooxygenase pathway and, 744, 744f receptor mimetics, 758, 763t prostate cancer, 511, 912 protamine, 23, 393, 400t, 906t protease protein therapeutics and, 901t viral proteins and, 659 proteasome inhibitors, 710, 714t structure and function, 701–705 protectins, 748, 749–751f, 759 protein C, 380 protein therapeutics and, 900t, 903t protein diagnostics, 897b, 912, 913–914t protein kinase R (PKR), immune system modulation, 668 protein S, hemostasis and, 380 protein therapeutics, 895–897 case study, 896 challenges for, 912–915 conclusion and future directions, 915 functional classification of, 897b group I, enzymes and regulatory proteins, 897–904, 899–901t, 901–904t, 905–906t group II, targeted proteins, 904–911, 907–910t, 910t group III, protein vaccines, 911–912, 911t group IV, protein diagnostics, 912, 913–914t protein(s) as drug receptors, 2–3 in medicine, 897–912

structure, 2–3, 4f vaccines, 897b, 911–912, 911t prothrombin G20210A mutation, 381 prothrombin time (PT), 388–389 protirelin, 478t proton pump inhibitors cytochrome P450 enzymes and, 52t metabolism of, 815b peptic ulcer disease and, 813–816, 813f, 818t protozoa, 637–638 protracted withdrawal syndrome, 289 protriptyline, 221t proximal tubule, 337, 338, 338f proxyfan, 772 PRPP. See phosphoribosyl pyrophosphate prucalopride, irritable bowel syndrome and, 218, 224t pseudoephedrine, 139, 143t Pseudomonas aeruginosa, 604 psoralen isomers, 885 PSVT. See paroxysmal supraventricular tachycardia psychedelic agents, drugs of abuse and, 286t psychological dependence, mechanisms, 285–286 psychostimulants drugs of abuse and, 286t physical dependence, 300–301 psychotic depression, 212 PT. See prothrombin time PTH. See parathyroid hormone PTHrP. See parathyroid hormone-related peptide pulmonary drug delivery, 918–919 pulmonary embolism, 517 pulmonary fibrosis, bleomycin and, 691 pulmonary toxicity, drug-induced, 67 pump-based hypertension, 439 Pure Food and Drugs Act, 861 purified protein derivative (PPD) test, 912 purine analogues, 695t, 723t DNA and, 686 purine derivatives, 675, 677t purine metabolism inhibitors, 684–685, 694t purine metabolism, physiology, 837–839, 838f purine ribonucleotide synthesis, 675 putamen, 98 PXR. See pregnane X receptor pyrantel pamoate, 642, 648t pyrazinamide, 612, 617t, 719 pyrethroid insecticides, 887 pyridostigmine, 121, 128t pyrilamine, 771, 774t pyrimethamine, 565, 576, 577, 579t, 720–721 malarial plasmodia and, 636 pyrimidine analogues, 723t pyrimidine(s) analogues, 695t DNA and, 686 ribonucleotide synthesis, 675–676

Q QRS complex, 405b, 405f QT interval, 405b, 405f quadruple therapy, H. pylori, 817 quantal dose-response relationships, 19–20, 20f quaternary structure, polypeptides and, 3, 4f quazepam, 171 clinical uses for, duration of action, 172t GABAA receptors and, 183t quetiapine, 205t schizophrenia and, 200 quinagolide, 471 quinapril, 349t quinidine, 51t, 410–411, 418t, 644t cytochrome P450 enzymes and, 51t malarial plasmodia and, 633 oxidation/reduction enzyme and, 73t quinine, 644t malarial plasmodia and, 633 quinolones, 587, 596t, 717t cytochrome P450 enzymes and, 52t quinone, malarial plasmodia and, 635 quinupristin, 572, 593f, 594, 598t

950 Index

R RAAS. See renin-angiotensin-aldosterone system rabeprazole, 813, 818t race, drug metabolism and, 53 raclopride, 198f radioactive iodide, thyroid gland and, 488t raloxifene, 24, 514 drug summary table, 521t, 559t SERMs and, 552 structure of, 552f raltegravir, 662, 672t ramipril, 349t randomization of subjects, clinical trials and, 864 randomized controlled trial (RCT), 872 ranibizumab, 910t ranitidine, 772, 775t, 818t peptic ulcer disease and, 812 RANK ligand (RANKL), calcium homeostasis, 542 RANK ligand (RANKL) antagonists, 555, 559t ranolazine, cardiac rhythm and, 416, 421t rapamycin, 710, 714t r-APC. See recombinant activated protein C raphe nuclei, 101, 101f serotonin and, 208 RAR. See retinoic acid receptor rasagiline, 195, 202t rasburicase protein therapeutics and, 905t uric acid metabolism and, 842, 845t RAS/MAP kinase pathway inhibition, 709, 714t rate of equilibration, partial pressure in alveoli and, 249f rational drug design, 6 ravuconazole, 567, 623–624 RCT. See randomized controlled trial receptor agonists adrenergic, 136t, 139–140 cholinergic, 122–124, 123f, 123t, 124t receptor antagonists, 21 adrenergic, 140–142 cholinergic, 119t, 124–126 selective estrogen receptor modulators, 513–515 types, 21f receptor binding assays, 855 receptor guanylyl cyclases, 10f, 12 receptor regulation, mechanism of, 15t receptor serine/threonine kinases, 12 receptor tyrosine kinases, 11–12 cancer and, 701t receptor tyrosine phosphatases, 12 recombinant activated protein C (r-APC), 392, 399t recombinant human bone morphogenic protein 2 (rhBMP-2), 904t recombinant human bone morphogenic protein 7 (rhBMP-7), 904t recombinant human erythropoietin (rhEPO), 783 recombinant human G-CSF, 784–785 recombinant human IL-11 (rhIL-11), 785 recombinant human thrombopoietin (rhTPO), 785, 788t recombinant insulin, 896 recombinant tissue plasminogen activator (t-PA), 393, 399t recurrent disease treatment, cancer and, 726 red man syndrome, 64, 607 5␣-reductase inhibitors, 513 re-entrant electrical pathways, 407–408 normal vs., 407f reflex tachycardia, 360 refractory cancers, treatment, 726 refractory period, 14 refractory state, 8, 14, 88 regular insulin, 533, 538t relapse, addiction and, 291 relative risk, drug study and, 876 release factors termination and, 586 viral proteins and, 651 relievers, asthma treatment, 827 remifentanil, 261t, 269, 275, 281t

remodeling. See bone remodeling remoxipride, 198f REMS. See Risk Evaluation and Mitigation Strategies renal excretion, drugs and, 36–37, 36f renal parenchymal disease, 440 renal sodium (Na⫹) reabsorption, agents decreasing, 344–347 renal sympathetic nerves, 337 renal toxicity, drug-induced, 66 renin, 334 inhibitors, 342–343, 349t, 444 renin-angiotensin system blockers, vascular tone and, 366 renin-angiotensin-aldosterone system (RAAS) extracellular fluid volume, 500 inhibitors of, 342 modulation, 444 volume regulators and, 334–335, 334f renovascular disease, 440 repaglinide, 539t repeat-dose toxicity studies, 857 repolarization, inhibitors, 413–414 class III antiarrhythmic drugs, drug table, 420t reproduction case study, 506 pharmacology, 505–523 reproduction hormones drug summary table, 520–523t male and female, future directions with, 519 physiology of, 505–510 reproductive tract disorders, 510t pathophysiologic process, 510–512 reserpine, 138, 143t, 209, 213, 443 resolution, inflammatory response and, 738 resolvins, 759 biosynthesis, 747f, 748, 749–751f respiratory physiology, concepts, 245 rest angina, 362 resting membrane potential, 84–86, 402 electrochemical basis, 85f, 86f relative contribution of K⫹ and Na⫹, 86f restitution, gastric acid and, 809 retapamulin, 572, 594–595, 598t reteplase, 393 drug summary table, 399t protein therapeutics and, 903t STEMI, 454 reticular activating system, 99, 101f, 117 retinoblastoma protein (pRB), 701 retinoic acid (vitamin A), 73t oxidation/reduction enzyme and, 73t teratogenesis and, 68 retinoic acid receptor (RAR), 786 retinoid X receptor (RXR), 68, 482 retrosynthetic analysis, 857, 857f reuptake inhibitors, 215–217 reversal potential, 91 reverse cholesterol transport, 318, 319f, 320 reverse transcriptase (RT), 659 reverse triiodothyronine (rT3), 480 reversible antagonist, 21, 21f Reye’s syndrome, 756 rFSH. See follitropin rhBMP-2. See recombinant human bone morphogenic protein 2 rhBMP-7. See recombinant human bone morphogenic protein 7 rhEPO. See recombinant human erythropoietin rheumatoid arthritis, 753, 906 rhIL-11. See interleukin-11; recombinant human IL-11 rhodanese, 883 rhTPO. See recombinant human thrombopoietin ribavirin, 667–668, 673t ribonucleotide reductase, 676 ribonucleotide reductase inhibitors, 685, 695t ribonucleotide reduction, thymidylate synthesis and, 676 30S ribosomal subunit, antimicrobial drugs and, 585–586

ribosomes, 581 ridogrel, 763t rifabutin, 596t cytochrome P450 enzymes and, 51t rifamycin B and, 587, 587f rifampin, 39, 485, 596t, 719 cytochrome P450 enzymes and, 50, 52t immune responses, immunotoxicity and, 64 metabolic drug interactions, 54 rifamycin B and, 587, 587f rifamycin B, 587 rifamycin derivatives, transcription inhibitors as, 587–588 rifapentine, cytochrome P450 enzymes and, 51t rilonacept, 800, 805t, 908t riluzole, 185t rimantadine, 568, 670t viral uncoating and, 653–655, 654f rimonabant, 269, 301 risedronate, 552, 559t Risk Evaluation and Mitigation Strategies (REMS), 862 risperidone, 205t cytochrome P450 enzymes and, 51t schizophrenia and, 200 ritonavir, 574, 672t cytochrome P450 enzymes and, 50, 51–52t development of, 665b, 666f HIV and, 662 rituximab, 65, 712, 715t, 801, 806t, 906, 907t rivastigmine, 121, 122t, 129t rizatriptan, 217, 223t, 278 RNA, 581 viral life cycle and, 650 RNA polymerases, 581, 583 rocuronium, 126, 131t rofecoxib (Vioxx), 78, 271, 276, 757 drug study and, 874, 877 withdrawal of, 879 roflumilast, 833 romiplostim, 788t, 914 protein therapeutics and, 902t ROMK channel, 339 ropinirole, 194, 202t ropivacaine, 157, 159 rosiglitazone (Avandia), 536, 540t, 874 rosuvastatin, 325–326, 330t roundworms. See nematodes RT. See reverse transcriptase rT3. See reverse triiodothyronine rufinamide, 232t, 235, 239t R-warfarin, cytochrome P450 enzymes and, 52t RXR. See retinoid X receptor ryanodine receptor, 254, 425

S SA nodal cells. See sinoatrial nodal cells salicylate, 755–756 saline diuresis, 554b salmeterol, 140, 145t, 828, 834t salmon calcitonin, 559t salvage pathway, 837 saquinavir, 51t, 53, 662, 672t sarcoendoplasmic reticulum calcium ATPase. See SERCA sarcolemma, cardiac myocyte contraction and, 425t sarcomere, cardiac myocyte contraction and, 425t sargramostim, 784–785, 788t protein therapeutics and, 902t satumomab pendetide, 913t saturation kinetics, 42, 42f saxagliptin, 536, 539t saxitoxins, 885 scavenger receptor class B, type I (SR-BI), 318 SCF. See stem cell factor schizophrenia central dopamine pathways and, 190 criteria for, 196b dopamine receptors and, 189 dopamine systems and, 186

Index 951

pathophysiology, 195–197 pharmacology, 197–200 scopolamine, 106, 124, 130t secobarbital cytochrome P450 enzymes and, 52t GABAA receptors and, 183t second messenger, regulated ion channels, 7, 8t, 9 second pain, 149, 150f secondary generalized seizures, pathophysiology of, 228–229, 229f secondary hemostasis, 372, 375–379 secondary hyperlipidemia, 323–324, 323t secondary hyperparathyroidism, 548, 550, 556–557 secondary hypertension, 439 secondary osteoporosis, 548 secondary prevention, statins and, 324 secondary structure, amino acid and, protein structure of, 3, 4f secretins, 106, 913t secretory phase, menstrual cycle, 509 seizure propagation, pathways of, 229f seizure(s). See also secondary generalized seizures brain and, 225–227 epileptic, classification of, 228t pathophysiology of focal, 227–228 primary generalized, 229–230, 229f secondary generalized, 228–229, 229f pharmacologic classes and agents, 231–235, 232t selection bias, 877 selective estrogen receptor modulator. See SERM selective factor Xa inhibitors, 391 drug summary table, 398t selective serotonin reuptake inhibitors. See SSRIs selective toxicity, 564, 887 selegiline, 139, 144t, 195, 202t serotonin degradation and, 214, 221t sensitization, 285 sensory nerve stimulation, 766 sensory transduction, 265–267, 266f sequential blockade, 578 SERCA (sarcoendoplasmic reticulum calcium ATPase), calcium storage/release and, 425 serine-protease inhibitors, 393, 400t SERM (selective estrogen receptor modulator), 24 bone homeostasis disorders and, 552 drug summary table, 521t, 559t reproduction hormones and, 513–515, 513t tissue specificity, model for, 514f sermorelin, 470, 477t serotonergic neurotransmission, pharmacology of, 207–224 serotonin (5HT), 101, 207, 208 degradation inhibitors, 214–215 drug summary table, 220–221t metabolic cycle, 208 monoamine theory of depression, 212–213 neurotransmitters and, 102, 103t pain and, 268 pathways, future directions, 219 receptor agonists, 217 drug summary table, 223t receptor antagonists, 217–218 drug summary table, 223–224t receptors, signaling mechanisms, 210, 210t regulation, 208–210, 209f, 210f reuptake inhibitors, 215–217 drug summary table, 221t storage inhibitors, 213–214 drug summary table, 220t synthesis of, 105, 105f, 208–210, 209f varicosities and, 208 serotonin syndrome, 139, 216 serotonin transporter (SERT), 210 serotonin-norepinephrine reuptake inhibitors. See SNRIs SERT. See serotonin transporter sertaconazole, 623, 627t Sertoli cells, gonadal hormone action and, 508–509 sertraline, 216, 221t cytochrome P450 enzymes and, 52t

serum sickness, 62 sevelamer, 560t sevoflurane, 249, 254, 255, 260t cytochrome P450 enzymes and, 52t sex hormone-binding globulin (SHBG), 506 SHBG. See sex hormone-binding globulin shotgun approach, 848, 853 shunting, 167 SIADH (Syndrome of inappropriate ADH secretion), 474 sickle cell anemia, 779 side effects, 56 signal transduction antagonists, 707–710 molecules, 12 cancer, 699–715 sildenafil, 362, 369t cytochrome P450 enzymes and, 51t pharmacodynamic drug-drug interactions, 61 silica, toxicity of, 891–892 simvastatin, 78, 325–326, 330t single nucleotide polymorphisms (SNPs), 71, 828 single-blind trials, 866 single-source divergent neuronal systems, 99, 100–102, 100f, 101t sinoatrial nodal cells (SA nodal cells) action potential, ion currents and, 403f action potential phases, ventricular myocytes and, 404t firing cycle of, phases, 402 resting ventricular muscle action potential, 403f sinus tachycardia, 409b sirolimus, 797–798, 798f, 804t, 922 STEMI, drug-eluting stent and, 454 sirolimus-eluting stents, 797–798 sitagliptin, 536, 539t skeletal muscle toxicity, drug-induced, 66–67 skin rashes, immune responses, immunotoxicity and, 64 SLC. See human solute linked carrier slow-reacting substance of anaphylaxis (SRS-A), 826 slow-wave sleep, 117, 230 SNAREs, synaptic vesicles and, 91–92, 91f sNDA. See supplemental NDA SNPs. See single nucleotide polymorphisms SNRIs (Serotonin-norepinephrine reuptake inhibitors), 216–217, 222t sodium bicarbonate, 816, 819t sodium channel inhibitors, 231–232, 236–237t sodium channel(s), local anesthetics and, 147, 153, 154f sodium excretion, renal control of, 337–338, 337f sodium nitrate, 883 sodium nitroprusside, 360–361, 361f, 368t sodium polystyrene sulfonate, 485 sodium pump, 424 sodium retention cirrhosis and, mechanisms, 341–342, 342f heart failure and, mechanisms, 341 sodium stibogluconate, leishmaniasis and, 640, 647t sodium thiosulfate, 883 sodium-calcium exchange, 424 solid organ rejection, 790 solifenacin, 125, 130t solvent activation, drug delivery and, 921 solvent/gas partition coefficient, 242b, 243f, 244b somatic gene recombination, 733 somatic nervous system, 96 somatostatin, 106, 529–530, 540t ␦-cells, 524 gastrin, 524 somatostatin receptor ligands (SRLs), 470 somatostatin-secreting D cells, gastric acid and, 808 somatotropin, protein therapeutics and, 899t somatrem, 477t somatrem, protein therapeutics and, 899t somatropin, 470, 477t somatropin, protein therapeutics and, 899t sonophoresis, 919 sorafenib, 711t, 714t sotalol, cardiac rhythm and, 414, 420t

space of Disse, 316 spare receptors, 19, 24–25, 25f sparteine, 73 specific lymphocyte-signaling inhibitors, 796–798, 803–804t specificity, drug-receptor binding, 6 spectinomycin, 596t spinal cord anatomy, 96, 97t, 99, 99f descending inhibitory regulation in, 268–269 local inhibitory regulation in, 268–269 opioid receptor agonists and, mechanism of action, 274f periphery to, conduction from, 267 spinal cord dorsal horn, transmission in, 267–268, 267f spinal nerves, 96 spironolactone, 347, 352t, 442t, 500, 504t, 515, 522t heart failure and, 460–461, 460t spiroperidol, 198f spontaneous pain, 269 spontaneous reports, pharmacoepidemiology and, 875 sporozoites, 630 squalene epoxidase inhibitors, 622, 626t SR-BI. See scavenger receptor class B, type I SREBP2. See sterol regulatory element binding protein 2 SRLs. See somatostatin receptor ligands SRS-A. See slow-reacting substance of anaphylaxis SSRIs (Selective serotonin reuptake inhibitors), 105, 216 drug summary table, 221t, 309t drug-herb interactions, 62 melancholic depression, 211 psychotic depression, 212 ST elevation myocardial infarction (STEMI), 449 clinical management, 453–454 St. John’s wort cytochrome P450 enzymes and, 51t, 53 drug-herb interactions, 62 ST segment, 405b, 405f stabilizers, 858 stable exertional angina, nitrates and, 451 Staphylococcus aureus, 39f, 572, 587, 601, 607–608 starvation state, 525–526 state-dependent binding, 8 state-dependent ion channel block, 408 statins cytochrome P450 enzymes and, 51t LDL lowering and, 324–325, 325f on-target adverse effects, 58 statistical review, IND and, 867 statistical significance, interpreting, 878 stavudine, 671t steel factor, 779 stem cell factor (SCF), 708, 779 STEMI. See ST elevation myocardial infarction stereochemistry, 4 steroid 21-hydroxylase, 501 steroid hormones, 12 steroids, oxidation/reduction enzyme and, 73t 14␣-sterol demethylase fungus and, 622–624 inhibitors, 627t sterol regulatory element binding protein 2 (SREBP2), 324 Stevens-Johnson syndrome, 64 Streptococcus pyogenes, 594 streptogramin B, 592 streptogramins, 572, 594, 598t streptokinase, 62, 392, 849, 898 drug summary table, 399t protein therapeutics and, 905t STEMI, 454 Streptomyces, 691 streptomycin, 589, 596t, 719–720 striatum, 98, 189 structural proteins, antineoplastic drugs and, 13 subject bias, 864 substance dependence. See Addiction substance P, 155, 267, 821

952 Index substantia nigra pars compacta, 98, 101, 189, 192, 192f subthalamic nucleus, 191 succinylcholine, 123, 129t, 261t drug metabolism and, 52, 72 general anesthesia and, 256 sucralfate, 816, 819t sufentanil, 275, 280t suicide substrate inhibition, 608 suicide substrates, 4 sulbactam, 609, 615t, 721 sulconazole, 623, 627t sulfa drugs, bacteria and, 575–576 sulfadiazine, 575, 576, 579t sulfadoxine, 579t, 721 malarial plasmodia and, 636, 645t sulfadoxine-pyrimethamine, 568 antimalarial drug resistance and, 636 malarial plasmodia and, 636, 645t sulfalene, 579t sulfalene-pyrimethamine, malarial plasmodia and, 636, 645t sulfamethoxazole, 575, 576, 579t, 720 conjugation enzyme and, 73t sulfanilamide, 579t sulfinpyrazone, uric acid excretion and, 842, 845t sulfonamide derivatives, 346 sulfonamides, 576, 579t, 720 conjugation enzyme and, 73t DHFR inhibitors and, synergy of, 577–578 immune responses, immunotoxicity and, 64 sulfones, 576, 579t sulfonylureas, 535, 538t sulfuric acid, 885 sulindac, 756, 761t sulpiride, 198f sumatriptan, 217, 223t, 278 sunitinib, 711t, 715t supercoils, DNA strands and, 581 superior cervical ganglion, 95f, 96 superior mesenteric ganglion, 96 supplemental NDA (sNDA), 870 supplements, 870–871 suprofen, cytochrome P450 enzymes and, 52t suramin, African trypanosomiasis and, 639, 646t surround inhibition, neurons, 227 S-warfarin, cytochrome P450 enzymes and, 52t sympathetic nervous system, 95–96, 95f sympathetic tone, down-regulation of, 442–443 sympatholytic, 138 sympathomimetic amines, heart failure and, 462 sympathomimetics, 138, 138f, 431t synapses, 89 synapsin, 91 synaptic cleft, 91 synaptic neuromodulators, 267 synaptic transmission, stages, 90f synaptic vesicles, 89, 91f syndrome of inappropriate ADH secretion. See SIADH synergistic drugs aminoglycosides, 589 combinations, 720–721 interactions, quantification, 718–719, 719f synergy, 718 synthetic agonists, 275, 280–281t systemic absorption, local anesthetics, 155–156 systemic mastocytosis, 708 systemic vascular resistance, equation, 353 systolic heart failure, 455–456

T T cell activation pathway, 735f T cells, 733–735, 734f T lymphocytes, 821 T wave, 405b, 405f T3. See triiodothyronine T4. See thyroxine TAC, 156 tachykinins, 106, 821 tachyphylaxis, 14, 301

tacrine, 121, 129t cytochrome P450 enzymes and, 52t tacrolimus, 796–797, 797f, 804t cytochrome P450 enzymes and, 51t oxidation/reduction enzyme and, 73t tactile mechanoreceptors, 148 tadalafil, 362, 369t TAL. See thick ascending limb tamoxifen, 24, 50, 511, 514, 521t, 699, 723t carcinogenesis, drug therapy and, 67 cytochrome P450 enzymes and, 52t drug metabolism and, 74 oxidation/reduction enzyme and, 73t pharmacogenetics, 75f tamsulosin, 141, 145t tapeworms. See cestodes tardive dyskinesia, 198 target product profile, 864 target-centered drug design, 848 taxanes, 692–693, 723t tazobactam, 609, 721 TBG. See thyroid binding globulin TCAs. See tricyclic antidepressants TCE. See trichloroethylene TD50. See median toxic dose technetium fanolesomab, 914t tegaserod, IBS and, 218 tegaserod, irritable bowel syndrome and, 224t teichoic acids, 601 teicoplanin, 607–608, 614t immune responses, immunotoxicity and, 64 telavancin, 614t telithromycin, 592, 597t telmisartan, 350t telomerase, 678 telomerase inhibitors, 693 telomere biology, 681–682, 681f telomeres, 678 temafloxacin, off-target effects and, 60 temazepam, 171 clinical uses for, duration of action, 172t GABAA receptors and, 183t temozolomide, 687, 696t temporal lobe, 97, 97f temsirolimus, 710, 714t tenecteplase, 393, 898 drug summary table, 399t protein therapeutics and, 903t STEMI, 454 teniposide (VM-26), 691, 697t tenofovir, 671t teratogenesis, due to drug therapy, 67–69 terazosin, 141, 145t, 371t, 443 terbinafine, 622, 626t terbutaline, 140, 145t, 828, 834t terconazole, 623, 627t terfenadine, off-target effects, 59 teriparatide, 556, 560t protein therapeutics and, 903t terlipressin, 344 terodiline, 125, 130t tertiary structure, amino acids and, 3, 4f tesamorelin, 477t testicular cancer, antineoplastic combination chemotherapy and, 726 testosterone cypionate, 519, 523t testosterone enanthate, 523t hypogonadism, 519 male contraception and, 518 testosterone patches, hormone replacement and, 519 testosterone undecanoate, contraception, 518 tetanic fade, 115 tetanus toxin, GABAergic transmission and, 169t tetracaine, 156, 158, 161t tetracyclines, 591–592, 597t, 719 30S ribosomal subunits and, 597t H. pylori, 817 malarial plasmodia and, 635–636, 645t unfavorable drug combinations, 721 tetrahydrocannabinol (THC), 301 5␣-tetrahydrodeoxycorticosterone (THDOC), 168

tetrahydrofolate (THF), 675 tetrahydrozoline, 139, 144t 3,5,3⬘,5⬘-tetraiodothyronine (T4), 480 tetrodotoxin, 89 TFPI. See tissue factor pathway inhibitor TH. See tyrosine hydroxylase TH cells. See T-helper cells thalamocortical projections, 228 thalamus, 98 thalidomide, 60, 711, 715t THC. See tetrahydrocannabinol THDOC, 5␣–tetrahydrodeoxycorticosterone thecal cells, gonadal hormone action and, 508 T-helper (TH) cells, 768 theophylline, 436t, 828f, 829, 835t therapeutic dosing, 39–42, 40f, 41f, 42f therapeutic index (TI), 25–26, 241–242, 564 therapeutic ratio (TR), 25–26 therapeutic window, 25–26 THF. See tetrahydrofolate thiabendazole, 642, 648t thiazides, 346–347, 352t, 440t, 441, 442t thiazolidinediones (TZD), 536, 540t thick ascending limb (TAL), 335, 337–339, 339f thioamines, 486 thiocyanate, 485, 488t thioguanine, 695t conjugation enzyme and, 73t DNA structure, 684f thiopental, 173, 261t clinical uses for, duration of action, 174t GABAA receptors and, 183t intravenous anesthesia and, 255 thioperamide, 772 thiopurine S-methyltransferase (TPMT), 72–75, 75f thioridazine, 198f, 204t thiotepa, 687, 696t thiothixene, 198f, 204t thioxanthenes, schizophrenia and, 197 thoracolumbar system, 95 threshold potential, 86, 88 thrombin, 374 protein therapeutics and, 903t thrombocytopenia, 785 thrombolytics, 392–393 drug summary table, 399t STEMI, 453–454 thrombomodulin, 380 thrombopoiesis, 782 thrombopoietin (TPO), 779, 782, 785, 788t thrombosis, 372 drug summary table, 395–400t pharmacology of, 372–394 protein therapeutics and, 903t, 909t thromboxane A2 (TxA2), 373 thromboxane antagonists, 758, 763t thromboxanes, cyclooxygenase pathway and, 744–745 thrombus, 380 thymidine kinase, 655 thymidylate synthase, 621, 676, 684 inhibitors, 621, 684, 694t thymine, 581 thyroglobulin, 481 thyroid binding globulin (TBG), 482 thyroid function, drugs testing, 478t thyroid gland case study, 481 diseases, future treatment, 487 pathophysiology, treatment agents, 484 pharmacology of, 480–487 physiology, 480–484 thyroid hormone bone mineral metabolism and, 547–548 effects on target tissues, 482–483 homeostasis, drugs affecting, 487 metabolism, 482 receptor actions, 483f release inhibitors, drug table for, 488t replacements, drug table, 488t structure and metabolism of, 481f

Index 953

synthesis, 480–482, 482f pharmacologic interventions, 485f synthesis, storage, release, 482f thyroid peroxidase, 481, 482f thyroid-stimulating hormone (TSH), 484, 912, 913t anterior pituitary gland, 467, 472 thyroid-stimulating immunoglobulin (TsIg), 484 thyrotropin (TSH), 472, 478t, 913t thyrotropin-releasing hormone (TRH), 106, 483–484 thyroxine (T4), 480 TI. See therapeutic index tiagabine, 182t, 232t, 239t GABA metabolism and, 169 GABAergic transmission and, 169t ticarcillin, 609, 611, 615t ticlopidine, 383–384, 395t tigecycline, 572, 592, 597t time constant, global equilibration and, 245 time-dependent bactericidal agents, 717–718, 718f timolol, 141t, 142, 146t cytochrome P450 enzymes and, 51t tinidazole, malarial plasmodia and, 639, 646t tinzaparin, 391, 398t tiotropium, 124, 130t, 827, 834t tipifarnib, 709 tipranavir, 662, 672t tirofiban, 386, 396t tissue autonomic ganglionic blockade and, 119t compartments, general anesthetics and, 246, 246f mass, volume of distribution and, 33 tissue factor, 372–374 tissue factor pathway inhibitor (TFPI), 380 tissue plasminogen activator (t-PA), 380 protein therapeutics and, 903t tissue schizonts, 630 tissue toxicity, 62–69 TLRs. See toll-like receptors TMA. See butyl trimethyl ammonium TNF-␣. See tumor necrosis factor-␣ tobacco, 299 peptic ulcer disease, 816 toxicity of, 888–889 tobramycin, 589, 596t tocilizumab, 800, 805t, 908t tolazamide, 538t tolbutamide, 538t tolcapone, 195, 203t tolerance, 54 drug dose and, 285 mechanisms, 287, 288f, 290f opioids and, 273 toll-like receptors (TLRs), 732 tolterodine, 125, 130t tolvaptan, 350t, 479t tonic inhibition, local anesthesia and, 153–155, 155f tonic phase, seizure propagation, 229 tonic-clonic seizures, 228, 229f tophi, 839 topical anesthesia, 156, 157b topiramate, 232t, 239t, 345 addiction and, 305 topoisomerase inhibitors, 596t DNA and, 691–692 drug summary table, 697t topoisomerases bacterial DNA, 582–583 IV, 583, 587 type I, 582, 691 type II, 582, 691 DNA supercoiling and, 584f topotecan, 691, 697t torsades de pointes, 407, 409b, 413–414 torsemide, 346, 351t, 442t tositumomab, 712, 715t, 910t toxic effects, 56 toxic epidermal necrolysis, 64 toxic metabolites, 50 toxic plants, 885 toxicology case study, 882

conclusion and future directions, 883 drug discovery and development, 856–857 environmental, 881–892 t-PA. See recombinant tissue plasminogen activator; tissue plasminogen activator TPH. See tryptophan hydroxylase TPMT. See thiopurine S-methyltransferase TPO. See thrombopoietin TR. See therapeutic ratio trade name, drugs, 869 tramadol, 269, 276–277, 280t trandolapril, 349t tranexamic acid, 393, 400t transcellular biosynthesis examples, 752f routes, eicosanoids and, 752 transcription, 596t inhibitors, 596t rifamycin derivative and, 587–588 viral life cycle, 650 transcription factors, 6, 12 transcription regulators, 12 transdermal drug administration, 30t, 31, 919 transduction, 572 transformation, DNA and, 568, 572 transient receptor potential (TRP), 266 transitional anesthetics, 257 translation inhibitors, 588–595, 588t malarial plasmodia and, 635–636, 645t viral life cycle, 650 transmembrane ion channels, 7–9, 8t transmembrane receptors, with enzymatic cytosolic domains, 10–12, 11f transmission mode, neurons and, 230 transmitter metabolism, reuptake and, 92 transpeptidase enzymes, bacterial cell wall and, 600 transpeptidases, cell wall synthesis and, 604 transplantation, 790–792 tranylcypromine, 139, 144t trastuzumab, 708, 713t, 724t, 906 travoprost, 763t trazodone, 217, 223t trematodes (Flukes), 640 tremor (T) syndrome, 887 tretinoin, 723t, 786, 789t TRH. See thyrotropin-releasing hormone triamcinolone, 497, 498f, 503t, 830, 835t triamterene, 347, 352t, 442t triazolam, 171 clinical uses for, duration of action, 172t cytochrome P450 enzymes and, 51t GABAA receptors and, 183t triazoles, 622–624, 627t unfavorable drug combinations, 721 trichloroethylene (TCE), 892 tricyclic antidepressants (TCAs), 105, 139, 215–216, 221t antiadrenergic effects, 216 anticholinergic effects, 216 antihistaminergic effects, 216 drug metabolism and, 53 drug summary table, 282t, 309t trientine, 884 trifluoperazine, 198f, 204t trifluperidol, 198f trifluridine, 671t trihexyphenidyl, 125, 195, 203t 3,5,3⬘-triiodothyronine (T3), 480, 481f trilostane, 499, 499t, 504t trimethaphan, 126, 131t, 443 trimethoprim, 565, 576, 577, 579t, 720 trimipramine, 221t tripelennamine, 774t triple therapy, H. pylori, 817 triptans, 217, 278 troglitazone, toxicity and, 56 troleandomycin, cytochrome P450 enzymes and, 51t tropomyosin, cardiac myocyte contraction and, 425t troponin complex, cardiac myocyte contraction and, 425t

trospium, 125, 130t trovafloxacin, off-target effects and, 60 TRP. See transient receptor potential TRPV1 cation channels, 266 TRPV2 cation channels, 266 Trypanosoma brucei gambiense, 639 Trypanosoma cruzi, 640 trypsin, 904t tryptophan hydroxylase (TPH), 208, 209 TSH. See thyroid-stimulating hormone TsIg. See thyroid-stimulating immunoglobulin T-type calcium channel, 230 tuberculosis, antimicrobial combination therapy, 719–720 tuberoinfundibular pathway, 191 tuberomammillary nucleus, 101, 101f tubocurarine, 126, 131t, 256, 261t tubuloglomerular feedback mechanism, 335 tumor lysis syndrome, 842 tumor necrosis factor-␣ (TNF-␣), 798–799, 798f, 804t tumor(s) initiation and promotion, 889f pharmacologic classes and agents, 707–712 tumor-specific monoclonal antibodies, 715t TxA2. See thromboxane A2 type I 5⬘-deiodinase, 482 type I alpha-interferon, 902t type II 5⬘-deiodinase, 482 type III 5-deiodinase, 482 typical antipsychotics schizophrenia and, 197 typical depression, 211 tyramine, 138 MAO inhibition and, 188 toxicity, 214 tyrosine hydroxylase (TH), 132, 186–187, 209 tyrosine kinase-associated receptors, 12 TZD. See thiazolidinediones

U U fibers, 228 UA/NSTEMI. See unstable angina/non-ST elevation myocardial infarction ubiquinone, malarial plasmodia and, 632, 635 ubiquitin-proteasome pathway, 701–705, 703f UDP-glucuronyl transferase (UDPGT), 47 UDPGT. See UDP-glucuronyl transferase UGN. See natriuretic peptide uroguanylin UMP. See uridylate uncoating. See viral uncoating uncompetitive antagonists, 7 underfill theory, cirrhosis, Na⫹ retention and, 341, 342f unfractionated heparin, 389, 391 coagulation factor inactivation and, 390f drug summary table, 397t unitary hypothesis, 256 unstable angina, 362, 452–453 unstable angina/non-ST elevation myocardial infarction (UA/NSTEMI), 449, 452–453 unstable plaques, acute coronary syndromes and, 448 uptake model, 244–250 applications of, 250–253, 251f uracil, DNA structure, 684f urate crystals, 839–840, 840f ureido penicillins, 611 uric acid, 839 excretion, agents increasing, 842, 845t metabolism, agents enhancing, 842, 845t synthesis, agents decreasing, 841–842, 844t uricase, uric acid metabolism and, 842 uricosuric agents, 842 uridylate (UMP), 675–676 urofollitropin (FSH), 479t anterior pituitary gland, 474 urokinase, protein therapeutics and, 903t use-dependent block, 88 ustekinumab, 805t, 908t

954 Index

V ⫹

⫹

vacuolar H -ATPase (vH ATPase), 338 vagomimetic, 122 vagus nerve, 96 valacyclovir, 658, 670t valdecoxib, 64, 271, 276, 757 valganciclovir, 670t valproic acid, 232t, 237t bipolar affective disorder and, 218 seizures and, 234, 237t valsartan, 350t, 371t van der Waals’ forces, 3–4 vancomycin, 8, 39f, 607–608, 614t, 718 immune responses, immunotoxicity and, 64 red man syndrome and, 64 vancomycin-resistant enterococcus (VRE), 572, 607 vandetanib, 707, 711t vardenafil, 362, 369t varenicline addiction and, 305 drug dependence and, 309t variant angina, 450 varicella zoster virus (VZV), 655 vascular capacitance, vascular resistance and, 353–354 vascular endothelial growth factor (VEGF) inhibitors, 711, 711t receptors, 705–706, 706t vascular endothelium, 356 vascular resistance-based hypertension, 439–440 vascular smooth muscle cells Ca2⫹ for, 356f contraction and relaxation, mechanism, 354–356, 356f sites in, vasodilators and, 358f vascular tone autonomic nervous system and, 357–358 cardiovascular physiology parameters and, 355t case study, 354 drug summary table, 368–371t future directions, 366–367 modulation, 443–444 pharmacologic agents/classes and, 358–366 pharmacology of, 353–367 regulation of, 356–368 vasoactive intestinal polypeptide (VIP) asthma, 821 vasoconstriction, 373 vasodilators, 355, 766 heart failure and, 461 vascular tone, 358, 358f vasopressin (ADH), 334. See also SIADH antagonists, 479t CNS and, 98 receptor agonists, 344 receptor antagonists, 344 vasopressin receptor 2 antagonists, 350t vatalanib, 711t vecuronium, 126, 131t, 261t VEGF. See vascular endothelial growth factor venlafaxine, 216, 222t, 277 atypical depression and, 212 cytochrome P450 enzymes and, 51t venodilators, heart failure and, 461 venous capacitance, 354 vental tegmental area (VTA), 189–190 ventilation, change effects in, anesthetic and, 251, 251f ventilation-limited anesthetics, 249 ventilation/perfusion mismatch (V/Q mismatch), anesthesia induction and, 252 ventral horn, 96 ventral roots, 96 ventral tegmental area (VTA), 189–190, 289–291 ventricular action potential, ion currents and, 404f ventricular fibrillation, 409b ventricular tachycardia (VT), 409b

verapamil, 363, 364t, 370t, 405, 443 cardiac rhythm and, 415, 421t cytochrome P450 enzymes and, 51t vertical transmission, 572 very-low-density lipoproteins (VLDL) lipoproteins and, 315 remnants, 316 secondary hyperlipidemia and, 323–324, 323t vesamicol, 111, 120, 128t vesicular monoamine transporter (VMAT), 133, 187 vesnarinone, 436t vessel dilation, inflammatory response, 737 vessel-poor group (VPG), anesthesia and, 246 vessel-rich group (VRG), anesthesia and, 246 vH⫹ ATPase. See vacuolar H⫹-ATPase VHL protein. See von Hippel-Lindau protein vidarabine, 671t vigabatrin, 182t, 232t, 234, 238t GABA metabolism and, 169–170 GABAergic transmission and, 169t seizures and, 235 vinblastine, 692, 698t combination chemotherapy, 726 vinca alkaloids, 692, 723t vincristine, 692, 698t, 723t combination chemotherapy, 726 Vioxx. See rofecoxib VIP. See vasoactive intestinal polypeptide viral attachment and entry inhibitors, 651–653 viral attachment inhibition, 670t viral genome replication inhibition, 655–662 viral infections case study, 650 drug summary table, 670–673t pharmacology, 649–669 viral integration inhibitors, 672t viral life cycle, 650–651, 651f viral maturation, 650–651 inhibition, 662 viral release inhibition, 664–665, 672t viral replication, physiology of, 649–651 viral uncoating inhibitors, 670t viral life cycle and, 650 Virchow’s triad, 380, 380f virions, 650 viruses, host cells and, 568 vitamin D and analogues, 560t hypoparathyroidism, 546–547, 557 vitamin D-dependent rickets, 557 vitamin K mechanism of action, 387 vitamin K epoxide reductase complex 1 (VKORC1), 77 VKORC1. See vitamin K epoxide reductase complex 1 VLDL. See very-low-density lipoproteins VM-26. See teniposide VMAT. See vesicular monoamine transporter voglibose, 538t voltage-clamping, 87 voltage-dependent function, cellular communication and, 82 voltage-gated Ca2⫹-selective channels, 354 voltage-gated channels, 7, 8t voltage-gated L-type Ca2⫹-channels, 355 voltage-gated sodium channel, 153, 154f volume capacity, global equilibration and, 245 volume of distribution (Vd), of drug, 33–34 volume regulation case study, 333 drug summary table, 349––352t pharmacology of, 332–348 physiology of, 332–340 volume regulators, 334–337 agents modifying, 342–344 volume sensors, 333–334 volume-based hypertension, 440

volume-contraction alkalosis, 346 von Hippel-Lindau protein (VHL), 705–706 von Willebrand factor (vWF), 373 voriconazole, 567, 623, 624, 627t VP-16. See etoposide VPG. See vessel-poor group V/Q mismatch. See ventilation/perfusion mismatch VRE. See vancomycin-resistant enterococcus VRG. See vessel-rich group VT. See ventricular tachycardia VTA. See ventral tegmental area vulnerable plaques, acute coronary syndromes, 448 vWF. See von Willebrand factor VZV. See varicella zoster virus

W warfarin, 4, 12, 77f, 397t anticoagulation effect drugs diminishing, 389t drugs enhancing, 389t clinical uses, 387–389 drug metabolism and, 53 mechanism of action, 387, 388f oxidation/reduction enzyme and, 73t pathway-based pharmacogeneticspharmacogenomics, 76–77 weak positive allosteric agonists, 171, 171f wheal-and-flare reaction, 62, 766 white matter, 97–98, 97f WHO (World Health Organization), tuberculosis treatment, 720 withdrawal case study, 285 mechanisms, 288f syndromes, 289 withdrawal, pharmacoepidemiology and, 874–875, 875t Withering, William, 422 WNT signaling, nuclear factor-kappa B (NF␬B) pathways and, 704, 704f Wolff-Chaikoff effect, 486 Women’s Health Initiative study, hormone replacement, 518, 518t workplace exposures, 891 World Health Organization. See WHO worms. See helminths

X xanthine oxidase, 838 xenobiotics, 43 x-ray crystallography, 853

Y yeasts, 618 yohimbine, 141, 145t

Z zafirlukast, 764t zalcitabine, 671t zanamivir, 568, 664, 672t zero-order kinetics, 37 ziconotide, 133, 267 zidovudine (AZT), 568, 659, 671t zileuton, 764t, 830, 836t ziprasidone, 200, 205t zoledronate, 552, 559t Zollinger-Ellison syndrome, 811 zolmitriptan, 217, 223t, 278 zolpidem, 171, 171–172, 183t zona fasciculata, 489 zona glomerulosa, 489 zona reticularis, 489 zonisamide, 232t, 239t zotarolimus, 454, 804t, 922 zotarolimus-eluting stents, 797–798