Advances in Pharmacology Volume 22

Advances in

Pharmacology Vohme 22

Advisory Board R. Wayne Alexander

Maureen Howard

Harvard Medical School Brigham and Women's Hospital Department of Medicine Cardiovascular Division Boston, Massachusetts

Director of Immunology DNAX Research Institute of Molecular and Cellular Biology Palo Alto, California

K. Frank Austen

Department of Pharmacology University of Virginia School of Medicine Charlottesville. Virginia

Harvard Medical School Brigham and Women's Hospital Department of Rheumatology and Immunology Boston, Massachusetts

Jay A. Berzofsky National Institutes of Health Bethesda, Maryland

Floyd E. Bloom Division of Preclinical Neuroscience Department of Basic and Preclinical Research Scripps Clinic and Research Institute La Jolla, California

Thomas F. Burks

Josep Larner

Anthony Y.H. Lu Department of Animal Drug Metabolism Merck, Sharp & Dohme Laboratories Rahway, New Jersey

Lawrence J. Marnett Department of Chemistry Wayne State University Detroit, Michigan

Bernard Moss Laboratory of Viral Diseases National Institutes of Health Bethesda, Maryland

Department of Pharmacology College of Medicine Health Sciences Center The University of Anzona Tucson, Arizona

Michael J. Peach

Anthony Cerami

Martyn T. Smith

Laboratory of Medical Biochemistry The Rockefeller University New York, New York

Julius J. Cohen Department of Physiology The University of Rochester Medical Center Rochester, New York

Joseph T. Coyle Division of Child Psychiatry The Johns Hopkins Medical Institutions Baltimore, Maryland

Morley Hollenberg Faculty of Medicine Department of Pharmacology and Therapeutics Health Sciences Centre The University of Calgary Calgary, Alberta. Canada

Department of Pharmacology University of Virginia School of Medicine Charlottesville, Virginia Department of Biomedical & Environmental Health Sciences The University of California, Berkeley Berkeley, California

August Watanabe Department of Cardiology Indiana University Medical Center Indianapolis, Indiana

Stephen Waxman Division of Neurology Yale University School of Medicine New Haven, Connecticut

Grant R. Wilkinson Division of Clinical Pharmacology Vanderbilt University School of Medicine Nashville. Tennessee

Advances in

Pharmacology V I

Volume 22

Edited by

J. Thomas August Department of Pharmacology Johns Hopkins University Baltimore, Maryland

M. W. Anders Department of Pharmacology University of Rochester Rochester, New York

Ferid Murad Pharmaceutical Products Division Abbott Laboratories Abhott Park, Illinois

Academic Press Harcoun Brace Jovanovich, Publishers San Diego New York Boston

London Sydney

Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1991 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 Unired Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road. London NWl 7DX

Library of Congress Catalog Card Number:

ISBN 0- 12-032922-0 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 91

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Contents

Contributors

ix

Acyclovir: Mechanism of Antiviral Action and Potentiation by Ribonucleotide Reductase Inhibitors John E. Reardon and Thomas Spector 1. Introduction 1 11. Acyclovir Transport 111. IV. V. VI. VII.

3 Metabolic Activation of Acyclovir 3 Mechanism of Antiviral Action 6 Potentiation of the Antiviral Action of Acyclovir 13 19 Resistance and Hypersensitivity to Acyclovir Conclusions 22 References 23

Rational Approaches to Osteoporosis Therapy Robert Marcus I. 11. 111. IV. V. VI. VII.

Introduction 29 Age-Related Changes in Bone Mass 32 34 Mechanisms of Bone Mass Regulation 37 Osteoporosis Experimental Approaches 47 Nonpharmacological Considerations 48 48 Conclusions References 49

Molecular Asymmetry and Its Pharmacological Consequences Kenneth M. Williams I. 11. 111. IV. V. VI.

Introduction 58 Pharmacokinetic Consequences of Chirality 64 Chirality and Polymorphic Drug Disposition 81 Pharmacodynamic Consequences of Chirality 88 Enantiomer-Enantiomer Interactions 97 I01 Enantioselective Drug-Enantiomer Interactions V

vi

Conknts

VII. VIII. IX. X. XI.

Enantiomeric Impurity and Potential Problems 105 Enantiomers as Biochemical Probes 108 Therapeutic and Regulatory Considerations 110 Therapeutic Drug Monitoring 119 Conclusions 120 References 120

Blood-Brain Barrier: Transport Studies in Isolated Brain Capillaries and in Cultured Brain Endothelial Cells Yoshinobu Takakura, Kenneth 1. Audus, and Ronald T. Borchardt I. Introduction 137 11. Establishment and Characterization of an in V i m Blood-Brain Barrier Model 138 111. In V i m Transport Studies 143 152 IV. In V i m Studies on Regulation of Blood-Brain Barrier Transport V. Summary 157 References 157

Protein Kinase Inhibitors: Probes for the Functions of Protein Phosphorylation John E. Casnellie I. Introduction

167

11. Inhibitors of Protein Serinerhreonine Kinases 111. Inhibitors of Protein nrosine Kinases 186

IV. Concluding Remarks References 194

169

193

Renin Inhibitors Hollis D. Kleinert, William R. Baker, and Herman H. Stein I. Introduction

207

11. Physical and Functional Properties of Renin 208 111. Biochemical Evaluation and Specificity of Renin Inhibitors

IV. Design and Structure of Renin Inhibitors V. Pharmacology of Renin Inhibitors 229 VI. Conclusion 241 24 1 References

214

210

Contents

vii

The Capacitative Model for Receptor-Activated Calcium Entry James W. Putney, Jr. I. 11. 111. IV.

Introduction 251 Epithelial Cells: Models for Studying Receptor-Activated Ca2+Signaling Mechanisms of CaZ+Entry 255 Conclusions 266 References 267

252

Calcium Channel Antagonists in the Prevention of Neurotoxicity Stuart A. Lipton I. Introduction 272 11. Potential Problems in Interpreting the Effectiveness of Calcium Channel Antagonists in Preventing Neurotoxicity 272 111. Types of Neurotoxicity Attenuated by Calcium Channel Antagonists 273 IV. The Hypothesis of Calcium-Associated Neuronal Damage 275 V. Potential Contribution of T, N, L, and P Types of Calcium Channels to Neurotoxicity 275 VI. Types of Voltage-Dependent Calcium Channel Antagonists 276 VII. In Vi'tro Models of Neurotoxicity and Effects of Calcium Channel Antagonists 279 VIII. In Vivo Animal Models of Ischemia and Effects of Calcium Channel Antagonists 283 IX. Early Human Trials and Prospects for Calcium Channel Antagonists in Preventing Neurotoxicity 288 X. Conclusions 290 References 29 1

New Directions in the Delivery of Drugs and Other Substances to the Central Nervous System Yvette Madrid, Laura Feigenbaum Langer, Henry Brem, and Robert Langer I. Introduction

299

11. Altering the Barrier

302 111. Altering Agents 304 IV. Circumventing the Barrier V. Conclusion 320 References 320

308

...

Contents

Vlll

Hormonal Regulation of Cytochrome P-450Gene Expression Johap Lund, Peter G. Zaphiropoulos, Agneta Mode, Margaret Warner, and Jan-Ake Gustafsson I. General Introduction 325 11. Cytochrome P-450 Nomenclature and Gene Structure 326 111. Transcriptional Regulation of Cytochrome P-450 Genes: Involvement of cisActing DNA Elements 327 IV. Hormonal Regulation of Cytochrome P-450 Involved in the Biosynthesis of Hormones 329 V. Regulation of Liver Cytochrome P-450 by Sex Steroids and Growth 337 Hormone 342 VI. Regulation of Cytochrome P-450 in the Prostate, Pituitary, and Brain 344 VII. General Conclusions VIII. Future Perspectives-Novel Endocrine Systems? 345 References 345 Index

355

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Kenneth 1. Audus (1 37),Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045 William R. Baker (204,Abbott Laboratories, Cardiovascular Research Division, Abbott Park, Illinois 60064 Ronald T. Borchardt (134, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045 Henry Brem (299), Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 2 1205 John E. Casnellie (164,Department of Pharmacology and Cancer Center, University of Rochester School of Medicine, Rochester, New York 14642 Jan-Ake Gustafsson (325), Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, S- 141 86 Huddinge, Sweden Hollis D. Kleinert (207),Abbott Laboratories, Cardiovascular Research Division, Abbott Park, Illinois 60064 Laura Feigenbaum Langer (2991,Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Robert Langer (299),Department of Chemical Engineering and Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Stuart A. Lipton (271),Laboratory of Cellular and Molecular Neuroscience. Department of Neurology, The Children’s Hospital, Beth Israel Hospital, Brigham and Women’s Hospital, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 021 15 Johan Lund (325), Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, S- 141 86 Huddinge, Sweden Yvette Madrid (299),Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139 Robert Marcus (29),Department of Medicine, Stanford University, Stanford, California 94305 and the Aging Study Unit, Geriatrics Research, Education & Clinical Center, Department of Veterans Affairs Medical Center, Palo Alto, California 94304 ix

X

Contributors

Agneta Mode (325),Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, S- 141 86 Huddinge, Sweden James W. Pulney, Jr. (251),Calcium Regulation Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 John E. Reardon (11, Division of Experimental Therapy, Wellcome Research Laboratories, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709

Thomas Spector (I), Division of Experimental Therapy, Wellcome Research Laboratories, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709 Herman H. Stein (207),Abbott Laboratories, Cardiovascular Research Division, Abbott Park, Illinois 60064 Yoshinobu Takakura (137),Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045 Margaret Warner (325),Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, S- 141 86 Huddinge, Sweden Kenneth M. Williams (57), Department of Clinical Pharmacology and Toxicology, St. Vincent’s Hospital, Sydney 2010, Australia and School of Physiology and Pharmacology, University of New South Wales, Sydney 2033, Australia Peter G.Zaphiropoulos (325),Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden

Acyclovir: Mechanism of Antiviral Action and Potentiation by Ribonucleotide Reductase Inhibitors John E. Reardon and Thomas Spector Division of Experimental Therapy Wellcome Research Laboratories Burroughs Wellcome Company Research Triangle Park, North Carolina 2 7709

1. Introduction 11. Acyclovir Transport 111. Metabolic Activation of Acyclovir A. Thymidine Kinase B. Cellular Kinases C . Cytoplasmic 5'-Nucleotidase IV. Mechanism of Antiviral Action A. Viral DNA Polymerase B. Human DNA Polymerases C . Mechanism-Based Affinity Chromatography of Viral DNA Polymerase V. Potentiation of the Antiviral Action of Acyclovir A , Properties of Viral Ribonucleotide Reductases B. Inhibition of Ribonucleotide Reductases C . Synergistic Inhibition of Virus Replication by Acyclovir and A 1 1 1OU VI. Resistance and Hypersensitivity to Acyclovir A. Thymidine Kinase Mutants B. DNA Polymerase Mutants C . Ribonucleotide Reductase Mutants VII. Conclusions References

1. Introduction Acyclovir (9-[(2-hydroxyethoxy)methyl]guanine, Zovirax) is the therapy of choice for treatment of initial, primary, and recurrent genital herpesvirus infecAdvances m Pharmacology. Volume 22

Copyright 0 1991 by Academic Press. Inc. A l l nghn of reproducuon in any form reserved

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John

E. Reardon and Thomas Spector

tions, herpes encephalitis, and herpes zoster, and for prophylactic suppression of recurrent genital herpes infections. In contrast to other nucleoside analogs, some of which are no longer used or are used only sparingly (Szczech, 1986; Shannon, 1984; Galasso, 1984), acyclovir has proved to be both a safe and efficacious treatment for herpesvirus infections. Adenosine analogs with the acyclic side chain of acyclovir (Fig. 1) were first synthesized for a structure-activity study of adenosine deaminase substrates (Schaeffer et al., 1971). Later, Elion et al. (1977) and Schaeffer et al. (1978) reported that acyclovir, the guanosine analog with the acyclic side chain, was a highly efficacious antiviral agent. Acyclovir was active against herpes simplex virus type 1 (HSV-I) replication, in a plaque reduction assay, with an ED,, of 0.1 pM. In contrast, acyclovir was relatively nontoxic to the host Vero cells, with an ED,, of 300 p M . Thus, the in vitro therapeutic index of 3000 suggested that acyclovir would be a safe and effective treatment for herpes virus infections. In vivo studies demonstrated the potent antiherpetic effect of acyclovir in mice infected intracerebrally with HSV-1, in rabbits with herpetic keratitis, and in guinea pigs with cutaneous herpes infections (Schaeffer et al., 1978). Acyclovir effectively inhibits in vitro replication of HSV- I , herpes simplex virus type 2 (HSV-2), and varicella zoster virus (VZV) and is currently used for clinical treatment of these viral infections. Although acyclovir exhibits in vitro activity against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) (Schaeffer et al., 1978; Drach, 1984), it is not currently approved for clinical use in the treatment of EBV and CMV infections. Herpesviruses encode for several enzymes that are isofunctional with cellular enzymes. Among these are thymidine kinase, DNA polymerase, and ribonucleotide reductase (reviewed in Kit, 1979). The early studies of Elion et al. (1977) indicated that (1) acyclovir is selectively phosphorylated by the viral thymidine kinase, (2) the triphosphate of acyclovir is an effective competitive inhibitor, with respect to dGTP, of the HSV-I DNA polymerase, and ( 3 ) acyclovir monophosphate is incorporated into DNA, resulting in termination of DNA synthesis. The mechanism of action and selectivity of acyclovir have been studied extensively. These studies have included investigations of the substrate specificity of the viral thymidine kinase and the mechanism of selective inhibition of the viral

‘0-0

J

Fig. 1 Structure of acyclovir.

Acyclovir

3

DNA polymerase by acyclovir triphosphate. Furthermore, a detailed understanding of the metabolic perturbations caused by acyclovir treatment has led to the development of a combination therapy in which the antiviral efficacy of acyclovir is potentiated by selective inhibitors of the viral ribonucleotide reductase. The development of acyclovir as a chemotherapeutic agent, its mechanism of antiviral action, and its potentiation by inhibition of viral ribonucleotide reductase have been reviewed (Elion, 1982, 1983, 1984, 1986, 1989; Brigden e t a l . , 1981; Hopkins and Furman, 1990; Furman et al., 1986; Spector and Fyfe, 1991). In light of the interesting mechanism of action of acyclovir and the clinical promise of an enhanced antiviral therapy, we were prompted to review this topic and to describe some of the recent data.

II. Acyclovir Transport The transport of acyclovir into human erythrocytes has been investigated by Mahony et al. (1988). The rate of influx of acyclovir is saturable, indicating that it is exclusively carrier-mediated. The kinetic evidence suggesting that acyclovir transport is mediated by the nucleobase transporter includes ( 1 ) adenine, hypoxanthine, and guanine compete with acyclovir for influx with K , values similar to their respective K , values as permeants, and (2) acyclovir competitively inhibits nucleobase influx with Ki values identical to its K,, value (260 pM) for influx. Evidence that acyclovir influx in human erythrocytes is not mediated by the nucleoside transporter includes ( 1 ) concentrations of 6-[(4-nitrobenzyl)thio]-9-BD-ribofuranosylpurine (NBMPR), dilazep, and dipyridamole which completely inhibit the nucleoside transport system have little effect on acyclovir influx, and (2) a 100-fold molar excess of several different permeants of the nucleoside transporter does not inhibit acyclovir influx. The nucleoside transport inhibitors NBMPR, dilazep, and dipyridamole enhance the in vitro antiviral activity of acyclovir (Mahony et d., 1988). A possible explanation for this observation is that these inhibitors impede the transport of thymidine, but not of acyclovir. Since thymidine and acyclovir compete for phosphorylation by the virally induced thymidine kinase (Section III,A) and the nucleoside transport inhibitors reduce the intracellular concentration of thymidine. they should enhance the phosphorylation of acyclovir.

111. Metabolic Activation of Acyclovir A. Thymidine Kinase Kit and Dubbs (1963) first demonstrated that HSV encodes for a thymidine phosphorylating activity. This unique kinase catalyzes the phosphorylation of a

4

John E. Reordon and Thomas Spector

series of nucleoside analogs, including 5-halogenated-2’-deoxycytidines (Cooper, 1973) and 5-alkyl- and 5-allyl-2’-deoxyuridine (Cheng et al., 1976). In contrast, these analogs are not appreciably phosphorylated in uninfected cells. This differential activity accounts for the selective antiviral effect of these compounds. Likewise, the selective antiherpetic effect of acyclovir suggested that it too was selectively phosphorylated by the virally encoded thymidine kinase. Elion er al. (1977) and Fyfe et al. (1978) demonstrated that the virally encoded thymidine kinase phosphorylates acyclovir. The evidence includes (1) the acyclovir phosphorylating activity of HSV- 1-infected Vero cells is separated from the host thymidine kinase activity by affinity chromatography on thymidine agarose, (2) thymidine inhibits acyclovir phosphorylation by the purified HSV- 1 thymidine kinase, (3) thymidine reverses the antiviral effect of acyclovir in HSV- 1-infected Vero cells, and (4) a temperature-sensitive thymidine kinasedeficient (TK-) HSV-1 mutant is resistant to acyclovir. The finding that the acyclovir phosphorylating activity of HSV- 1-infected Vero cells is separated from the host thymidine kinase activity by affinity chromatography suggested that the host cell thymidine kinase does not play a significant role in the phosphorylation of acyclovir. Further evidence includes (1) acyclovir is not a substrate for thymidine kinase purified from Vero cells (Keller et al., 1985) and is a very weak inhibitor of this enzyme with a K ivalue of 20 mM versus thymidine (Keller et al., 1981), (2) the trace phosphorylation of acyclovir in uninfected cells is not antagonized by thymidine (Funnan et al., 1986), and (3) host cells deficient in thymidine kinase phosphorylate acyclovir (Furman et al., 1980). The kinetics for HSV-1 and HSV-2 thymidine kinase are complex (Cheng, 1976). Both enzymes have a sigmoidal dependence on Mg2+-ATP with a Hill constant of 2 (at saturating concentrations of thymidine), indicating that there are two Mg2 -ATP binding sites on the enzyme. The K , values for Mg2 -ATP are 30 pM (HSV-1) and 70 fl(HSV-2). At saturating concentrations of Mg2 -ATP (2 mM), both enzymes have a hyperbolic dependence on thymidine concentration, with K , values of 0.59 pM (HSV-1) and 0.35 pM (HSV-2). Acyclovir exhibits a hyperbolic velocity versus substrate plot with HSV-2 thymidine kinase with a K , value of 88 pM and a relative V,,, 16% that of thymidine (Ellis et al., 1987). In contrast, a nonlinear dependence on acyclovir concentration is observed with HSV- 1 thymidine kinase. The concentration of acyclovir giving halfmaximal velocity is 100 pM (Fyfe et al., 1983). The nonlinear kinetic behavior of HSV-1 thymidine kinase with acyclovir makes comparison of kinetic constants with thymidine difficult; however, the velocity of acyclovir phosphorylation is 36% of that of thymidine at a fixed (1 mM) nucleoside concentration. Acyclovir exhibits hyperbolic kinetics with VZV thymidine kinase with a K , value of 890 pV, compared to 0.16 pA4 for thymidine, and a relative V,,, of 38% that of thymidine (Averett et al., 1991). Thus, the K , value for acyclovir with VZV thymidine kinase is 8-fold higher than with HSV-1 (apparent K,) or +

+

+

Acyclovir

5

HSV-2 thymidine kinase. This difference may, in part, explain the somewhat weaker effect of acyclovir on VZV replication.

B. Cellular Kinases Acyclovir monophosphate is phosphorylated at similar rates in extracts of infected and uninfected Vero cells. Thus, cellular enzymes appear to be responsible for the synthesis of acyclovir di- and triphosphate (Miller and Miller, 1980). Although HSV- 1 thymidine kinase phosphorylates thymidine 5’-monophosphate, the purified enzyme does not catalyze appreciable phosphorylation of acyclovir monophosphate (ACVMP) (Miller and Miller, 1980). Guanosine 5’-monophosphate (GMP) kinase is responsible for conversion of acyclovir monophosphate to the diphosphate. The kinetic constants for human erythrocyte guanosine 5’-monophosphate kinase are K , (GMP) = 22 @f, V,,, (GMP) = 110 kmol/min/mg, K , (ACVMP) = 330 @f, and V,,, (ACVMP) = 3.6 p,mol/min/mg (Miller and Miller, 1980). The level of GMP kinase in Vero cells is sufficient to account for six times the amount of acyclovir diphosphate production observed in HSV-1 infected cells exposed to acyclovir (Miller and Miller, 1980). Several cellular enzymes catalyze the conversion of acyclovir diphosphate to the triphosphate form. These include phosphoglycerate kinase, pyruvate kinase, phosphoenolpyruvate carboxykinase, nucleoside diphosphate kinase, succinylCoA synthetase, creatine kinase, and adenylosuccinate synthetase (Miller and Miller, 1982). The theoretical potential to phosphorylate acyclovir diphosphate in Vero cells greatly exceeds that required to account for the observed acyclovir triphosphate production (Miller and Miller, 1982).

C. Cytoplasmic 5‘-Nucleotidase The acyclovir triphosphate level in HSV-1-infected Vero cells after 7 hours exposure to 100 pA4 acyclovir is 40-fold greater than that observed in uninfected Vero cells (Elion et al., 1977). As described above, the enzyme responsible for the phosphorylation of acyclovir in herpes-infected cells is the viral thymidine kinase. However, the trace levels of phosphorylated acyclovir metabolites observed in uninfected cells indicate that a cellular enzyme is also capable of acyclovir phosphorylation. Cellular thymidine kinase does not catalyze the phosphorylation of acyclovir (Section 111,A). Further, neither deoxycytidine kinase from calf thymus nor adenosine kinase from rabbit liver catalyzes acyclovir phosphorylation (Elion et af., 1977). Surprisingly, cytoplasmic 5‘-nucleotidase catalyzes the phosphorylation of acyclovir (Keller et al., 1985). Cytoplasmic 5 ’ nucleotidase catalyzes the phosphorolysis of nucleoside 5’-monophosphates, preferentially inosine 5’-monophosphate, by a ping-pong mechanism involving a phosphoryl-enzyme intermediate (Worku and Newby, 1982). The enzyme readily catalyzes the reversible transfer of a phosphate moiety between the enzyme and

6

John E. Reardon and Thomas Spector

inosine. Acyclovir is capable of substituting for the normal acceptors, inosine or water, resulting in the transfer of a phosphate moiety from inosine 5’-monophosphate to acyclovir. The K, value for acyclovir is 90 mM and the maximum rate of acyclovir phosphorylation is 2-5% of the rate of inosine 5’-monophosphate phosphorolysis. This activity is sufficient to account for the first step in the formation of the trace levels of phosphorylated acyclovir metabolites in uninfected Vero cells treated with acyclovir (Keller et al., 1985).

IV. Mechanism of Antiviral Action A. Viral DNA Polymerase Viral DNA synthesis is inhibited 20, 55, and 100% by incubation of HSV-Iinfected Vero cells with 0.1, 1, or 10 pJ4 acyclovir, respectively, for 24 hours (Furman et al., 1979). In contrast, 100 pJ4 acyclovir is required for 50% inhibition of DNA synthesis in actively growing uninfected Vero cells. DNA synthesized by HSV- I-infected cells in the presence of acyclovir remains near the top of an alkaline sucrose gradient after centrifugation (McGuirt et al., 1984). These studies suggest that acyclovir triphosphate is a substrate for the viral DNA polymerase and that chain-terminating incorporation of acyclovir monophosphate into the viral DNA occurs. Further, the sedimentation characteristics of the DNA are not changed after prolonged incubation of the cells in an acyclovir-free medium. These data suggest that the proofreading 3’-exonuclease activity of the viral DNA polymerase does not excise the 3’-terminal acyclovir monophosphate moiety. The inability of the 3 ’-exonuclease to excise acyclovir monophosphate from the 3’-primer terminus was confirmed with purified HSV-1 DNA polymerase (Derse et al., 1981). Inhibition of purified HSV- 1 DNA polymerase by acyclovir triphosphate has been studied in detail using activated calf thymus DNA as the template-primer. Acyclovir triphosphate causes a time-dependent loss of enzymatic activity in the presence of activated calf thymus DNA and the four deoxynucleoside 5 ’-triphosphates (Fig. 2) (Furman et al., 1984). The kinetic data reveal that acyclovir triphosphate competes favorably with dGTP to form a Michaelis complex with the polymerase-template-primer complex. The K , value for acyclovir triphosphate is 4 nM, compared to 200 nM for dGTP. After formation of the Michaelis complex, apparent inactivation of the enzyme occurs with a first-order rate constant of 0.24 min-I. No loss of activity occurs when either enzyme or template-primer is omitted from a preincubation mixture including acyclovir triphosphate, dCTP, dATP, and dGTP. This suggests that enzyme catalysis is a requirement for inactivation. Further, the apparent inactivation of the polymerase is not reversed by addition of template-primer (Furman et al., 1984). Addition of

Acyclovir

7 25

pM ACVTP

5

10

15

20

MINUTES

Fig. 2 Progressive inhibition of HSV-I DNA polymerase by acyclovir triphosphate. The incorporation of [3H]dGMP into activated calf thymus DNA from 5 pA4 [?H]dGTP in the presence of 100 phf dATP, dCTP, and dTTP was measured in the presence of acyclovir triphosphate at the indicated concentrations. (Reproduced from Furman et ul.. 1984, by permission.)

enzyme to the inactivated polymerase reaction mixture causes a resumption of DNA synthesis which exhibits a similar first-order decay of the rate. Finally, when enzyme inactivated by acyclovir triphosphate in the presence of activated calf thymus DNA, dATP, and dCTP is isolated by rapid gel filtration, addition of fresh template-primer and the four deoxynucleoside 5 '-triphosphates does not result in a recovery of polymerase activity (Furman ef al., 1984). Thus, acyclovir triphosphate appears to possess the kinetic qualifications of a suicide substrate for HSV-I polymerase. The mechanism of the apparent inactivation of HSV-I DNA polymerase by acyclovir triphosphate has been elucidated with defined sequence template-primers (Reardon and Spector, 1989). The model template-primer, 20:9 mer (Fig. 3), accepts dGTP (or acyclovir triphosphate) as the first nucleotide to be incorporated. This simplified DNA substrate permits analysis of the turnover of dGTP or acyclovir triphosphate in the absence of the other deoxynucleoside 5'-triphosphates. When acyclovir triphosphate is incubated with the viral polymerase and the 20:9 mer, rapid and complete incorporation of acyclovir monophosphate into the DNA is observed (Fig. 4A). Acyclovir triphosphate exhibits hyperbolic kinetics on this template-primer with a K , value of 2.6 p& and a relative V,,, value of 15. In comparison, the K,, value for dGTP is 2.3 rJ.n and the relative

8

John E. Reardon and Thomas Spector 3'-AGC GTC GAG CGA TTC CCA AA-5' S-TCG CAG CTC-3' 20:9 mer

3-AGC GTC GAG CGA TTC CCA AA-5' 5'-TCG CAG CTC G-3' 3'-dGMP-20:9 mer (20:lO mer)

3-AGC GTC GAG CGA TTC CCA AA-5' S-TCG CAG CTC "Ga~,"-3' 3'-ACVMP-20:9 mer

Fig. 3 Defined sequence template-primers. The nomenclature refers to the length of the temp1ate:primer. "G,,," indicates acyclovir monophosphate at the 3'-terminus of the primer. Additions

100

None

~

2o 0

2 2

4 6 4 6 TIME (min)

0 0

100 liM dCTP

10

None 100 pM dATP

100 )IM dCTP added at 2 man

100 PM dCTP

TIME (rnin)

Fig. 4 Effect of added dNTPs on HSV-I DNA polymerase utilization of acyclovir triphosphate as a substrate. The incorporation of [side chain 2-3H] acyclovir monophosphate into 15 phf 20:9 mer from 1.5 phf [side chain 2-3H] acyclovir triphosphate was measured in the presence of the indicated additions. (A) 2.1 units of HSV-I DNA polymerase. (B) 0.53 units of HSV-I DNA polymerase. (Adapted from Reardon and Spector, 1989, by permission.)

9

Acyclovir

V,,, is 100. Furthermore, acyclovir triphosphate is a competitive inhibitor with a K , value of 2.5 fl versus dGTP (J. E. Reardon, unpublished data). Thus, acyclovir triphosphate is, by itself, a conventional substrate and not a mechanism-based inactivator of the viral polymerase. HSV- 1 DNA polymerase-catalyzed incorporation of acyclovir monophosphate into 20:9 mer is inhibited by dCTP (the next nucleotide encoded by the template) (Fig. 4A and B). Addition of dCTP to an ongoing reaction also results in an immediate onset of inhibition. In contrast, neither dATP nor dTTP causes significant inhibition of the incorporation of acyclovir monophosphate into the 20:9 mer (Fig. 4B). Thus, the nucleotide encoded by the template for incorporation immediately after acyclovir monophosphate is a specific inhibitor of the reaction. From initial velocity analysis, the inhibition of acyclovir monophosphate incorporation into the 20:9 mer by dCTP is uncompetitive versus acyclovir triphosphate and the K , value is 76 nM.In contrast, the K , value for dCMP incorporation into the 3'-dGMP-20:9 mer (20:lO mer, Fig. 3) is 2.6 fl.Thus, dCTP binding to the 3'-acyclovir monophosphate terminal 20:9 mer is 30-fold tighter than the apparent binding constant (K,) with the 3'-dGMP-20:9 mer (20: 10 mer). Whether this difference reflects the difference between the K , and K , values for 2'-deoxynucleoside 5'-triphosphate binding to the viral polymerase is not known. A kinetic model consistent with the data described above is shown in Fig. 5. The uncompetitive inhibition pattern for dCTP inhibition of acyclovir monophosphate incorporation is consistent with formation of a dead-end complex (Segel, 1975). This inhibition is a unique form of induced substrate inhibition (Cleland, 1979). Induced substrate inhibition is observed in an ordered Bi-Bi mechanism with an inhibitor which is competitive with the first substrate. On binding of the inhibitor to the enzyme, the inhibitor induces substrate inhibition by the second

PPi

E-20:9 rneredGTP dGTP

') E*20:9 mer

4

dCTP

PPi

E-20:10 r n e r u E.20:ll rner

-

20:9 rner

L -E

AMpJ

E-20:9 mer-ACVTP

PPi E.ACVMP-2O:O rner

dCTP [E*ACVMP90:9 rner-dCTP] "Dead-End Complex"

Fig. 5 Kinetic scheme describing the induced substrate inhibition of HSV-I DNA polymerase (Adapted from Reardon and Spector. 1989, by permission.)

10

John E. Reardon and Thomas Spector

substrate. Substrate inhibition occurs because the enzyme-inhibitor-second substrate ternary complex is catalytically incompetent and therefore a dead-end complex. In the present case, enzyme turnover generates the 3’-acyclovir monophosphate-20: 9 mer that (due to the processive nature of HSV-1 polymerase) induces inhibition by dCTP, the next nucleotide substrate encoded by the template. Complete induced substrate inhibition requires a compulsory ordered mechanism for substrate binding and is therefore not an unexpected type of inhibition to observe with polymerases (Fisher and Korn, 1981; Wang and Korn, 1982; Bryant et al., 1983). Low levels of acyclovir triphosphate do not inhibit HSV-I DNA polymerasecatalyzed incorporation of [3H]dGMP into the 20:9 mer. If, however, dCTP is also present, a first order loss of enzyme activity, dependent on the concentration of acyclovir triphosphate, is observed (Fig. 6). dCTP does not affect the rate of dGMP incorporation in the absence of acyclovir triphosphate. This kinetic pattern mimics the kinetics expected for a suicide substrate and explains the kinetic profile of Furman et al. (1984) discussed above (Fig. 2). Further, the finding by Furman et al. (19841, that enzyme inactivated by acyclovir triphosphate in the presence of activated calf thymus DNA, dATP, and dCTP is not reactivated after rapid gel filtration, is consistent with an induced substrate inhibition mechanism. Since dATP and dCTP are present in both the inactivation and reactivation incubation mixtures, the polymerase is maintained in the dead-end complex. The

[Acyclovir Triphosphate] OlMi

04

Y

/

08

/

16

32 2

L

a

12

16

20

TIME (rnin)

Fig. 6 Apparent time-dependent inhibition of HSV-I DNA polymerase by acyclovir triphosphate. The incorporation of [8-3H]dGMP into the 20:9 mer from 100 phf [8-3H]dCTP was measured in the presence of 1 .O mM dCTP and acyclovir triphosphate at the indicated concentrations. (Adapted from Reardon and Spector, 1989, by permission.)

Acyclovir

11

enzyme is reactivated when dATP and dCTP are omitted from the reactivation mixture (Reardon and Spector, 1989). Incorporation of acyclovir monophosphate into the 20:9 mer (Fig. 4A) does not result in significant product inhibition. Consequently, the 3’-acyclovir monophosphate-20:9 mer is not, by itself, a potent inhibitor and the apparent K , value for the 3’-acyclovir monophosphte-20:9 mer of 0.62 p M is comparable to the apparent K,,, value of 0.88 pM for the nonterminated 20:9 mer (Reardon and Spector, 1989). If dCTP is present, potent inhibition is observed. Thus, the 3’acyclovir monophosphate-20:9 mer, either added exogenously or generated enzymatically by turnover of acyclovir triphosphate as a substrate, induces potent inhibition of the viral polymerase by dCTP, the next encoded nucleotide. Acyclovir monophosphate-terminated activated calf thymus DNA has been reported to be a potent inhibitor of HSV-I polymerase (Derse ef al., 1981). However, all four deoxynucleoside 5’-triphosphate substrates were included in the assay. Thus, the relatively weak binding of the 3’-acyclovir monophosphateterminal-primer sites to the polymerase followed by tight-binding of the next encoded dNTP could account for the low K , value. Acyclovir triphosphate is also a substrate for HSV-2 DNA polymerase (Reid er a / . , 1988). The K,, value for acyclovir triphosphate is 8.4 and the relative V,,,, value is 17, using the 20:9 mer as the template-primer (J. E. Reardon, unpublished data). In contrast, the K , value for dGTP is 3.2 p,M and the relative V,, value is 100 (J. E. Reardon, unpublished data). The Ki value for dCTP inhibition of acyclovir monophosphate incorporation into the 20:9 mer is 290 nM (J. E. Reardon, unpublished data). Thus, the K,, value for acyclovir triphosphate is 3-fold higher and the K, for dCTP is 4-fold higher with HSV-2 polymerase compared to HSV- 1 polymerase. This may, in part, explain the somewhat weaker inhibition of HSV-2 DNA polymerase by acyclovir triphosphate (St. Clair et al., 1980) and the slightly weaker in vitro inhibition of HSV-2 replication by acyclovir (Crumpacker et a/., 1979; McLaren e f a/., 1982; Barry et al., 1985).

B. Human DNA Polymerases Acyclovir triphosphate interactions with human DNA polymerases are in sharp contrast to those with the viral enzymes. Polymerase a is inhibited by acyclovir triphosphate with a competitive K , value of 1.9-2.8 phi’ (versus dGTP) with activated calf thymus DNA as the template-primer (St. Clair et al., 1980). In contrast, the K , value for inhibition of HSV-I DNA polymerase is 0.004 (Furman et al. , 1984). The K,, values for acyclovir monophosphate incorporation into the 20:9 mer are 24 for polymerase a and and 2.3 p.M for HSV-1 polymerase (Reardon, 1989). Induced substrate inhibition of polymerase a is not detected using activated calf thymus DNA as the template-primer. Further, dCTP (the next encoded nucleotide) is only a weak inhibitor of polymerase a-catalyzed incorporation of acyclovir monophosphate into the 20:9 mer, with a K ivalue of

12

John E. Reardon and Thomas Spector

2.3 pM. This K , value is 30-fold weaker than the corresponding Ki value with HSV-1 polymerase, and is the same as the K, value for incorporation of dCMP into the 3'-dGMP-20:9 mer (20: 10 mer). Time-dependent inhibition of dGMP incorporation into the 20:9 mer, in the presence of acyclovir triphosphate and dCTP, is also observed (Reardon, 1989). This is consistent with reports of induced substrate inhibition of polymerase cr. by 3'-dideoxy-TMP-terminal template-primers (Atkinson et al., 1969; Fisher and Korn, 1981). It is unlikely that induced substrate inhibition of polymerase cr. contributes to the weak toxicity of acyclovir in vitro. Acyclovir is neither a substrate nor an inhibitor of polymerase p (Derse er al., 1981; Reid et al., 1988; Reardon, 1989). Thus, in addition to the selective phosphorylation of acyclovir by herpes thymidine kinase, the selective inhibition of herpes DNA polymerase contributes to the safety and efficacy of acyclovir as an antiviral agent.

C. Mechanism-Based Affinity Chromatography of Viral DNA Polymerase The finding that herpes DNA polymerases are subject to induced substrate inhibition has led to the development of a method for affinity chromatography of the enzyme (Reardon, 1990). The affinity resin is a 3'-acyclovir monophosphateterminal hook template-primer coupled to Affi-Gel 15 through a hexanolamine phosphate linkage to the 5'-end of the DNA (Fig. 7). By itself, this 3'-acyclovir

__

CC

I

1) SequenaseIACVTP 2) Phenol Extraction

3) Ethanol Precipitation 4) Sephadex G-25, 50 rnM KPi, pH 8.0,2 mM EDTA

C CCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCCCCCC-PO~-(CHZ)~-NH~ G GGGGGGG GGGGGGGGGG Gam GG 1) AffiGel-15, RT, 2 hours 2) Wash

cc

C CCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCCCCCC-P04-(CH&-NH G GGGGGGG GGGGGGGGGG G m GG

Fig. 7 Preparation of affinity resin for purification of HSV-I DNA polymerase. (Reproduced from Reardon, 1990, by permission.)

13

Acyclovir

monophosphate-terminal template-primer is a relatively potent inhibitor of HSV-1 polymerase with a Kivalue of 6 nM versus the 20: 10 mer (Fig. 3). This Ki value, one-hundredth that of the 3'-acyclovir monophosphate-terminal-20:9mer, is consistent with the high affinity of the enzyme for poly(dC)-oligo(dG) (J. E. Reardon, unpublished data). The inhibition by the 3'-acyclovir monophosphate-terminal hook template-primer is greatly enhanced by dGTP, the next nucleotide encoded by the template. HSV-1 polymerase is eluted from the affinity resin with a salt gradient, in the absence of dGTP and Mg2 . The elution position of the polymerase, 0.4 M NaCl, is the same as from a DNA-cellulose column, suggesting that the column behaves as a simple DNA-agarose affinity column (Fig. 8). However, in the presence of dGTP and Mg2+, the column becomes a mechanism-based affinity column and HSV-1 polymerase is retained even during a 1 M NaCl wash. On removal of dGTP and Mg2 from the buffer, the enzyme is eluted by a linear salt gradient (Fig. 8). HeLa polymerase (Y is eluted by a 1 M NaCl wash in the presence of dGTP and Mg2+ . Polymerase f3 is not retained by the column. Thus, the susceptibility of HSV polymerase to induced substrate inhibition provides a unique mechanism-based approach to purification of the enzyme. +

+

V. Potentiation of the Antiviral Action of Acyclovir Inhibition of viral DNA polymerase by acyclovir triphosphate is central to the antiviral action of acyclovir (Section IV,A). The success of acyclovir triphosphate as an inhibitor of the viral polymerase is due to its surprisingly effective competition with dGTP for binding to the enzyme and to the subsequent formation of a dead-end complex between the next encoded nucleotide and the polymerase-3'-acyclovir monophosphate-terminal template-primer complex. An undesirable consequence of inhibition of the polymerase is the accumulation of dGTP, as well as the other nucleotide substrates, in the infected cell. This could reduce the ability of acyclovir triphosphate to compete with dGTP for binding to the viral polymerase, which would reduce the antiviral effect of acyclovir. In fact, dGTP concentrations are increased in HSV- 1-infected Vero cells treated with acyclovir (Furman et al., 1982). In uninfected cells, the extensive feedback control of mammalian ribonucleotide reductase would be expected to maintain all deoxynucleoside 5 '-triphosphate levels in homeostasis (Thelander and Reichard, 1979; Hunting and Henderson, 1982). Herpes simplex and varicella zoster vimses encode ribonucleotide reductases that are not subject to allosteric control (reviewed in Spector, 1985, 1989). While this lack of regulation of viral ribonucleotide reductases may limit the antiviral efficacy of viral DNA polymerase inhibitors, it distinguishs them from their isofunctional mammalian counterpart and makes them attractive chemotherapeutic targets.

14

John E. Reardon and Thomas Spector

40

10

0

Fraction # 40

E

10

0

0

5

I 0

15

20

25

30

Fraction # Fig. 8 Affinity chromatography of HSV-I DNA polymerase. (A) HSV-I DNA polymerase was applied to the column equilibrated with elution buffer [50 mM Tris-HCI, pH 8.5 (4°C). 100 mM ammonium sulfate, 10% glycerol, 1 mM dithiothreitol] and eluted with a salt gradient as indicated. (B) HSV-I DNA polymerase was applied to the column in affinity buffer (elution buffer + 1 mM dGTP and 5 mM MgCI2). The column was washed and then eluted with a salt gradient in elution buffer, as indicated. (Reproduced from Reardon, 1990, by permission.)

A. Properties of Viral Ribonucleotide Reductases Mammalian ribonucleotide reductase, the enzyme responsible for 2'-deoxynucleotide synthesis, is exquisitely regulated by an elaborate allosteric feedback control mechanism that limits deoxynucleotide pool sizes in mammalian cells (Thelander and Reichard, 1979; Hunting and Henderson, 1982). HSV-1 is capa-

15

Acyclovir

ble of replication in the presence of high thymidine concentrations, suggesting that the virus encodes a ribonucleotide reductase not subject to allosteric control (Cohen, 1972). Subsequent studies with ammonium sulfate-fractionated extracts of infected cells verified that HSV-I and HSV-2 induce a ribonucleotide reductase (Ponce de Leon er al., 1977). A one-column procedure for removal of substrate-diverting enzymatic activities provides an enzyme preparation suitable for kinetic studie.; (Averett et al., 1983). Purified HSV- 1 (Averett et al., I983), HSV-2 (Averett et al., 1984), and VZV (Spector et al.. 1987) ribonucleotide reductase are not subject to the control mechanisms exhibited by their mammalian counterpart. The 2’-deoxynucleoside 5’-triphosphates, at physiological concentrations, do not inhibit the enzymatic reduction of either CDP or ADP (Averett et af., 1983). Further, the viral enzymes are not stimulated by ribonucleoside triphosphates. Since each of the nucleoside diphosphates competitively inhibits the reduction of the other substrates with a K , value similar to its K,, value, reduction of all four nucleoside diphosphates appears to be catalyzed at a common site !Averett et al., 1983, 1984). Acyclovir diphosphate is a weak competitive inhibitor, with a K , value of 350 fl. Acyclovir triphosphate is a very weak inhibitor of the purified enzyme (Averett et al., 1983).

B. Inhibition of Ribonucleotide Reductases Brockman er ul. ( 1970) first reported that heterocyclic thiosemicarbazone inhibitors of mammalian ribonucleotide reductase also inhibited HSV replication. Several series of 2-acetylpyridine thiosemicarbazones have subsequently been reported to show a selective antiviral effect on herpes-viruses (Shipman et al., 1981). The mechanism of action appears to be selective inhibition of viral ribonucleotide reductase (Spector et al., 1985, 1989; Turk et al., 1986). 2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A1 1 IOU) (Fig. 9) is a particularly potent inhibitor that has been studied in depth (Spector et al., 1989), and will serve as the model for the present discussion. An explanation for the selectivity of A1 110U inhibition of the viral ribonucleotide reductases, relative to their mammalian counterpart, was revealed by Porter et al. ( 1 990). A 1 1 IOU forms a very stable complex with iron. A1 1 10U, in either the free or iron-complexed form, is a potent inactivator of HSV-I

Fig. 9 Structure of A1 I IOU

16

John E. Reordon and Thomas Specfor

ribonucleotide reductase. Mammalian ribonucleotide reductase is weakly inhibited by iron-complexed AlllOU and was unaffected by free A111OU. Interestingly, the combination of free A1 1 1OU and iron-complexed A1 1 IOU has a synergistic effect on the rate of inactivation of the viral enzyme. The combination also has a synergistic effect on the human ribonucleotide reductase, however, the second-order rate constant for inactivation of the human enzyme is one-tenth that observed with the viral enzyme (Fig. 10).HSV- 1 ribonucleotide reductase inactivated by the combination of free A1 110U and iron-complexed A1 110U can be reactivated upon removal of the inhibitors and replenishing the iron. Thus, it appears that A 1 1 1OU removes the catalytically essential iron from the enzyme active site and that iron-complexed A1 1 IOU facilitates the process.

C. Synergistic Inhibition of Virus Replication by Acyclovir and A1 110U A1 1 IOU inhibits HSV-I , HSV-2, and VZV replication in vitro with an IC,, of about 1.5 pA4 (Spector et al., 1989), which is similar to the concentration required to inactivate the viral ribonucleotide reductase. Acyclovir inhibits viral replication with an IC,, of 0.1 pA4 (Section I,A) (Elion et al., 1977). Combination of the two drugs results in a synergistic effect on virus replication. A doubledrug isobologram illustrating the synergistic effect of acyclovir and A1 1 IOU against HSV-2 replication in vitro is shown in Fig. 11. The combination of acyclovir and A1 110U shows a statistically significant synergistic effect against cutaneous HSV- 1 and HSV-2 infections in the immunodeficient nude (athymic) mouse model, the hairless immunocompetent mouse model, and the guinea pig model (Ellis et al., 1986, 1989;Lobe et al., 1991).The combination therapy is also effective against several acyclovir-resistant HSV- 1 strains partially deficient in thymidine kinase and against a thymidine kinase mutant with a reduced acyclovir phosphorylating activity (Ellis et al., 1989). Synergy was not demonstrable against another acyclovir-resistant virus which expresses a DNA polymerase with diminished sensitivity to acyclovir triphosphate inhibition. Interestingly, while the combination therapy is very efective against this virus strain, A1 110U by itself produces unusually potent efficacy, thus reducing the possibility of statistically significant synergistic improvement (Ellis et al., 1989). The underlying cause of the synergism is revealed upon examination of the metabolic perturbations induced by incubating either A1 1 IOU,acyclovir, or the combination of these compounds with HSV-1 infected Vero cells (Spector er al., 1989).Acyclovir causes an elevation of the dGTP concentration (Table I), presumably due to inhibition of the viral polymerase by acyclovir triphosphate. The dATP concentration is elevated as well. In contrast, when both drugs are present, dGTP pool levels are depressed below those found in untreated, uninfected cells

17

Acyclovir

A

Inhibitor bM)

none

40

E

(AIIIOU)2Fe+' (1)

&20

0 -

AllIOU (2)

0

a

lo

30

TIME, minutes

300

tB

,

Inhibitor bM) none AllIOU (20)

200

(AlllOU),Fe"

(10)

100 (AlllOU),Fe+' (5) AlllOU (10)

0

0

10

20

TIME, minutes Fig. 10 Inhibition of herpes and human ribonucleotide reductase. Effects of A1 1 lOU, A1 I 10UzFe complex, and the combination of A1 11OU + A l l IOU2Fe complex on the reduction of ["TICDP by HSV-1 (A) and human (B) ribonucleotide reductase. Reaction temperature was lowered from 37°C (human enzyme) to 30°C (HSV-Ienzyme) to observe the rapid rates of inactivation. (Reproduced from Porter et a / . . 1990. Published with permission of the copyright holder, Pergamon Press, plc, Oxford.)

John E. Reardon and Thomas Spector

18

0

0.2

0.6

0.4

0.8

1.0

[AlllOUI ICeI ( A 3 1 1 w

Fig. 11 Synergistic inhibition of HSV-2 replication by Al I IOU and acyclovir. The concentration of each inhibitor was covaried and the inhibition of plaque formation assessed. The FIC60(AC-) is the ratio of the concentration of acyclovir required to inhibit plaque formation by 60% in the presence of a fixed concentration of A l l IOU. The x-axis is the ratio of the fixed concentration of Al I IOU to the concentration of Al I IOU that produced 60% inhibition of plaque reduction in the absence of acyclovir. ICW values were used because >50% inhibition was observed at some of the combination doses. The dashed line shows the theoretical plot for independent inhibitors. (Reproduced from Spector et 01.. 1989, by permission.)

Table I Effects of A l l IOU on Deoxynucleoside Triphosphate and Acyclovir Triphosphate Levels0.h Treatment Al 1 IOU

(W) None None 2

2

Acyclovir (pM) None 10 None 10

Intracellular pools (pmolilO6 cells)

dCTP

dTTP

dATP

dGTP

ACVTP

Ratio ACVTPidGTP

20 21

225 240 260 260

20 82 20 33

16 68 8.0 6.4

2.6

0.038

16 16

.-

23

-

3.6

"Data from Spector er a/.(1989). Wonfluent cultures of Vero cells were infected with 10 PFUicell of HSV-1 (Patton strain). After 1 hour at 37°C. the medium was replaced with fresh medium with or without A1 1 IOU andlor [side chain 2-3H] acyclovir, as indicated. After an additional 8 hours, the cells were collected and the intracellular levels (in pmol/106 cells) of the deoxynucleoside 5'-triphosphates and acyclovir triphosphate (ACVTP) were determined.

Acyclovir

19

and dATP levels are also reduced. Surprisingly, when both drugs are present, acyclovir triphosphate levels are increased approximately 10-fold. It is not clear why inhibition of the viral ribonucleotide reductase increases the intracellular acyclovir triphosphate concentration. A723U, another inhibitor of herpes ribonucleotide reductase decreases thymidine excretion into the media of HSV-infected cells (Karlsson and Harmenberg, 1988). This suggests that the intracellular thymidine nucleoside/nucleotide pool is reduced. Thus, the ability of thymidine to compete with acyclovir for phosphorylation by the viral thymidine kinase would be reduced, resulting in increased levels of acyclovir monophosphate. As discussed in Section III,B, the acyclovir monophosphate and acyclovir diphosphate phosphorylation potential of Vero cells greatly exceeds that required for production of the observed levels of acyclovir triphosphate in herpesvirus-infected cells. Therefore, increased synthesis of acyclovir monophosphate should lead to increased production of the di- and triphosphates as well. AlllOU does not significantly affect the levels of the pyrimidine deoxynucleoside triphosphates (Table I). Since all four ribonucleotide diphosphates compete for reduction at the same active site (Section V,A), a selective inhibition of the reduction of purine deoxynucleotides is unlikely. Thus, while the total pyrimidine deoxynucleoside/deoxynucleotide pool is probably reduced in A I 1 IOU-treated cells, levels of the pyrimidine deoxynucleoside triphosphates remain constant. This may reflect a more efficient salvage pathway for pyrimidine nucleotides (reviewed in Reichard, 1988). As discussed in Section IV,A, the apparent time-dependent inactivation of the viral DNA polymerase is due to formation of a dead-end complex on binding of the next encoded deoxynucleoside triphosphate to the polymerase-3'-acyclovir monophosphate-template-primer complex. For this reason, it is fortunate that A1 1 IOU administration does not uniformly reduce the levels of all four deoxynucleoside triphosphates, but rather that pyrimidine nucleotide pool levels remain sufficiently high to ensure trapping of the polymerase in a dead-end complex.

VI. Resistance and Hypersensitivity to Acyclovir Prior to the emergence of acquired immune deficiency syndrome (AIDS), reports of clinical resistance to acyclovir were rare. Drug-resistant virus strains in the clinical setting have arisen both via selection of a preexisting resistant strain in an individual infected with multiple virus strains and by mutation of sensitive predecessor strains (Erice et al., 1989). Resistance of herpesviruses to acyclovir can develop rapidly in v i m . One passage of the virus in the presence of acyclovir may result in the development of resistance (Field et al., 1980). The mechanisms of acyclovir resistance include ( I ) deletion of.the viral thymidine kinase gene (TK-) (Bums et al., 1982; Crumpacker et al., 1982; Wade et al., 1983), (2)

John E. Reardon and Thomas Spector

20

alteration of the viral thymidine kinase resulting in less efficient phosphorylation of acyclovir (TK+) (Darby et al., 1981; Larder et al., 1983; Ellis er al., 1987), and (3) alteration of the viral DNA polymerase resulting in weaker inhibition by acyclovir triphosphate (Schnipper and Crumpacker, 1980; Derse et al., 1982; Gibbs et al., 1988). Viral mutants in which there is a complete loss of viral thymidine kinase gene expression (TK-) are not as virulent in animal models and establish latent infections less efficiently (Field and Wildy, 1978; Field and Darby, 1980; Tenser et al., 1981). Since this mechanism of resistance is at the level of gene expression, it will not be discussed further. In contrast, viral mutants that retain the thymidine kinase phenotype (TK+), are of more clinical concern due to their virulence in animal models (Darby et al., 1981; Larder et al., 1983).

A. Thymidine Kinase Mutants In most cases, mutants expressing the TK+ phenotype isolated from clinical specimens are mapped to the thymidine kinase gene and result in expression of an enzyme with altered kinetic properties. Such mutants have also been isolated in vitro after passage of wild-type virus in the presence of acyclovir (Darby et al., 1981). Acyclovir-resistant thymidine kinase mutants exhibit an impaired ability to phosphorylate acyclovir. These mutant enzymes may or may not also have impaired thymidine phosphorylating activity. Further, selective effects on both K, and V,,, values have been observed. For example, the HSV-1 (strain SC16) variants shown in Table I1 induce thymidine kinase mutants with different kinetic properties. Varients S 1 and Tr7, isolated after passage of the virus in the presence of acyclovir, exhibit increased K , values for thymidine and increased

Table II Substrate Kinetic Properties of Mutant Thymidine Kinasesa

Virus strain of HSV-I

SC 16(wt) SI

B3

Tr7

K , (w) Thymidine 0.2 10

0.3 2.5

K , (PW

Ki(lwU)

Relative velocityb (%I)

BVdU

Acyclovir

Thymidine

BVdU

0.1 5 4.5

200 2200 225 2500

100 100 100 100

120 I29 42 I45

1.5

Acyclovir

39
30
aData from Larder ef al. (1983). hRates of phosphorylation, measured using either thymidine (30pM), acyclovir (500@), or (E)-5-(2-bromovinyl)-2’-deoxyuridine (BVdU) (42pV)are expressed as a percentage of the thymidine phosphorylation rate for each enzyme. CNomeasurable phosphorylation.

Acyclovir

21

K , values for both acyclovir and (E)-5-(2-bromovinyl)-2’-deoxyuridine (BVdU). However, only the relative rate of phosphorylation of acyclovir is reduced. In contrast, with variant B3, isolated after passage of the virus in the presence of BVdU, only the kinetics of BVdU binding and phosphorylation are affected. These data demonstrate that some mutants may exhibit altered kinetic properties solely for the nucleoside analog that is used to generate the variant virus. The three mutants described above map to distinct regions of the thymidine kinase gene. Darby et al. (1986) have proposed a model for the three-dimensional structure of the enzyme in which these three regions of the primary structure combine to form the nucleotide-binding domain of the enzyme.

B. DNA Polymerase Mutants The first TK+ acyclovir-resistant HSV-I mutant reported was isolated in virro and expressed a DNA polymerase less susceptible to inhibition by acyclovir triphosphate (Coen and Schaffer, 1980). Reports of polymerase mutations conferring clinical resistance to acyclovir are rare (Collins et al., 1989). Gibbs et al. (1988) have isolated and sequenced a series of laboratory-derived HSV- 1 polymerase mutants with altered drug sensitivity. Each mutant has a different amino acid change that is located in one of four distinct regions of the polymerase gene. Three of these regions show sequence similarity to other viral and mammalian polymerases. Other polymerase mutations conferring altered drug sensitivity phenotypes (Larder et al., 1987; Knopf, 1986; Tsurumi et al., 1987; Collins et al., 1989) have amino acid changes in one of the four regions described by Gibbs et al. (1988). This suggests that the three-dimensional structure of the polymerase juxtaposes these four regions of the primary sequence in the nucleotidebinding domain of the active site. Elevated K , values have been reported for acyclovir triphosphate (or other nucleoside triphosphate or pyrophosphate analogs) with polymerases purified from mutant viruses (Derse et al., 1982). However, there is no detailed understanding of the kineticlmechanistic consequences of a polymerase mutation. This will undoubtably be a fruitful area of research in the near future.

C. Ribonucleotide Reductase Mutants As discussed in Section V,C, inhibition of herpes ribonucleotide reductase by A1 1 IOU potentiates the antiviral effect of acyclovir in vitro and in vivo. This potentiation results from both increased acyclovir triphosphate and reduced purine nucleotide pool levels that allows for more effective inhibition of the viral DNA polymerase by the dGTP analog acyclovir triphosphate. Based on these studies,it was predicted by Coen er al. (1989) that herpes ribonucleotide reductase mutants would exhibit hypersensitivity to acyclovir. This was confirmed by constructing two HSV- 1 ribonucleotide reductase mutants. One contains an in-

22

John E. Reardon and Thomas Spector

sertion of the Escherichia coli lac Z gene (hrR3) and the other contains a 2.9 kilobase-pair deletion (ICP6A), both in the ribonucleotide reductase gene. The viruses with these null mutants are five to seven times more sensitive to acyclovir than both the wild-type virus [HSV-1 (KOS)] and a recombinant virus (ICP6Af -3. I ) in which the deleted sequence was restored (Coen et a l . , 1989). Interestingly, the ribonucleotide reductase mutants are equally hypersensitive to both acyclovir and aphidicolin. Aphidicolin inhibits the incorporation of all four 2’-deoxynucleoside 5’-triphosphates into DNA (Frank et al., 1984). If the aphidicolin permeability of cells infected with both the wild-type and mutant strains of the virus are equivalent, then the hypersensitivity to aphidicolin would be solely the result of more efective inhibition of 2’-deoxynucleoside 5’-triphosphate binding to the viral polymerase due to the reduced deoxynucleotide pool levels. Equivalent hypersensitivity to acyclovir suggests that acyclovir triphosphate levels may not be increased in cells infected with the mutant virus strains. This would be in contrast to the results with A1 110U-treated HSV-I infected cells in which acyclovir triphosphate levels were increased 10-fold and only the deoxypurine nucleotide levels were significantly decreased (Table I). An examination of deoxynucleotide pool levels in cells infected with hrR3 or 1CP6A and the effects of acyclovir and A1 I1OU on these pool levels may provide additional insight into the mechanism of potentiation by ribonucleotide reductase inhibitors.

VII. Conclusions The safety and efficacy of acyclovir as an antiviral agent are due to its selective interactions with virally induced enzymes. Acyclovir is selectively phosphorylated by the viral thymidine kinase and then is converted to its triphosphate form by cellular enzymes. Acyclovir triphosphate is an efficient substrate for the virally encoded DNA polymerase. Incorporation of acyclovir monophosphate at the 3’-primer terminus results in chain termination. Further, inhibition of the polymerase is induced by relatively tight binding of the next encoded nucleotide to the polymerase-3’-acyclovir monophosphate terminal-template-primer complex, resulting in the formation of a dead-end complex (induced substrate inhibition). In contrast, human polymerase ci is only weakly inhibited by binding of the next encoded nucleotide to the polymerase-3’-acyclovir monophosphate terminal-template-primer complex. In addition, human polymerase f3 is not inhibited by nor does it utilize acyclovir triphosphate as a substrate. Thus, a detailed knowledge of the mechanism of action of acyclovir provides a molecular basis for understanding its antiviral safety and efficacy. The antiviral efficacy of acyclovir is potentiated by inhibition of the virally encoded ribonucleotide reductase by thiocarbonohydrazones. These inhibitors

Acyclovir

23

cause a decrease in the dGTP pool size and an increase in the concentration of acyclovir triphosphate in herpesvirus-infected cells, thereby increasing the effectiveness of acyclovir triphosphate as a competitive inhibitor with respect to dGTP for the virally encoded DNA polymerase. The synergism between thiocarbonohydrazones and acyclovir has been demonstrated both in vitro and in vivo. Hopefully, this combination therapy will prove useful in the future treatment of human disease.

References Atkinson, M. R., Deutscher, M . P.. Kornberg, A . , Russell, A . F., and Moffatt, J. G . (1969). Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2‘,3’dideoxyribonucleotide. Biochemistrv 8, 4897-4904. Averett. D. A , , Lubbers, C., Elion. G. B., and Spector, T. (1983).Ribonucleotide reductase induced by herpes simplex type 1 virus: Characterization of a distinct enzyme. J . B i d . Chem. 258, 983 I 9838. Averett. D. A , , Furman. P. A.. and Spector, T. (1984). Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1 . J . Virol. 52, 981-983. Averett. D. A , , Kosralka. G . W., Fyfe. J. A . , Roberts, G . B., Purifoy. D. J. M., and Krenitsky, T. A . ( 1991 1. 6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus. Antimicrub. Agents Chemother. 35, 8.5 1-857. Barry, D. W., Nusinoff-Lehrman, S . , Ellis, M . N., Biron. K. K., and Furman, P. A. (1985). Viral resistance, clinical experience. Scund. J . Inf2c.r Dis., .Strpp/. No. 47, 155- 164. Brigden, D . , Fiddian, P.. Rosling. A . E., and Ravenscroft, T. (1981). Acyclovir-A review of the preclinical and early clinical data o f a new antiherpes drug. Antiviral Res. 1, 203-212. Brockman. R. W., Sidwell. R. W.. Arnett, G . . and Shaddix, S . (1970). Heterocyclic thiosemicarbazones: Correlation between structure. inhibition of ribonucleotide reductase, and inhibition of DNA viruses Proc. Soc. E.xp. B i d . Med. 133, 609-614. Bryant, F. R . , Johnson. K . A . , and Benkovic, S.J . (1983). Elementary steps in the DNA polymerase I reaction pathway. Biochemistry 22, 3537-3546. Burns, W. H.,Saral. R . , Santos, G. W.. Laskin. 0. L., Leithan. P. S . , McLaren, C . , and Barry, D. W. (1982). Isolation and characterization of resistant herpes simplex virus after acyclovir therapy. Lancet I , 42 1-423 Cheng, Y.-C. (1976). Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type 11: Substrate specificity and kinetic behavior. Biorhirn. Biophss. Acra 452, 370381. Cheng. Y.-C., Doniin, B . A.. Sharma. R . A . , and Bobek, M . (1976). Antiviral action and cellular toxicity of four thymidine analogues: 5-Ethyl-. S-vinyl- 5-propyl-, and S-allyl-2’-deoxyuridine. Antimicrob. Agents Chemother, 10, 1 19- 122. Clcland, W. W. (1979). Substrate inhibition. Methods t’rrz~mol.63A, 500-513. Coen. D. M.. and Schaffer, P. A. (1980). Two dihtinct loci confer resistance to acycloguanosine in herpes simplex virus type 1 . Proc. Natl Acad. Sci. U.S.A. 77, 2265-2269. Coen. D. M . , Goldstein. D. J., and Wellcr, S. K. (1989). Herpes simplex virus ribonucleotlde reductase mutants are hypersensitive to acyclovir. Antimicrob. Agents Chemother. 33, 13951399.

Cohen, G. H. ( 1972). Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus. J . Virol. 9,408-418. Collins, P., Larder. B. A . . Oliver. N. M . . Kemp. S . , Smith. I . W.. and Darby, G . (1989). Charac-

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terization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir. J . Gen. Virol. 70, 375-382. Cooper, G. M. ( 1973). Phosphorylation of 5-bromodeoxycytidine in cells infected with herpes simplex virus. Proc. Narl. Acad. Sci. U.S.A. 70, 3788-3792. Crumpackper, C. S . , Schnipper, L. E., Zaia, J. A , , and Levin, M. J. (1979). Growth inhibition by acycloguanosine of herpesviruses isolated from human infections. Antimicrob. Agents Chemother. 15, 642-645. Crumpackper, C. S . . Schnipper, L. E., Marlowe, S. I., Kowalsky, P. N., Hershey, B. J . , and Levin, M. J. (1982). Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. N. Engl. J , Med. 306, 343-346.) Darby, G . , Field, H. J . , and Salisbury, S . A. (1981). Altered substrate specificity of herpes simplex virus thymidine kinase confers acyclovir-resistance. Nature (London) 289, 8 1-83. Darby, G . , Larder, B. A , , and Inglis, M. M. (1986). Evidence that the “active center” of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide. J . Gen. V i r d 67, 753-758. Derse, D., Cheng, Y.-C., Furman, P. A,, St. Clair, M. H.. and Elion, G. B. (1981). Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethy1)guanine triphosphate. J . B i d . Chem. 256, I 1447- I 145 I . Derse, D., Bastow, K. F., and Cheng, Y.-C. (1982). Characterization of the DNA polymerases induced by a group of herpes simplex virus type 1 variants selected for growth in the presence of phosphonoformic acid. J . Biol. Chem. 257, 10251- 10260. Drach, J. C . (1984). Purine nucleoside analogs as antiviral agents. In “Targets for the Design of Antiviral Agents” (E. DeClercq and R. T. Walker, eds.), pp. 221-258. Plenum, New York. Elion, G. B. (1982). Mechanism of action and selectivity of acyclovir. Am. J . Med. 73, Suppl. A, 713. Elion, G . B. (1983). The biochemistry and mechanism of action of acyclovir. J . Anrimicrob. Chemother. 12, Suppl. B, 9-17. Elion, G. B. (1984). Acyclovir. In “Antiviral Drugs and Interferon: The Molecular Basis of Their Activity” (Y. Becker, ed.), pp. 71-88. Nijhoff, The Hague. Elion, G . B. (1986). History, mechanism of action, spectrum and selectivity of nucleoside analogs. In “Antiviral Chemotherapy: New Directions for Clinical Application and Research” (J. Mills and L. Corey, eds.), pp. 118- 138. Elsevier, New York. Elion, G . B. (1989). The purine path to chemotherapy. Scirnce 244, 41-47. Elion, G. B., Furman, P. A , , Fyfe. J. A,, de Miranda, P., Beauchamp, L., and Schaeffer, H. J. (1977). Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl)guanine. Proc. Narl. Acad. Sci. U.S.A. 74, 5716-5720. Ellis, M. N . , Lobe, D. C . , and Spector, T. (1986). Combination antiviral chemotherapy for treatment of HSV-I infection in athymic mice. ICAAC, 26th, New Orleans, La. p. 21 I , Abstr. No. 625. Ellis, M. N., Keller, P. M., Fyfe, J . A., Manin, J. L., Rooney, J. F., Straus, S. E., NusinoffLehrman, S . , and Barry, D. W. (1987). Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered Substrate specificity. Antimicrob. Agenrs Chemother. 31, I 1171125. Ellis. M. N.. Lobe, D. C . , and Spector, T. (1989). Synergistic therapy by acyclovir and A1 I IOU for mice orofacially infected with herpes simplex virus. Anrimicrob. Agents Chemother. 33, I6911696. Erice, A., Chou, S . , Biron, K. K . , Stanat, S . C . , Balfour, H. H . , Jr., and Jordan, M. C . (1989). Progressive disease due to ganciclovir-resistant cytomegalovirus in immunocompromised patients. N. EngI. J . Med. 320, 289-293. Field, H. J., and Darby, G . (1980). Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo. Anrimicrub. Agents Chemother. 17, 209-216.

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Field, H. J., and Wildy. P.(1978). The pathogenicity of thymidine kinase deficient mutants of herpes simplex virus in mice. J . H y g . 81, 267-277. Field. H. J.. Darby, G . , and Wildy, P. (1980). Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus. J . Gen. Virol. 49, 115- 124. Fisher, P. A., and Korn, D. (1981). Ordered sequential mechanism of substrate recognition and binding by KB cell DNA polymerase a . Biochemistry 20, 4560-4569. Frank, K. B . , Derse, D. D., Bastow, K. F., and Cheng, Y.-C. (1984). Novel interaction of aphidicolin with herpes simplex virus DNA polymerase and polymerase-associated exonuclease. J . B i d Chem. 259, 13282- 13286. Furman, P. A.. St. Clair, M. H.. Fyfe, J. A., Rideout, J. L., Keller. P. M . , and Elion, G . B. (1979). Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate. J . Virol. 32, 72-77. Furman, P. A., McGuirt, P. V.. Keller. P. M.. Fyfe, J . A., and Elion. G. B. (1980). Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Virology 102, 420-430. Furman. P. A . , Lambe, C. U . , and Nelson, D. J (1982). Effect of acyclovir on the deoxynucleoside triphosphate pool levels in Vero cells infected with HSV-I. Am. J . Med. 73, Suppl. A, 14-17. Furman, P. A., St. Clair, M. H., and Spector. T. (1984). Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase. J . B i d . Chem. 259, 9575-9579. Furman, P. A., Spector. T., and Fyfe, J. A. (1986). Acyclovir: Mechanism of action. In “Human Herpesvirus Infections” (C. Lopez and B. Roizman, eds.), pp. 129-140. Raven, New York. Fyfe, J. A,. Keller, P. M.. Furman, P. A., Miller, R. L., and Elion, G. E. (1978). Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethy1)guanine. ./. B i d . Chem. 253, 8721-8727. Fyfe, J. A., McKee. S. A . , and Keller. P. M. (1983). Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2’-deoxyuridine.Mu/. Pharmacol. 24, 3 16-323. Galasso. G . J. (1984). Antiviral agents: Why not a “penicillin” for viral infections? I n “Targets for the Design of Antiviral Agents” (E. DeClercq and R. T. Walker, eds.), pp. 337-362. Plenum, New York. Gibbs, J. S . . Chiou, H . C., Bastow, K. F., Cheng. Y.-C., and Coen, D. M. (1988). Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proc. Natl. Acad. Sci. U.S.A. 85, 6672-6676. Hopkins, S., and Furman, P. ( I 990). Molecular basis for the antiviral activity of acyclovir. In “Acyclovir Therapy for Herpesvirus Infections” (D. A. Baker, ed.), pp. 1-13. Dekker, New York. Hunting, D., and Henderson, J. F. (1982). Models of the regulation of ribonucleotide reductase and their evaluation in intact mammalian cells. CRC Cril. Rev. Biochem. 13, 325-348. Karlsson, A., and Harmenberg, J. (1988). Effects of ribonucleotide reductase inhibition on pyrimidine deoxynucleotide metabolism in acyclovir-treated cells infected with herpes simplex virus type 1 . Anrimicroh. Agenfs Chemother. 32, I 100- I 102. Keller, P. M . , Fyfe, J. A , , Beauchamp, L., Lubbers, C. M., Furman. P. A , , Schaeffer, H. J., and Elion, G . B. (1981). Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities. Biorhem. Pharmacnl. 30, 307 1-3077. Keller, P. M . , McKee, S. A,. and Fyfe, J. A. (1985). Cytoplasmic S’hucleotidase catalyzes acyclovir phosphorylation. J . B i d . Chem. 260, 8664-8667. Kit, S . (1979). Viral-associated and induced enzymes. Pharmacol. Ther. 4, 501-585. Kit, S . . and Dubbs. D. R. (1963). Acquisition of thymidine kinase activity by herpes simplex infected mouse fibroblast cells. Biochem. Biophys. Res. Commun. 11, 55-59. Knopf, C . W. (1986). Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 1 strain Angelotti. N i d e i c Acids Rrs. 14,8225-8226.

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Larder, B. A,, Derse, D., Cheng, Y.-C., and Darby, G . (1983). Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus: Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase. J . Biol. Chem. 258, 20272033. Larder, B. A., Kemp, S . D., and Darby, 0 . (1987). Related functional domains in virus DNA polymerases. EMBO J . 6, 169-175. Lobe, D. C., Spector, T., and Ellis, M. N. (1991). Synergistic topical therapy by acyclovir and A l l IOU for herpes simplex virus induced zosteriform rash in mice. Anriviral Res. 15, 87-100. Mahony, W. B., Domin, B. A., McConnell, R. T., and Zimmerman, T. P. (1988). Acyclovir transport into human erythrocytes. J . Biol. Chem. 263, 9285-9291. McGuirt, P. V., Shaw, 1. E., Elion, G . B., and Furman, P. A. (1984). Identification of small DNA fragments synthesized in herpes simplex virus-infected cells in the presence of acyclovir. Anfimicrob. Agenrs Chemorher. 25, 507-509. McLaren, C., Sibrack, C. D., and Barry, D. W. (1982). Spectrum of sensitivity to acyclovir of herpes simplex virus clinical isolates. Am. J . Med. 73, Suppl. A, 376-378. Miller, W. H., and Miller, R. L. (1980). Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase. J . Biol. Chem. 255, 7204-7207. Miller, W. H., and Miller, R. L. (1982). Phosphorylation of acyclovir diphosphate by cellular enzymes. Biochem. Pharntacol. 31, 3879-3884. Ponce de Leon, M., Eisenberg, R. J., and Cohen, G . H. (1977). Ribonucleotide reductase from herpes simplex virus (types 1 and 2) infected and uninfected KB cells: Properties of the partially purified enzymes. J . Gen. Virol. 36, 163-173. Porter, D. J. T., Hamngton, J. A , , and Spector, T. (1990). Herpes simplex virus type I ribonucleotide reductase: Selective and synergistic inactivation by Al I IOU and its iron complex. Biochem. Phurmacol. 39, 639-646. Reardon, J. E. (1989). Herpes simplex virus type 1 and human DNA polymerase interactions with 2’deoxyguanosine 5’-triphosphate analogues: Kinetics of incorporation into DNA and induction of inhibition. J . Biol. Chem. 264, 19039-19044. Reardon, J. E. (1990). Herpes simplex virus type 1 DNA polymerase: Mechanism-based affinity chromatography. J . Biol. Chem. 265, 71 12-71 15. Reardon, J. E., and Spector, T. (1989). Herpes simplex virus type 1 DNA polymerase: Mechanism of inhibition by acyclovir triphosphate. J . Biol. Chem. 264, 7405-741 1. Reichard, P. (1988). Interactions between deoxyribonucleotide and DNA synthesis. Annu. Rev. Biochem. 57, 349-374. Reid, R., Mar, E.-C., Huang, E.-S., and Topal, M. D. (1988). Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviradae, human a and human f3 polymerases. J . Biol. Chem. 263, 3898-3904. Schaeffer, H. J., Gunvara, S . , Vince, R . , and Bittner, S. (1971). Novel substrate of adenosine deaminase. J . Med. Chem. 14, 367-369. Schaeffer, H. J., Beauchamp, L. P., de Miranda, P., Elion, G. B . , Bauer, D. J . , and Collins, P. ( 1978). 9-(2-Hydroxyethoxymethyl)guanine activity against viruses of the herpes group. Nature (London) 272, 583-585. Schnipper, L. E., and Crumpacker, C. S. (1980). Resistance of herpes simplex virus to acycloguanosine: Role of viral thymidine kinase and DNA polymerase loci. Proc. Norl. Acad. Sci. U.S.A. 77, 2270-2273. Segel, I. H.(1975). “Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and SteadyState Enzyme Systems,’’ p. 143. Wiley, New York. Shannon, W.M. (1984). Mechanisms of action and pharmacology: Chemical agents. In “Antiviral Agents and Viral Diseases of Man” ( G . J. Galasso, T. C. Merigan, and R. A. Buchanan, eds.), 2nd Ed., pp. 55-121. Raven, New York.

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Shipman, C . , Jr., Smith, S. H., Drach, I. C . , and Klayman, D. L. (1981). Antiviral activity of 2acetylpyridine thiosemicarbazones against herpes simplex virus. Antimicrob. Agents Chemother. 19, 682-685. Spector, T. (1985).Inhibition of ribonucleotidc reductases encoded by herpes simplex viruses. Pharmacol. Ther. 31, 295-302. Spector, T. (1989). Ribonucleotide reductases encoded by herpes viruses: Inhibitors and chemotherapeutic considerations. In “Inhibitors of Ribonucleotide Diphosphate Reductase Activity” (J. G. Cory and A. H. Cory, eds.), Sect. 128, pp. 235-243. Pergamon, New York. Spector, T., and Fyfe, J. A. (1991). Synergy and antagonism in polymerase-targeted antiviral therapy: Effects of deoxynucleoside triphosphate pool modulation on prodrug activation. In “Synergism and Antagonism in Chemotherapy” (D. C. Rideout and T. C. Chou. eds.), pp. 285309. Academic Press, San Diego, California. Spector. T., Averett, D. R.. Nelson, D. J . , Lambe, C. U . , Morrison, R. W., Jr., St. Clair, M. H . , and Furman, P. A. (1985). Potentiation of antiherpetic activity of acyclovir by ribonucleotide reductase inhibition. Proc. Nail. Acad. Sci. U.S.A. 82, 4254-4257. Spector, T., Stonehuerner, J. G . . Biron, K . K., and Averett, D. R . (1987). Ribonucleotide reductase induced by varicella zoster virus: Characterization and potentiation of acyclovir by its inhibition. Biochem. Pharmacol. 36, 4341-4346. Spector, T., Harrington, J. A., Morrison, R. W., Jr., Lambe, C. U., Nelson, D. J., Averett, D. R., Biron, K., and Furman, P. A. (1989). 2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1 1 IOU), a potent inactivator of ribonucleotide reductases of herpes simplex and varicella zoster viruses and a polentiator of acyclovir. Proc. Narl. Acad. Sci. U.S.A. 86, 10511055. St. Clair, M. H., Furman. P. A., Lubbers. C. M.. and Elion, G . B. (1980). Inhibition of cellular (Y and virally induced deoxyribonucleic acid polymerases by the triphosphate of acyclovir. Antimicrob. Agents Chemother, 18, 741 -745. Szczech, G. M. ( 1986). The toxicity of nucleoside analogs. In “Antiviral Chemotherapy: New Directions for Clinical Application and Research” (J. Mills and L. Corey, eds.), pp. 204-225. Elsevier, New York. Tenser, R. B., Ressel, S., and Dunstan, M. E. (1981). Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: Correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation. Virolop 112, 328-341. Thelander, L., and Reichard, P. (1979). Reduction of ribonucleotides. Annu. Rev. Biochem. 48, 133- 158. Tsurumi, T., Maeno, K., and Nishiyama, Y. (1987). A single-base change within the DNA polymerase locus of herpes simplex type 2 can confer resistance to aphidicolin. J . Virol. 61, 388-394. n r k , S. R., Shipman, C., Ir., and Drach, J. C. (1986). Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone. Biochem. Pharmacol. 35, 1539- 1545. Wade, J. C . , McLaren, C., and Myers, J. D. (1983). Frequency and significance of acyclovirresistant herpes simplex VINS isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J . Infect. Dis. 148, 1077-1082. Wang, T. S.-F., and Korn, D. (1982). Specificity of the catalytic interaction of human DNA polymerase p with nucleic acid substrates. Biochemistr.y 21, 1597- 1608. Worku, Y., and Newby, A. C. (1982). Nucleoside exchange catalyzed by the cytoplasmic 5’nucleotidase. Biochem. J . 205. 503-5 10.

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Rational Approaches to Osteoporosis Therapy Robert Marcus Department of Medicine Stanford University Stanford, California 94305 and the Aging Study Unit Geriatrics Research, Education & Clinical Center Department of Veterans AfSairs Medical Center Palo Alto, Cal(fornia 94304

I. Introduction

11.

111.

IV.

V. VI , VII.

A . Skeletal Organization B. Bone remodeling Age-Related Changes in Bone Mass A. Acquisition of Peak Bone Mass B. Age-Related Bone Loss C. Relationship between Mineral Density and Bone Strength Mechanisms of Bone Mass Regulation A . Physical Activity B. Calcium Nutritional State C . Endocrine Reproductive Status D. Regulation of Bone Mass: A Global View Osteoporosis A . Prevention of Osteoporosis B. Therapy of Established Osteoporosis Experimental Approaches Nonphmacological Considerations Conclusions References

1. Introduction Among the characteristic changes in body composition that accompany normal human aging, loss of bone continues to provoke interest and study. Osteoporosis is considered to be an end result of this loss, and constitutes a major and growing public health problem for older women and men in Western society. In this article, I review pharmacological approaches to limiting bone loss and restoring bone mass. Because effective intervention strategies must be founded on an understanding of the factors that regulate bone mass and the mechanisms by which they operate, I first summarize human skeletal organization, the central Advrrncrs m Phrrrmocobgv. Volume 22 Copyright 0 1Y91 by Acadcmlc Press. Inc. All rights 01 reproduction iii any lonn reserved

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role of bone remodeling, the acquisition and maintenance of bone mass through life, and the structural characteristics of osteoporosis.

A. Skeletal Organization The skeleton can be considered as two compartments, peripheral and central. The peripheral skeleton constitutes 80% of skeletal mass, and is composed of compact plates, or lamellae, of cortical bone organized about central nutrient canals. Bones of the central, or axial skeleton consist of trabecular, or cancellous bone contained within a thin cortical shell. Trabecular bone is a honeycomb of vertical and horizontal bars that are filled with red marrow and fat. It is localized for the most part in the vertebral bodies, the pelvis, and in the proximal femur. The metaphyseal ends of long bones also contain trabecular bone, but no red marrow in the adult. Vertebral bodies contain about 35% trabecular bone by weight (Nottestad el al., 1987) and about 70% by volume, which greatly exceeds the trabecular component of peripheral bones. The increased surface area of trabecular bone accounts for the fact that changes in bone mass due to altered turnover are earlier and more impressive in the axial skeleton.

B. Bone Remodeling Bone participates in three fundamental activities: Modeling refers to the process by which the characteristic shape of a bone is achieved. Repair is the regenerative response to fracture. Remodeling is a continuous cycle of destruction and renewal that is carried out by individual, independent “bone remodeling units,” illustrated in Fig. 1. Normally, 90% of bone surfaces are inactive, covered by a thin layer of lining cells. Remodeling is initiated by hormonal or physical signals that cause marrow precursor cells to cluster on the bone surface, where they fuse to become multinucleated osteoclasts and dig a cavity into the bone. In cortical bone this appears as a resorption tunnel within a Haversian canal. On trabecular surfaces it is a scalloped area called a Howship’s lacuna. The resorption front leaves a cavity about 60 pm deep. The extent of deepest resorption appears as a cement line, a region of poorly organized collagen fibrils, as opposed to the osteon itself, which shows lamellar collagen deposition. Coupled to resorption, bone formation ensues when local release of chemical mediators attracts preosteoblasts into the resorption cavity. These mature into osteoblasts that replace the missing bone by secreting new collagen and matrix constituents. Matrix production is initially rapid, the new osteoid seam approaching 20 Fm in thickness when mineral deposition begins. Mineralization catches up to matrix deposition with time, and the new bone becomes fully mineralized.

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Fig. 1 The remodeling cycle: (a) resting trabecular surface; (b) multinucleated osteoclasts dig a cavity of approximately 20 pm; (c) completion of resorption to 60 p n by mononuclear phagocytes; (d) recruitment of osteoblast precursors to the base of the resorption cavity; (e) secretion of new matrix by osteoblasts; ( f ) continued secretion of matrix, with initiation of calcification; and (g] completion of mineralization of new matrix. Bone has returned to quiescent state, but a small deficit in bone mass persists. (From Marcus, 1987. with permission.)

Resorption and formation are complete within 8- 12 weeks, several additional weeks being required to complete mineralization. If the replacement of resorbed bone were completely efficient, bone would be restored to its initial state on termination of a remodeling cycle. However, remodeling, like most biological processes, is not entirely efficient, so that a small bone deficit persists on completion of each cycle. The consequence of this inefficiency is age-related bone loss, a normal, predictable phenomenon, that begins shortly after cessation of linear growth. Alterations in remodeling activiry represent the jinal pathway through which diverse stimuli, such as dietary or hormonal insuficiency, affect the rate of bone loss. A change in overall bone remodeling rate can reflect several distinct perturbations in the components of the remodeling cycle. These include a change in the activation, or birthrate, of remodeling units, in the resorptive capacity of individual units, or in the rnagnitude of bone formation response. Since bone remodeling leads to progressive bone loss, one may ask what useful function it serves. Current evidence suggests that remodeling fulfills a scavenger role, by which areas of fatigue microdamage are removed and replaced by intact lamellar bone. Although the inefficiency of this process may diminish bone mass and strength in the long run, short-term protection against routine wear is provided. For a more detailed discussion of bone remodeling, the reader is referred to the review by Marcus ( 1987).

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II. Age-Related Changes in Bone Mass A. Acquisition of Peak Bone Mass Bone density and fracture risk in later years are determined by the maximal bone mineral content at skeletal maturity, called the peak bone mass, and by the subsequent rate of bone loss. Major increases in bone mass, accounting for about 60% of final adult levels, take place during the years of adolescence, particularly during the 2- or 3-year period of highest growth velocity. It is frequently stated that bone mass continues to increase into the third decade. Conclusive evidence on this issue is not available, but such late consolidation of bone mass may vary with the particular skeletal site. Recent data suggest that axial bone mass reaches an earlier maximum, perhaps in late adolescence (Gilsanz et al., 1988). Peak bone mass is determined by multiple factors. It appears that inheritance accounts for much of the variance in bone mass of healthy young people (Pocock et al., 1987; Dequeker ef al., 1987; Seeman et al., 1989). Other independent predictors include muscle strength, physical activity, circulating estrogen and androgens, and dietary calcium (Buchanan et al., 1988b; Kanders et al., 1988; Snow-Harter et al., 1990). Expressed as total grams of bone mineral, peak bone mass is generally higher in men than in women. However, when bone mass is corrected for differences in body size, it appears that peak bone mineral density may be equivalent in men and women (Kelly et al., 1990b).

B. Age-Related Bone Loss It has become axiomatic that bone is lost with age. Careful radiographic measurements of metacarpal cortical thickness by Cam (1966) described a characteristic trajectory of bone mass throughout life. By this model, bone is gained during adolescence, reaches a plateau level during the third decade, and remains stable until about age 50, after which progressive loss occurs. A similar trajectory occurs for men and women, and for all ethnic groups. For cortical bone, this model has been repeatedly confirmed by newer methods. For the axial skeleton, evidence suggests that loss begins earlier than for cortical bone. This evidence includes direct measurement of postmortem and biopsy material and absorptiometric studies. Although there is consensus that early loss of trabecular bone occurs, controversy persists over its timing, over the presence of menopausal acceleration, and whether all regions of trabecular bone are similarly affected. Biopsy results document a significant premenopausal decline in trabecular bone mass of the iliac crest that appears to begin as early as the third decade (Meunier et al., 1973; Marcus et al., 1983; Mosekilde et al., 1987; BirkenhagerFrenkel et al., 1988), with a predicted rate of bone loss of about 0.7% per year.

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At that rate, cumulative bone loss over a span of 30 years could amount to 25% of original trabecular bone volume even before menopause is reached. Densitometric measurements of the spine have been variably interpreted to suggest linear and nonlinear loss of bone with age. Madsen (1977) and Riggs et al. (1 98 1) both described a single negative correlation of lumbar spine mineral with age, with a slope in women of about 1% per year. Men also lost bone with age, but the slope was less than half that observed for women. Other absorptiometric data have varied, some in support of early axial loss (Buchanan et a l . , 1988a), and some failing to show such a change (Rosenthal et a f . , 1989). These discrepancies are not understood, but they may reflect the cross-sectional nature of the data and the fact that the sample size for detecting subtle changes in regression slope exceeds that of any reported study. Most studies suggest a transient period of accelerated cortical and axial bone loss at (Krolner and Pors Nielsen, 1982; Firooznia et a l . , 1984; Cann et al., 1985; Aloia et a l., 1985) or a few years immediately prior to the menopause (Ribot et al., 1988; Block et a l . , 1989).

C. Relationship between Mineral Density and Bone Strength It is generally acknowledged that bone strength is strongly related to its mineral density (Carter and Hayes, 1977). Thus, it is not surprising that most patients with osteoporotic fractures have low bone mass compared to age-matched nonfractured control subjects. However, major overlap is seen in the bone densities of fracture patients and controls, regardless of measurement technique. In prospective studies (Hui et al., 1988; Cummings et a l . , 1990; Ross et a / . , 1990), progressive increases in fracture incidence can be demonstrated as levels of bone mass decrease. Individuals in the lowest quartile of bone mineral may have a 2to 3-fold increase in hip fracture over time (Cummings et a l . , 1990). Here again, many people in that category may never actually sustain a fracture, so fracture risk must also depend on factors beyond simple bone mineral density. Extraskeletal factors such as susceptibility to falls have been frequently discussed. Less commonly appreciated are skeletal factors other than global bone mineral density that influence bone competence. These include bone material and geometrical properties. Material properties include the brittleness and ultimate breaking strength of bone material. Although mineral content is a primary determinant of material properties, factors such as the accumulation of cement lines over time, crystal maturation, and changes in collagen cross-linking may all diminish bone strength with age. The major geometrical influence on the competence of long bones is the distribution of bone mass about its bending axis. This can be expressed mathematically as a term called the cross-sectional moment of inertia. Briefly summarized, the further away the mass of an object is located from the central bending axis, the greater is its strength. An interesting example of this phenomenon in

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men is age-related expansion of the femoral diameter. Here, an increase in moment of inertia compensates for the loss of mass and maintains bone strength. This compensation appears not to occur in women (Martin and Atkinson, 1977; Ruff and Hayes, 1988). In the axial skeleton, changes in trabecular microstructure are highly relevant to fracture risk. In young people, trabecular bone consists of thick vertical plates that are connected by thinner horizontal trabeculae. Parfitt et al. (1983) and Weinstein and Hutson (1987) showed that the space between adjacent trabeculae increases with age, reflecting the loss of entire trabecular elements. They suggested that this loss was the result of trabecular perforation during resorption. Presumably, perforated trabeculae offer no scaffold for new bone formation to take place. Thus, loss of trabeculae with age leads to deficient horizontal trabecular connections, the consequence of which is a substantial decrease in the capacity of vertical trabeculae to withstand bending stress. The role of trabecular connectivity is central to understanding why measurements of bone mineral density do not adequately predict fracture risk for individuals. Two people may have the same low mineral density; in one this reflects diffusely thin trabeculae with maintenance of normal trabecular connections, while the other shows porous bone with reduced trabecular connections. The first person may have a trivial fracture risk, while the bones of the second person may be in extreme jeopardy. Current noninvasive tools for clinical assessment of bone mass do not distinguish between these two situations. Until methods are developed to make such distinctions, the utility of bone density measurements to predict individual fracture risk will be limited.

111. Mechanisms of Bone Mass Regulation Alterations in remodeling dynamics are the common mechanism by which bone mass is regulated. Primary regulators include physical activity, reproductive endocrine status, and calcium nutriture. The contributions of these three areas to bone mass are summarized here.

A. Physical Activity Although the mechanisms remain to be elucidated, bone adapts to the loads applied to it (Wolffs Law), giving rise to the view that increased physical activity is a potential means to improve bone mass (Marcus and Carter, 1988). This adaptation includes an increase in density when mechanical loading increases, and a loss of bone density when customary loads are removed. Loss of bone with immobilization, such as with spinal cord injury, is a frequent and challenging clinical problem. Loss of bone with extended space flight emphasizes the critical

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35

role of gravitational stress to the maintenance of bone mass (Mack et al., 1967). Since muscle contraction is the dominant source of skeletal loading, it is not surprising that significant relationships exist between indices of bone and muscle mass (Doyle rt al., 1970), and between bone mineral density and muscle strength at specific sites (Snow-Harter et al., 1990; Sinaki et al., 1986; Bevier er al., 1989). There is strong support for the notion that bone mass of athletes exceeds that of sedentary individuals (Marcus and Carter, 1988) and that increased activity may slow age-related bone loss. One must interpret these cross-sectional analyses with caution, since genetic and musculoskeletal characteristics may distinguish “athletic” from “sedentary” individuals even before exercise training is initiated. A few intervention trials have examined the effects of exercise on bone density. Once again, interpretation requires caution, since most of these studies have not been randomized trials, and several have involved training routines suited for cardiovascular fitness but perhaps inappropriate for skeletal loading. Nonetheless, the results generally confirm that bone density increases with exercise training. The magnitude of increased bone mass from exercise training has been disappointing. In young and middle-aged individuals, several months to a year of serious training has increased spine mineral density by only a percent or so, whereas a training program for elderly women (Dalsky et al., 1988) increased spine mineral by several percent. Although exercise may reduce the rate of bone loss, it is likely that training regimens necessary to achieve major gains in bone mass may exceed the capacity of most people. For example, it is highly improbable that an individual whose bone mass is in the lowest 5 percentile could ever be restored to the 50th or even the 20th percentile by exercise alone. For additional discussion of physical activity and bone mass, the reader is referred to the paper by Marcus and Carter (1988).

B. Calcium Nutritional State The skeleton contains 99.5% of body calcium stores. It provides a mineral reservoir to support plasma calcium levels at times of need. The relationship of dietary calcium to skeletal integrity is complex, and will not be detailed here. However, a few summary statements are in order. The daily recommended dietary calcium intake for adolescent boys and girls is 1200 mg, a figure derived primary from balance studies (National Research Council, 1989). Since the 2 or 3 years that define the pubertal growth spurt are accompanied by deposition of 50% of final bone mass, dietary inadequacy limits bone formation far more at this time than at other times of life. Consumption figures indicate that the calcium intake of American men corresponds reasonably well to recommended levels at most ages, but that median intakes for girls are well below target levels by age 1 1 , and never recover (Carroll et al., 1983).

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Thus, calcium undernutrition probably has an important influence on peak bone mass in women. During the third through fifth decades, growth has stopped and robust compensatory mechanisms permit rapid adaptation to even severe dietary restriction. Calcium nutritional state appears not to affect the rate of bone loss during this period (Riggs et al., 1987), so it is unlikely that calcium supplementation will exert important beneficial effects on bone mass at this time. It is ironic that this population receives the greatest portion of calcium supplement advertising. At menopause, accelerated bone loss reflects loss of endogenous estrogen and has little relationship to dietary calcium. Riis et al. (1987) showed only modest effects on bone loss when early-menopausal women were given extra calcium. After age 60, the early effects of estrogen deficiency have dissipated, and the compensations to accommodate dietary inadequacy are less efficient. At this time proper attention to calcium intake, be it from diet or supplements, is rational, and has been shown to have beneficial effects on bone turnover and mass (Recker and Heaney, 1985; Need et al., 1987; Dawson-Hughes et al., 1990).

C. Endocrine Reproductive Status Formidable evidence supports a critical role for gonadal function in the acquisition and maintenance of bone mass. Hypogonadal boys and girls show important deficits in cortical and trabecular bone mineral, and loss of endogenous androgen or estrogen during adult life leads to accelerated loss of bone mineral. In women, loss of estrogen has dual effects. Decreased efficiency of intestinal and renal calcium handling increases the calcium requirement (Heaney et al., 1977). Recent evidence shows that estrogen directly affects bone cell function (Eriksen et al., 1988; Komm et al., 1988), and this interaction may underlie the accelerated bone loss of early estrogen deficiency. In terms of bone remodeling, estrogen deficiency permits osteoclasts to resorb bone with greater efficiency. This may lead to trabecular perforation. As discussed below, estrogen replacement at menopause protects bone mass and affords significant protection against osteoporotic fractures (Horsman et al., 1977; Recker et al., 1977; Hutchinson et al., 1979; Lindsay et al., 1980; Weiss et al., 1980). The degree to which this protection reflects direct actions of estrogen on bone cells is not known, since estrogen replacement also increases circulating levels of 1,25-(OH), vitamin D, improving the efficiency of intestinal calcium absorption in menopausal women (Gallagher et al., 1980; Cheema et al., 1989). The skeletal role of androgens is less well understood. Testosterone deficiency is a major cause of osteoporosis in men, in whom replacement therapy restores bone mass. Specific testosterone receptors have been recently demonstrated in normal human osteoblasts (Colvard et ul., 1989). Whether direct actions on bone cells fully account for the effects of androgen on bone mass is unknown. Androgens also increase muscle mass, so

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37

effects on bone may be secondary to the increased mechanical loading that would accompany increased muscle bulk. Finally, circulating androgens make a significant contribution to peak bone mass in women.

D. Regulation of Bone Mass: A Global View Optimal acquisition and maintenance of bone require sufficiency in physical activity, reproductive status, and nutrition. Deficiency in one area is not compensated by excessive attention to another. Women athletes who become hypogonadal lose bone despite frequent high-intensity exercise (Drinkwater et al., 1984; Marcus et al., 1985). Therapeutic specificity is a venerable medical tradition. Just as it would be inappropriate to treat iron deficiency with folic acid, it is equally futile to prescribe calcium to an immobilized patient, or to prescribe exercise and calcium rather than giving estrogen at menopause.

IV. Osteoporosis Osteoporosis is a condition of low bone mass that results in fractures. Characteristic fracture syndromes have been described, including vertebral compression, Colle’s fracture of the distal radius, and subcapital and intertrochanteric fractures of the proximal femur, the so-called hip fracture. In addition, individuals with osteoporosis show generalized skeletal fragility, so that fractures at other sites, such as ribs and long bones, are very common. Prior to the introduction of noninvasive bone mass measurements, osteoporosis was a clinical and radiological diagnosis that could be made only after obvious skeletal damage had occurred. Diagnosis based on the presence of fracture proved unworkable because it was too restrictive, excluding individuals whose bones were jeopardized but had not yet fractured. It is currently fashionable to define osteoporosis more inclusively, as a critical reduction in bone mass to the point that fracture vulnerability increases. In this sense, osteoporosis is analogous to anemia defined as a low red blood cell mass. Although this is a useful definition, it introduces problems of specificity. Bone mineral content does predict fracture risk but accounts for only a portion of this risk, as discussed above. Osteoporosis is generally considered to be of two broad categories, primary and secondary. Patients with secondary osteoporosis sustain bone loss because of systemic illness or medication. A partial listing of secondary osteoporotic states appears in Table I . The most successful approach to secondary osteoporosis is prompt resolution of the underlying cause, for example, successful treatment of thyrotoxicosis. However, mechanisms of secondary osteoporosis can all be related in terms of disordered bone remodeling, so that the same therapeutic strategies may apply to these conditions as are appropriate for primary osteoporosis.

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liable I Representative Examples of “Secondary” Osteoporosis with Characteristic Change\ in Dynamic Histomorphometry Cause Glucocorticoids Hyperthyroidism Immobilization Heparin Hypervitaminosis A Anticonvulsants

BRU birthrate”

Reqorption

Formationh

Probably increased Increased Early increase Probably increased Probably increased Probably increased

Normal to increased Increased Increased then normal Probably increased Increased Increased

Decreased Increase& Decreased Uncertain Unceratin Increased‘

oRefers to activation, or birthrate, of bone remodeling units. bRefers to total bone-forming activity. “Despite an increase in overall formation, osteoblastic function for individual bone remodeling units may be depressed.

In 1948, Albright and Reifenstein concluded that primary osteoporosis was composed of two separate entities, one related to menopausal estrogen loss, and the other to aging. Support for this concept has been published by Riggs and associates ( 1982), who proposed that “primary” osteoporosis represents two fundamentally different conditions: type I osteoporosis, loss of trabecular bone after menopause, and rype I1 osteoporosis. age-related loss of cortical and trabecular bone in men and women. Whereas the type I disorder is theoretically related to lack of estrogen, type I1 osteoporosis would reflect long-term remodeling efficiency, dietary adequacy, and intestinal and renal function. Compelling proof has not been presented that these two entities are truly distinct. Iliac crest biopsies from women with “type I osteoporosis” showed no consistent or unique histomorphometric features (Eriksen et al. , 1990). It has been widely assumed that osteoporosis must reflect excessive bone loss, but some patients with apparent “type I” osteoporosis on the basis of vertebral compression and a low axial bone mass for age may represent inadequate bone acquisition rather than excessive loss. It is also possible that differences in fracture patterns from one patient to the next are more indicative of the tendency to fall and the types of falls that are experienced rather than fundamental differences in bone physiology. Resolution of this issue will require additional longitudinal study. Although many osteoporotic women undoubtedly have experienced excessive menopausal bone loss, it may be more appropriate to consider osteoporosis as the result of multiple physical, hormonal, and nutritional factors acting alone or in concert, rather than to become wedded to an unvalidated theoretical model.

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39

A. Prevention of Osteoporosis A rational strategy to prevent osteoporosis follows from the considerations outlined above. Regular physical activity of reasonable intensity can be endorsed for all age groups. For growing children and adolescents, adequate attention to dietary calcium is important if peak bone mass is to reach the level appropriate for genetic endowment and habitual mechanical loading. Further attention to nutritional support may be required in the seventh decade and beyond, and might take the form of increased food calcium or as calcium and/or vitamin D supplementation. For women at menopause, timely administration of estrogen is the most powerful intervention for preserving hone mass and protecting against fracture. Indeed, at any age, prevention or correction of hypogonadism is an important consideration. No therapy or combination of approaches can guarantee freedom from osteoporotic fracture. However, with appropriate lifelong attention to physical activity, nutritional state, and reproductive hormone status, important reductions in subsequent fracture risk can be achieved.

B. Therapy of Established Osteoporosis In this section, I use the term “established osteoporosis” to indicate patients having at least one nontraumatic fracture of the spine, forearm, or hip. This usage avoids the question of whether a person whose bone mineral density is one or two standard deviations below predicted levels necessarily has bone fragility. As discussed above, a decreased bone mass is associated with increased fracture risk, but even at very low levels does not necessarily lead to fractures. I consider it premature to recommend treatment (as opposed to preventive intervention) based simply on bone mass determinations in patients who have not sustained a fragility fracture. Pharmacological approaches to managing osteoporotic patients fall into two groups (Table 11). The first consists of agents that constrain the rate of bone loss. These are thought to work primarily by decreasing the rate of bone resorption, and are frequently referred to as “antiresorptive” drugs. The second group consists of agents that promote bone formation.

1 . Antiresorptive Therapy a. Calcium and Vitamin D The important nutritional role for calcium has been discussed above. Although the use of calcium supplement is often considered a means to increase dietary calcium, I shall consider it a pharmacological intervention. This distinction is admittedly artificial, hut it is justified, since mononutrient supplementation may produce effects that are not seen with dietary adjustments. The rationale for supplemental calcium differs with the time of life.

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Robert Marcus

Table I! Pharmacological Approaches to Osteoporosis Antiresorptive Calciumivitamin D Calcitriol Estrogen Calcitonin Bisphosphonate

Bone forming

Experimental"

Fluoride Androgen Parathyroid hormone

Growth hormone (IGF-1) TG F- P Coupling Factors Sequenced therapy (ADFR)

WiF-I, Insulin-like growth factor I ; TGF-P, transforming growth factor p; ADFR, activate-depress-free-repeat.

For adolescents, one hopes to supply adequate substrate calcium for bone accretion. Although assessments of calcium intake suggest important relationships with peak bone mass and subsequent fracture risk (Matkovic et al., 1979; BlackSandler et al., 1985; Holbrook ef ul., 1988; Kelly et al., 1990a), convincing interventional data that supplemental calcium promotes adolescent bone acquisition have not yet appeared. In the elderly, the goal is to suppress the parathyroid axis and thereby decrease the rate of bone turnover. Supplemental calcium clearly suppresses biochemical markers of bone turnover, such as immunoreactive parathyroid hormone and urinary hydroxyproline, and also has a beneficial effect on bone mass (Recker et al., 1977; Horowitz et al., 1984). Controversy surrounds the importance of calcium during the early years after menopause. Although the paper by Riis et al. (1987) is frequently cited to support the argument that calcium is not effective, the data actually show a significant reduction in cortical bone loss in calcium-treated women compared to a placebo group. This result is surprising, since average calcium intakes of Danish women approximate 1100 mg/day. Thus, an even greater effect on cortical bone might be found for North American women, whose average calcium intakes are only about 500 mg/day (Carroll et al., 1983). Numerous salts of calcium are available for human use, the most frequently prescribed being the carbonate. Other preparations include the lactate, gluconate, and citrate salts as well as hydroxyapatite and powdered bone. Lead contamination of some lots of bone powder diminishes the acceptability of this product. Evidence has been presented that calcium citrate is more efficiently absorbed than other salts, and may actually be advantageous for skeletal protection (Dawson-Hughes et al., 1990). However, absorption efficiency for most of the commonly prescribed calcium products is reasonable (Goddard et al., 1986; Sheikh et al., 1987),and for many patients cost and palatability outweigh modest differences in efficacy. The relative insolubility of calcium carbonate has led to

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41

concern that individuals with poor gastric acidity will fail to absorb this compound; however, Recker ( 1985) established that sufficient acidity is present in food to permit reasonable absorption, even for achlorhydric patients. Traditional dosing of calcium is about 1000 mg/day, nearly the amount present in a quart of milk. Added to the 400-500 mg of dietary calcium that typifies the diet of elderly men and women, this provides a total daily intake of about 1500 mg. Heaney and Recker (1986) have argued that more than this amount may be necessary to overcome endogenous intestinal calcium losses, but daily intakes of 2000 mg or more are frequently attended by constipation. Calcium supplements are most often taken with meals. The rationale for taking a portion of the supplement at bedtime is to suppress late night increases in parathyroid hormone secretion; however, the utility of this method has not been fully evaluated. b. Vitamin D Considerable evidence supports the view that vitamin D status is commonly jeopardized in elderly men and women (Omdahl et al., 1982; Chapuy et af., 1983). The mechanisms for this decline are multiple, and include decreased exposure to sunlight, impaired cutaneous production of the vitamin (MacLaughlin and Holick, 1985), and inadequate renal production of calcitriol [ 1,25-(OH), vitamin D] (Slovik et al., 198 I ; Tsai et al., 1984). People living at Northern latitudes are particularly at risk for marginal vitamin D deficiency, since the component of the ultraviolet spectrum that stimulates production of vitamin D may not be present in ambient sunlight during the winter months (Webb et al., 1988). The definition of vitamin D adequacy remains ambiguous. Healthy, sunlight-replete men and women of all ages maintain blood 25-(OH) vitamin D levels of 20-30 ng/ml. Levels below 10 ng/ml are associated with osteomalacia. It is considered likely that intermediate values, 10-20 ng/ml, provide only marginally adequate substrate for calcitriol production and lead to compensatory hypersecretion of parathyroid hormone to maintain blood calcium levels. By this formulation, higher levels of circulating parathyroid hormone would support calcium homeostasis at the expense of increased bone turnover and aggravated bone loss. Although this is an attractive model to explain observed age-related abnormalities in the vitamin D-parathyroid axis, it has not been fully validated. Nonetheless, the common occurrence of marginal vitamin D status in older men and women justifies a recommendation for a modest increase, perhaps to 1000 IU/day, in vitamin D intake for that population. In particular, frail elderly individuals who live in Northern latitudes and residents of nursing homes would probably benefit from such a recommendation. c. Calcitriol Although calcitriol is categorized here as an antiresorptive compound, it is important to realize that this polar metabolite of vitamin D has potent effects on osteoblast function, and may also stimulate bone formation. The use of calcitriol as a pharmacological agent for osteoporosis is distinct from assurring vitamin D nutritional adequacy. Here, the rationale is directly to suppress parathyroid function and reduce bone turnover (Gallagher, 1990). Although

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calcitriol and another polar vitamin D metabolite, 1a-hydroxycholecalciferol, enjoy frequent use in other countries, particularly Japan (Fujita, 1990), experience in the United States remains limited and mixed. In one Japanese study (Shiraki et al., 1985). elderly women with osteoporosis showed increased radial mineral content when treated with either 1a-hydroxycholecalciferol or 1,24dihydroxycholecalciferol compared to controls. Similarly, Aloia et al. ( 1988) reported a statistically significant increase in spine and whole-body mineral density in subjects treated with calcitriol compared to a placebo group. However, Falch et al. (1987) found no difference in annual bone loss from the forearm between women treated with 0.5 pg/day of calcitriol compared to a control group given 400 IU vitamin D/day. Further, Ott and Chesnut (1989) reported no differences in spine mineral density or total body mineral in subjects treated with calcitriol plus calcium compared to calcium alone. In this study, no significant change in bone mineral status of the calcium controls was observed, and it is possible that the calcium supplement was itself sufficient to reduce bone loss. On the other hand, Jensen er al. (1985) reported that calcitriol appeared to accelerate the loss of vertebral height in a I-year controlled intervention trial. Gallagher and Goldgar (1990) recently published results of a 2-year randomized intervention trial in which subjects in the treatment arm received sufficient calcitriol to maintain blood and urine calcium values just below toxic levels. The calcitriol group showed maintenance and even increases in bone mineral compared to the placebo group, which lost bone from all measured sites. The power of this study was not adequate to show differences in fracture rate, but in a separate publication reflecting the combined 1-year experience from two medical centers, Gallagher and Riggs (1 990) reported that calcitriol significantly reduced vertebral fracture rate compared to placebo. The difference in results among these and other studies reflect the diversity of patients and administration schedules. It appears that higher doses of calcitriol are more likely to improve bone mass. However, this improvement is achieved at the risk of hypercalciuria and hypercalcemia (Gallagher and Goldgar, 1990; Aloia, 1990). Thus, close scrutiny of patients and dose modification is required. For the time being, polar metabolites of vitamin D remain a promising avenue for future study, but their toxicity makes it premature to endorse them for widespread use. Gallagher and Goldgar (1 990) suggest that restriction of dietary calcium may be an important means to reduce toxicity during calcitriol therapy. The fact that hypercalciuria and hypercalcemia are unusual complications of therapy in Japan may reflect relatively poor calcium intakes in that country. d. Estrogen Over the past 20 years, overwhelming evidence from randomized clinical trials has established a major role for menopausal estrogen replacement in the conservation of bone mass and protection against osteoporotic fracture (Lindsay et al., 1976, 1980; Horsman et al., 1977, 1983; Recker er al., 1977; Nachtigall et a l . , 1979; Hutchinson et al., 1979; Weiss et al., 1980;

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43

Civitelli el al., 1988a; Naessen et al., 1990). When estrogen is given as sole therapy, the minimum effective dose necessary for skeletal protection appears to be 0.625 mg/day of conjugated equine estrogens or its equivalent. Both oral and transcutaneous estrogen reduce biochemical evidence of bone turnover and conserve bone mass (Stevenson et al., 1990). Although both should provide adequate protection against fracture, experience with transcutaneous hormone is fairly recent and its effect on fracture is not known. For oral estrogen, protection against hip fracture appears to require at least 5 years to emerge (Weiss et al., 1980). Cessation of estrogen leads to acceleration in bone loss (Lindsay et al., 1978), so therapy, once initiated, should be long-term. Standard practice recommends the cyclic or continuous administration of progestational drugs to women with an intact uterus. The C , , progestins (e.g., medroxyprogesterone acetate) appear not to interfere with the skeletal effects of estrogen. The androgenic progestins (e.g., norethisterone) may actually preserve or increase bone mass and provide added skeletal benefit when given with estrogen (Christiansen and Riis, 1990). The optimal time to institute estrogen replacement is early menopause, when bone turnover accelerates. As time from last menses increases, the impact of initiating estrogen may be progressively reduced. However, it should be noted that the clinical trials that first convincingly demonstrated preservation of bone mass by estrogen included patients older than 60 years, and it would not be appropriate to withold estrogen replacement simply because a woman is more than an arbitrary number of years beyond menopause. Even for women beyond age 65, estrogen has extraskeletal actions that may be useful. For example, estrogen increases circulating levels of calcitriol (Gallagher et al., 1980; Cheema et al., 1989), which may improve intestinal calcium absorption. For women without a uterus, therapy is continuous, relatively uncomplicated, and can be offered to women of any age. However, many elderly women will not accept cyclic bleeding or other anticipated or real side effects of estrogen. Thus, initiation of estrogen therapy to elderly women, while rational, must be individualized. e. Calcitonin This peptide hormone is a powerful inhibitor of osteoclastic bone resorption, and is associated with modest increases in bone mass in patients with osteoporosis (Gruber et al., 1984; Gonzales et al., 1986; Mazzuoli et al., 1986). These increases are most impressive in patients whose intrinsic rate of bone turnover is high (Civitelli et al., 1988b), and in such patients increases may approach 10-15%, after which a plateau is reached. It is likely, therefore, that they represent simple reductions in the labile deficit of bone called the “remodeling space,” which is an overall reflection of remodeling activity. Since bone resorption and formation are normally coupled, long-term use of calcitonin leads ultimately to a reduction in bone formation. Calcitonin has not been shown to decrease the risk for fracture. In the United States, salmon and human calcitonins

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are available for prescription. Eel calcitonin is used more frequently in Japan. Two disadvantages of calcitonin are expense and the need for parenteral administration. Salmon calcitonin for nasal administration should be available soon. Many women are unable or unwilling to accept estrogen replacement on entering menopause. In this setting, calcitonin has been shown to afford reasonable preservation of bone mass (Reginster et al., 1987; MacIntyre et al., 1988). f. Bisphosphonates (Geminal Diphosphonates) These constitute another category of medications that are primarily anti-resorptive. These compounds have two phosphonic acid groups on the same carbon atom and mimic the structure of endogenous pyrophosphate. They differ from pyrophosphate, however, in their resistance to hydrolysis by alkaline phosphatase. The general structure of diphosphonates is shown in Fig. 2. In its simplest form, the alkyl groups R, and R, are hydrogen. This agent, methylene diphosphonate, is widely used in 99mTe diphosphonate scanning. Many bisphosphonates have been synthesized during the past 20 years. The sole bisphosphonate currently available in the United States is etidronate (sodium 1-hydroxyethylidene diphosphonate, Didronel), shown in Fig. 2, which has enjoyed wide use in the management of patients with Paget’s disease of bone. The mechanisms by which bisphosphonates inhibit bone resorption are not completely known. The compounds are deposited on bone surfaces regardless of whether the osteoid surface is mineralized or not. It is censidered likely that osteoclasts acquire the drug during bone resorption, leading to profound inhibition of osteoclast function. It is this suppression of bone resorption that makes biphosphonate an attractive candidate for use in osteoporosis. However, another effect of etidronate is inhibition of bone mineralization. Severe osteomalacia was observed in early Paget’s disease trials in which daily doses of 20 mg/kg were administered. Even with modest doses of 5- 10 mg/kg, focal osteomalacia is occasionally produced. Consequently, caution has been warranted in applying this drug to osteoporosis. One attempt to avoid the mineralization defect involves intermittent drug administration. The rationale of current treatment strategies is to provide sufficient drug to inhibit resorption, followed by a sustained interval off medication to permit normal mineralization. Two randomized intervention trials (Storm et al., 1990; Watts et

Diphospho nate

Etidronate

Fig. 2 Structural formulas of bisphosphonates.

Rational Approaches to Osteoporosis Therapy

45

al., 1990) recently found that cyclic etidronate (400 mg/day for 2 weeks, followed by 3 months off therapy) achieved modest increases in bone mass over several years, and that these increases were associated with a significant reduction in vertebral fracture compared to a placebo group. Even though cyclic administration may reduce the long-term consequences of etidronate, additional studies must be canied out before its long-term safety will be understood. Nonetheless, from the encouraging results to date, it appears that cyclic bisphosphonate will be a useful therapy. Although it remains premature to offer definitive views on the ultimate therapeutic role of bisphosphonates, it is reasonable to ask whether they constitute a satisfactory alternative to estrogen. My view is that bisphosphonates are not equivalent to estrogen, and women who can take estrogen should be encouraged preferentially to do so. Although etidronate has reduced the short-term incidence of vertebral compression fracture, in contrast to estrogen it has not been shown to decrease the risk for hip or other peripheral bone fractures. It is interesting to speculate whether addition of etidronate to estrogen may confer added benefit, but there is no published information in this regard. Many patients who cannot or will not take estrogen might benefit from bisphosphonate. These include women with prior history of breast cancer and men. Chronic glucocorticosteroid therapy is another clinical setting in which bisphosphonate therapy may prove to be very useful. One can anticipate that studies of these particular groups will be reported in the near future. In addition, a number of new diphosphonates are under study and should be ready for introduction in the near future. For example, Reginster et a / . ( 1989) reported that women in early menopause experienced maintenance of spinal bone mass when treated with tiludrinate (chloro-4-phenyl-thiomethylene bisphosphonate). One attractive feature of these newer agents is the fact that their antiresorptive potency is sufficiently great that adequate treatment can be accomplished with minimal or no apparent inhibition of mineralization. It is likely, therefore, that a newer diphosphonate will replace etidronate for long-term management of osteoporosis. g. Thiazide Diuretics Although not strictly antiresorptive, thiazides reduce urinary calcium excretion and help constrain bone loss in patients with hypercalciuria. Whether thiazides will prove useful in patients who are not hypercalciuric remains to be established, but recent epidemiological data suggest that they provide significant protection against the risk for hip fracture (LaCroix et a ( . , 1990).

2. Bone-Forming Agents a. Fluoride It has been known for many years that sodium fluoride increases bone volume, an effect that is due specifically to increased osteoblastic activity (Baylink el d . , 1970; Briancon and Meunier, 1981). In doses of 30-60

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mglday, fluoride increases trabecular bone mass in many, but not all patients. Until recently, much of the fluoride literature has been anecdotal or retrospective, not based on properly controlled therapeutic trials. Such literature has been interpreted to show that fluoride provides significant vertebral fracture protection. Concern has been expressed, however, that fluoride treatment may create an increased risk for hip fracture (Hedlund and Gallagher, 1989). Two recently published prospective clinical trials provide new insights into the utility of fluoride. Riggs et al. (1990) and Kleerekoper et al. (1989) both conducted randomized, double-blinded, placebo controlled protocols, in which bone mass and fracture occurrence were monitored over several years. Although fluoride was associated with significant increases in lumbar spine mineral density, neither study showed any protective effect against vertebral compression fractures. In addition, the study of Riggs et al. (1990) showed a significant increase in the occurrence of peripheral fractures and stress fractures in the fluoride group. These studies might be criticized for the high dose of fluoride that was used (75 mg/day), and a prospective trial in France (Mamelle et al., 1988) reported that 30-50 mg/day of sodium fluoride was associated with a decreased fracture risk. However, the results of Riggs et al. ( 1 990) and Kleerekoper et al. (1989) clearly show that increased bone mass is not synonymous with increased bone strength, and that if any dose of fluoride proves useful there is a fairly narrow therapeutic window. It is incumbent on the advocates of fluoride therapy to demonstrate its efficacy and safety in properly controlled clinical trials. Until that has been done, the general use of fluoride cannot be recommended. b. Androgen Testosterone deficiency is a major etiological factor for 0steoporosis in men, and replacement therapy significantly increases bone mass in such patients. In addition, chronic administration of androgens significantly improves bone mass in osteoporotic women, but therapy has been limited by virilizing side effects. Several reports suggest that nandrolone decanoate, 50-mg injection every 3 weeks, increases peripheral and axial bone mass without bothersome side effects in osteoporotic women (Geusens and Dequeker, 1986; Need et al., 1989). Adequate fracture data are not yet available, so it is impossible to make a judgment on the therapeutic role for this therapy. However, in view of the potential benefits to muscle as well as bone, in addition to its potential deleterious effects on lipoprotein metabolism and circulating HDL and LDL cholesterol, androgen clearly deserves additional careful study. c. Parathyroid Hormone The classical devastating effects of parathyroid hormone (PTH) on the skeleton of patients with severe hyperparathyroidism are well described. Because of improved diagnostic methods, most patients with primary hyperparathyroidism encountered today show very mild biochemical and structural abnormalities. Parathyroid bone disease is still seen on occasion, but it is most common to observe few, if any, skeletal changes. Although modest deficits in cortical bone mass are frequent, normal or even increased trabecular

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bone mass and architecture are typical (Parisien et al., 1990), and such patients appear not to suffer an increased fracture risk (Wilson et af., 1988) An anabolic effect of PTH on trabecular bone has been repeatedly observed, and can be produced experimentally by intermittent administration of the hormone (Tam et al., 1982). Consequently, several laboratories have examined the effects of PTH on bone mass in patients with osteoporosis (Reeve et al., 1980; Slovik et al., 1986). In these studies, PTH and its synthetic analog hPTH(1-34), have been found to increase axial bone mineral, although effects on cortical bone have been disappointing. When given as a single agent, hPTH 1-34 increased axial mineral, but reduced bone mineral content of the femur (Hesp et al., 1981). No change in whole-body calcium balance was observed, inviting the conclusion that mineral deposition in axial bone came at the expense of cortical stores. Reeve et af. (1990) recently showed that coadministration of hPTH( 1-34) with estrogen or synthetic androgen resulted in impressive gains in axial mineral without loss of cortical bone. One limitation with this literature to date is the lack of concurrent randomized controls or fracture data. Pending a formal randomized intervention trial, hPTH( 1-34) remains an attractive candidate for future studies but cannot yet be embraced as an established treatment.

V. Experimental Approaches It can be seen from this discussion that standard therapies, such as estrogen, calcium, and exercise, are successful primarily in maintaining, rather than increasing bone. Agents that stimulate new bone formation are either problematic or still experimental, and effective strategies to increase bone mass in patients with osteoporosis remain elusive. Thus, there has been considerable interest in developing new approaches to increase bone mass. One area of great excitement has been to manipulate the components of bone remodeling to achieve optimal stimulation of bone formation. Frost (1981) and others described methods to exploit the separate components of the bone remodeling cycle in sequence. This approach, called ADFR (activate-depress-free-repeat) or “coherence” therapy, has great theoretical interest, but has been applied only casually to date. In these protocols, agents that increase the synchronous birthrate of active remodeling units. such as PTH, inorganic phosphorus, calcitriol, or triiodothyronine, are given for several days, followed by compounds like bisphosphonate or calcitonin that inhibit osteoclast function and diminish resorption. This “depression” phase is followed by a drug-free interval to permit completion of bone formation, and the cycle is repeated. It is hoped that the combination of stimulated remodeling and inhibited resorption will allow bone formation to generate more bone than had been previously removed. Encouraging results have been reported (Anderson

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et al., 1984; Mallette et al., 1989), but the ultimate utility of this approach remains uncertain. One major problem with coherence therapy is that the regimens employed have all been developed in a completely empirical manner. The dose and timing of remodeling initiators necessary to commit a coupled bone formation response are unknown. Whether early administration of antiresorptive drugs attenuates the osteoblastic response appears to be an important question, but no published information is available on this point. A variety of peptide hormones and biologicals show potential in treating osteoporosis. Parathyroid hormone has been discussed. Transforming growth factor p has not been used in treatment protocols, but is attractive since it inhibits bone resorption and increases bone formation. Recombinant human growth hormone has been shown to elevate circulating levels of osteocalcin (Marcus et al., 1990; Brixen er a f . , 1990), a marker of bone formation. Thus, growth hormone or its surrogate, somatomedin (IGF-I), might also be useful to increase bone mass.

VI. Nonpharmacological Considerations A number of hygienic measures may be of use in reducing the risk of fracture for osteoporotic patients. Proper instruction in lifting technique may reduce strain on the spine. Proper footware and installation of safety features around the home may minimize the risk of falling. Such features include bathroom safety rails and night-lights, rails and lighting for stairways, and elimination of floor clutter. In many counties in the United States, home safety inspections by public health nurses can easily be arranged.

VII. Conclusions Recent years have seen considerable progress in our understanding of the normal processes by which peak adult bone mass is acquired and maintained, the physical, nutritional, and hormonal factors which regulate bone mass throughout adult life, and the critical role of reproductive hormonal replacement for maintaining bone mass within the early years after menopause. Moreover, it is now apparent that loss of bone quantity is not sufficient to explain increased fracture risk. Bone fragility depends not only on its material properties, of which mineral content is an important component, but also on its microarchitecture and geometry. Age-related trabecular bone loss is manifest not simply as a global loss of bone mass, but is characterized by a loss of trabecular connectivity and increased porosity. Traditional treatment modalities include estrogen, calcium, and exercise. Each

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shows significant positive effects on bone mass, and estrogen, in particular, exerts a profound influence on fracture risk. However, none of these has been shown to restore bone mass of osteoporotic patients to normal levels, and it appears likely that their primary utility will be preventive maintenance. Bisphosphonates may be useful for managing established osteoporosis, but restoration of lost bone remains elusive. Dramatic gains in bone mass can be achieved with sodium fluoride, but serious questions concerning bone quality and drug toxicity limit the clinical utility of this agent. Modem technology offers the hope that further progress may come about through the development of growth factors and other osteotropic compounds for clinical use.

Acknowledgment The author is supported in part by the Research Service of the Department of Veterans Affairs.

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Recker, R. R . (1985). Calcium absorption and achlorhydria. N . Engl. J . M e d . 313, 70-73. Recker, R. R., and Heaney, R. P. (1985). The effect of milk supplements on calcium metabolism, bone metabolism and calcium balance. Am. J . Clin. Nurr. 41, 254-263. Recker, R. R., Saville, P. D., and Heaney, R. P. (1977). Effect ofestrogens andcalcium carbonate on bone loss in postmenopausal women. Ann. Intern. Med. 87, 649-655. Reeve, J., Meunier, P. J., Parsons, J. A., Bernat, M., Bijvoet, 0. L. M., Courpron, P., Edouard, C., Klenerman, L., Neer, R. M., Renier, J. C., Slovik, D., Visman, F. J., and Potts, J. T., Jr. ( 1980). Anabolic effect of human parathyroid hormone fragment (hPTH 1-34) therapy on trabecular bone in involutional osteoporosis: report of a multi-centre trial. B r . M e d . J . No. 280. 1340-1344. Reeve, 1.. Davies, U. M., Hesp, R., McNally, E., and Katz, D. (1990). Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride. Br. M e d . J . No. 301, 314-318. Reginster, J. Y.,Denis, D., Albert, A., Deroisy, R.. Lecart, M. P., Fontaine, M. A , , Lambelin, P., and Franchimont, P. (1987). I-Year controlled randomised trial of prevention of early postmenopausal bone loss by intranasal calcitonin. Lancer ii, 1481- 1483. Reginster, J. Y., Lecart, M. P., Deroisy, R., Sarlet, N . , Denis, D., Ethgen, D., Collette, J., and Franchimont, P. (1989). Prevention of postmenopausal bone loss by tiludronate. Lancet ii, 14691471. Ribot, C., Tremollieres, F., Pouilles. J. M., Louvet, J. P., and Guiraud, R. (1988). Influence of the menopause and aging on spinal density in French women. Bone Miner. 5 , 89-97. Riggs, B. L., Wahner, H. W., Dann, W. L., Mazess, R. B., and Offord, K. P. (1981). Differential changes in bone mineral density of the appendicular and axial skeleton with aging. J . Clin. Invest. 67, 328-335. Riggs, B. L., Wahner, H. W., Seeman, E., Offord, K . P., Dunn. W. L., Mazess, R. B., Johnson, K. A., and Melton, L. J., 111 (1982). Changes in bone mineral density of the proximal femur and spine with aging. Differences between the postmenopausal and senile osteoporosis syndromes. J . Clin. Invest. 70, 716-723. Riggs, 8.L., Wahner, H. W., Melton, L. I., 111, Richelson, L. S., Judd, H. L., and O’Fallon, W. M. (1987). Dietary calcium intake and rates of bone loss in women. J . Clin. Invesl. 80, 979-982. Riggs, B. L., Hodgson, S . , O’Fallon, W. M., Chao, E. Y. S., Wahner, H. W., Muhs, J. M., Cedel, S. L., and Melton, L. J., 111 (1990). Effect of fluoride treatment on the fracture rate in postmenopausal women with osteoporosis. N . Engl. J . M e d . 322, 802-809. Riis, B., Thomsen, K.. and Christianssen, C. (1987). Does calcium supplementation prevent postmenopausal bone loss? A double-blind, controlled clinical study. N . Engl. J . M e d . 316, 173177. Rosenthal, D. I . , Mayo-Smith, W., Hayes, C. W., Khurana, J. S., Biller, B., Neer, R. M., and Klibanski, A. (1989). Age and bone mass in premenopausal women. J . B o n e M i n e r . Res. 4,533538. Ross, P. D., Davis, J. W., Vogel, J. M., and Wasnich, R. D. (1990). A critical review of bone mass and the risk of fractures in osteoporosis. Culcif: Tissue Int. 46, 149-161. Ruff, C. B., and Hayes, W. C. (1988). Sex differences in age-related remodeling of the femur and tibia. J. Orthop. Res. 6, 886-896. Seeman, E., Hopper, J. L., Bach, L. A., Cooper, M. E . , Parkinson, E., McKay, J., and Jerums, G . (1989). Reduced bone mass in daughters of women with osteoporosis. N . Engl. J . M e d . 320,554558. Sheikh, M. S . , Santa Ana, C. A., Nicar, M. J., Schiller, L. R., and Fordtran, J. S. (1987). Gastrointestinal absorption of calcium from milk and calcium salts. N . EngI. J . M e d . 317, 532536. Shiraki. M., Orimo. H., Ito, H., Akiguchi. I . . Nakao, J., Takahashi, R., and Ishizuka, S. (1985). Long-term treatment of postmenopausal osteoporosis with active vitamin D3, I-alpha-hy-

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droxycholecalciferol ( laOHD3) and I ,24 dihydroxycholecalciferol ( I ,24(OH)2D3).Endocrinol. Jpn. 32, 305-315. Sinaki, M.. McPhee. M. C.. Hodgson, S . E . , Merritt, I. M.. and Offord, K. P. (1986). Relationship between bone mineral density of spine and strength of back extensors in healthy postmenopausal women. Mavo Clin. Proc. 61, 116-122. Slovik, D. M., Adams, J. S., Neer, R. M., Holick. M. F.. and Potts, J. T., Jr. (1981). Deficient production of I .25-dihydroxyvitamin D in elderly osteoporotic patients. N . EngI. J . Med. 305, 312-374. Slovik, D. M., Rosenthal. D. I . , Doppelt, S . H.. Potts. J . T., Jr., Daly, M. A . , Campbell, J. A , , and Neer, R. A. (1986). Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1.25-dihydroxyvitamin D. J . Bone Miner. Rrs. 1, 377-381. Snow-Harter, C., Bouxsein, M.. Lewis. B., Charette, S . , Weinstein, P., and Marcus, R. (1990). Muscle strength as a predictor of bone mineral density in young women. J Bone Miner. Res. 5, 589-595. Stevenson, J. C.. Cust, M. P., Gangar, K. E . , Hillard, T. C., Lees, B . , and Whitehead, M. I. (1990). Effects of transdermal versus oral hormone rcplacenient therapy on bone density in spine and proximal femur in postmenopausal women. Luncet ii, 265-269. Storni. T., Thamsborg, G . . Steiniche, T., Genant, H. K., and Sorenson, 0. H. (1990). Effect of cyclical etidronate therapy on bone mass and fracture rate in women with postmenopausal osteoporosis. N . E q / . J . Mud. 322, 1265-1271. Tam, C. S . , Heersche, J. N. M.. Murray, T. M.. and Parsons, J. A. (1982). Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration. Endocrinology (Baltimore) 110, 506-5 12. Tsai, K . 4 . . Heath, H.. 111, Kumar. R., and Riggs. B. L. (1984). Impaired vitamin D metabolism with aging in women. Possible role in pathogenesis of senile osteoporosis. J . Clin. Invest. 73, 1668- 1672. Watts. N . B., Harris, S . T.. Genant, H. K., Wasnich. R . D., Miller, P. D.. Jackson. R . D., Licata, A . A , , Ross. P.. Woodson, G. C., Yanover, M. J.. Mysiw. W. J., Kohse, L., Rao, M . B., Steiger, P., Richmond, B.. and Chesnut, C. H.. 111 (1990). Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. N . Engl. J . Med. 323, 73-79. Webb, A. R.. Kline. L., and Holick. M F. (1988). Influence of season and latitude on the cutaneous synthesis of vitamin D3: response to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. J. Clin. Endocrinol. Metab. 67, 373-378. Weinstein, R. S . . and Hutson, M. S . (1987). Decreased trabecular width and increased trabecular spacing contribute to bone loss with aging. Bone 8, 137-142. Weiss. N . S . . Ure, C. L., Ballard, J. H.. Williams. A . R . , and Daling, J . R. (1980). Decreased risk of fractures of the hip and lower forearm with postmenopausal use of estrogen. N. Etigl. J . Med. 303, 1195-1 198. Wilson, R. 1.. Rao, S.. Ellis, B . , Kleerekoper, M., and Parfitt, A . M. (1988). Mild asymptomatic primary hyperparathyroidisni is not a risk factor for vertebral fractures. Ann. Intern. Med. 109, 959-962.

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Molecular Asymmetry and Its Pharmacological Consequences Kenneth M. Williams Department of Clinical Pharmacology and Toxicology St. Vincent’s Hospital Sydney 2010, Australia and School of Physiology and Pharmacology University of New South Wales Sydney 2033, Australia

1. Introduction Background and Nomenclature 11. Pharmacokinetic Consequences of Chirality A. Concepts B. Pharmacokinetics of Enantiomers 111. Chirality and Polymorphic Drug Disposition A . The Sparteine-Debrisoquin Phenotype B. The Mephenytoin Phenotype IV. Pharmacodynamic Consequences of Chirality A. Our Asymmetric Senses B. Receptors, Acceptors, and Chiral Discrimination C. Immunological Enantioselectivity D. Timolol and the P-Receptors V. Enantiomer-Enantiomer Interactions A. Protein and Tissue Binding B . Metabolic Interactions C. Pharmacodynamic Interactions VI. Enantioselective Drug-Enantiomer Interactions VII. Enantiomeric Impurity and Potential Problems VI11. Enantiomers as Biochemical Probes IX. Therapeutic and Regulatory Considerations A. Nonracemic Drugs B . Achiral Drugs C. Racemic Drugs D. Enantiomeric Drugs and the Considerations E. Racemate or Enantiomer? The Choice F. Bioavailability Studies X . Therapeutic Drug Monitoring XI. Conclusions References

Advancer m Pharmacolo~).Volume 22

Copyright 0 1991 by Academic

Press, Inc All nghts of rcproducllon In any form reserved

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Kenneth M. Williams

1. Introduction The chirality (handedness) of drugs and the consequences of this molecular asymmetry are among the most important basic concepts underlying drug disposition and action. It is thus a subject particularly suited to the editorial thrust of this series which is directed at the science that underlies pharmacology and therapeutics. It is a topic which has received increasing attention and there are a number of other reviews which can be read in conjunction with this article (Jenner and Testa, 1973, 1980; Ariens et al., 1983; Smith, 1984, 1989a; Simonyi, 1984; Williams and Lee, 1985; Ariens, 1986a,b; Drayer, 1986; Vermeulen, 1986; Mason, 1986, 1989; Testa and Mayer, 1988; Eichelbaum, 1988; Wainer and Drayer, 1988; Jamali et al., 1989). The following overview is selective and is based primarily on human data. It aims to develop some of the concepts which are basic to appreciating the significance of chirality for both pharmacokinetics and pharmacodynamics and the clinical consequences of enantioselective drug disposition. Selected illustrations are given, with the intent to emphasize that in many respects enantiomers frequently are distinct pharmacological entities, being more different from each other than drugs belonging to the same chemical or pharmacological class. This fact suggests, therefore, that, of all properties, chirality should not be ignored in the study of the phannacokinetics and pharmacodynamics of a drug, especially when that chiral drug is administered, as is frequently the case, as the racemic mixture, i.e., as an equal mixture of the enantiomers.

Background and Nomenclature The basic biochemical milieu is asymmetric presumably because “chiral homogeneity is the precondition for an economic and efficient metabolic turnover and biosynthesis in stereochemical ‘lock and key’ terms, like the universal adoption of right handed nuts and bolts or screws in the engineering industry for efficiency and economy of operation.” (Mason, 1988). Consequently, biological macromolecules, whether plasma proteins or proteins involved in the active transpott of substrates, enzymes, or neuronal receptors, frequently can distinguish between enantiomerically related drugs. The principle of chirality was one most elegantly studied by Pasteur in the nineteenth century (Geison and Secord, 1988) and followed the observations of another French scientist, Biot, that salts of tartaric acid, which were apparently identical by chemical analysis, were different in one respect, namely, in their effects on the plane of polarized light. Pasteur solved this paradox, demonstrating that the optically inactive salt of paratartaric acid (racemic acid, hence the term racemic), could be separated into two optical isomers (enantiomers), the “natural” form, which rotated the plane of polarized light to the right (dex-

59

Molecular Asymmetry

or +), and a previously unknown form, which rotated the light in an equal but opposite direction (fevorotatory, I, or -). The molecular basis for this asymmetry was described by van? Hoff, who recognized that when four different groups were attached to a tetravalent carbon (the most common form of molecular asymmetry), a pair of molecules which were mirror-image related was possible. The main contributors to the development of the concepts of the asymmetry of pharmacodynamic response are listed in Table I. Pasteur’s studies on the tartrates, including their optical resolution and the observation that a mold which grew effectively when fed a solution of the (-)-tartrate effectively stopped growing at 50% consumption of the tartrate if the racemic drug were the energy source, were the basis for his important conclusion that life was dependent on molecular asymmetry. “Molecular asymmetry . . . forms perhaps the only sharply defined boundary which can be drawn at the present time between the chemistry of dead and that of living matter.” This unique quality of life and the reasons for this quality have been the focus of much interest. Pasteur ( 1886) also described a clear difference in the pharmacodynamic response elicited by enantiomers, noting that there were two asparagines, one sweet and one insipid or tasteless. Importantly, he attributed this difference to an optically active substance in the “gustatory” nerve. Cushney ( 1908) demonstrated important differences in the pressor activities of (-)-adrenaline and (+)-adrenaline. The latter enantiomer was inactive and the racemate had half the activity of the natural (-)-enantiomer. He took this to indicate that the “receptive bodies” were themselves “optically active,” in keeping with Pasteur’s comments on the taste of the asparagines and from observations in chemistry that a chiral substance is required to differentiate between pairs trorotatory, d,

Table I Brief Historical Overview of the Development of the Concepts of the Asymmetry of Pharmacodynamic Response” ~~

Contributor

Year

Substance

Pasteur Cushney Chen Eason and Stcdnian Euler

I886 1908 1929 1933 I949

Asparagme Adrenaline, atropine Ephednnes Phenethylarnines Noradrenaline

“For further reading, refer to Drayer ( I 98th). from which this table was adapted.

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Kenneth M. Williams

of enantiomers. By 1925, significant data had begun to accumulate on the relative activities and disposition of enantiomers (Cushney, 1926). However, there was still confusion as to the significance of optical activity per se for pharmacological activity. Studies such as those of Chen et al. (1929), who used the stereoisomers of ephedrine to establish structure-activity relationships, were the basis for the important contribution of Easson and Stedman (1933). Easson and Stedman (1933) introduced the concept of the three-point interaction between a chiral drug and a receptor. Thus, they postulated that the maximum interaction of an enantiomer with a receptor surface at its binding sites B', C', and D' (IV; Fig. 1) can only occur for one of the enantiomeric pair (I). The distomer (11) can interact at only two sites. If asymmetry is lost, for example, by changing A of the eutomer (I) to B, then the now symmetrical substrate (HI), would not be different in activity to I. This concept was investigated by a study of adrenaline, where the important functional groups for receptor recognition of the eutomer, (-)-adrenaline, were the catechol (B), the hydroxyl function (C), and the nitrogen (D). Consistent with their hypothesis, the desoxy analog (3,4-dihydroxy-P-phenylethylmethylamine, epinine) had pressor activity not distinguishable from (+)-adrenaline. They made the important clarification that molecular asymmetry and the associated optical activity are without direct influence on the magnitude of the physiological activity of a drug, i.e., that molecular dissymmetry and structure should not be considered as separate characteristics.

A

A

I

B

I11

c IV Fig. 1 Easson-Stedman (1933) hypothesis. Illustrating the three-point attachment hypothesis for the binding of enantiomeric substrates (I, 11) and the nonasymmetric substrate (111) to the active sites (B', C', D') of the receptor surface. It was thus predicted that I11 and I would bind similarly to the receptor even though Ill was not asymmetric. (Reprinted with permission of the copyright holder, Portland Scientific Press, London.)

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Molecular Asymmetry

The three-point interaction which they studied has been extended to other direct agonists of adrenergic receptor subtypes (Patil et al., 1967; Ruffolo, 1983) but does not explain completely the action of some adrenoceptor agonists (Rice et al., 1987). On a final historical note, a mention should be given to the contributions of von Euler (1949), who established that the transmitter of sympathetic action was (-)-noradrenaline (rather than adrenaline), which was twice as active as racemic adrenaline. The configuration of an enantiomer in space cannot be determined from the direction in which it rotates polarized light. Rotation is a variable which can be altered by enantiomer concentration, pH, wavelength, and temperature. The absolute configuration was once assigned by relating the structure back to one of two reference compounds, serine, whose natural enantiomer was designated L , or glyceraldehyde, whose natural configuration was designated D. While this system was of use in describing the amino acids and sugars, it was not of sufficiently broad applicability. The system of Cahn et af. (1956) has been adopted. Using a series of sequence rules, each of the groups attached to a chiral carbon are given a priority (Fig. 2), the simplest rule being that the atom of highest atomic number takes the highest priority. The molecule is viewed with the atom of lowest priority distal to the viewer. If the order from highest to lowest priority is clockwise, the molecule is designated recfus (R),otherwise it is sinister (S). These examples illustrate several other important points. The naturally occurring enantiomers of alanine and cysteine are both designated L but, because of the sequence rules, alanine has the ( R ) and cysteine has the ( S ) configuration. The illustration also serves to make the important distinction between L and 1 or (+), which are not synonymous as is not uncommonly suggested in the literature. Enantiomers are described in this review by the descriptors ( R ) and (S), or (+) and (-) when the absolute configuration is unknown. The further examples

2

HOOC H 2 ~ - ? f . m l4H 3

3

HOOC H2k+*81H 4 2

CH3

(+),(L),(S)-alanine

CH2SH

(+),(L),(R)-cystelne

Fig. 2 The Cahn/lngold/Prelog (1956) nomenclature for absolute configuration. Each group attached to the asymmetric carbon (*) is assigned a decreasing priority according to a set of rules (Sequence Rules), The molecule is viewed such that the lowest priority moiety is distal to the observer. If the direction of rotation on moving from highest to lowest priority is anticlockwise, as for alanine, the molecule is designated “sinister” (S);if clockwise (cysteine), the designation is “rectus” (R).

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Kenneth M. Williams

(R)-haloxyfop

(S)- ib u prof e n

Fig. 3 The topographical similarity of the active enantiomers of the herbicide haloxyfop and the anti-inflammatory drug ibuprofen are illustrated. Despite their conformational similarity, the Sequence Rules assign the R and S configurations, respectively.

(Fig. 3) of the active enantiomers of the herbicide haloxyfop and the antiinflammatory drug ibuprofen, also emphasize that absolute configurations may be the same, despite opposite designations by the Sequence Rules. It should be noted that differences between the activities of enantiomers can vary both qualitatively and quantitatively. The more active enantiomer in one selected situation may be less active in another. Useful terminology to describe the activity of an enantiomer in eliciting a specific pharmacological response are the descriptors eutomer (more active) and distomer (less active) (Lehmann, 1982). Thus, (S)-ibuprofen is the eutomer and the (R)-enantiomer the distomer for inhibition of prostaglandin synthesis. However, in terms of effects on lipid biochemistries, @)-ibuprofen is the eutomer (Section II,B,3 ,c). A few further remarks on nomenclature are necessary. Enantiomers belong to the class of stereoisomers which are isomers where the constituent atoms are identically connected. If the molecules do not have a mirror-image relationship, they are termed diastereoisomers or diastereomers. Importantly, enantiomers have identical physical characteristics, while diastereomers are chemically distinct and can be separated by achiral techniques. This often becomes of importance when the molecule has more than one chiral center, such as labetalol. In this instance, if the molecule is inverted at one or more of the chiral centers, but not at all centers, the product is no longer enantiomerically related to its stereoisomer. A naturally occurring example of such “imperfectly isomeric bodies,” as Cushney (1926) has described them, is that of quinine and quinidine, stereoisomers which are still occasionally described as being enantiomerically related (Block e?a/., 1988), even when specifically chosen for the study of structureactivity relationships (Hill et d.,1988). Quinine (8S,9R)and quinidine (8R,9S) are in fact mirror images at the 8 and 9 chiral carbons but share the same

Molecular Asymmetry

63

qulnlne

quinidine

Fig. 4 The diastereomerically related drugs quinine (3R,4S,8S,9R) and quinidine (3R,4S,8R,9S). These drugs are not enantiomers.

stereochemistry at the other two asymmetric centers (3R,4S; Fig. 4). Consequently, physical characteristics of quinine and quinidine are different (Table 11). In particular, their solubilities in organic solvents and, therefore, their lipid solubilities are dissimilar. Differences in the pharmacokinetics of these diastereoisomers have been described (Notterman et al., 1986) and their pharmacodynamic actions are also dissimilar. Table / I Physicochemical Parameters for Quinine and Quinidine” Value

Molecular weight Melting point Optical rotation Solubility Water Alcohol Ether Benzene Chloroform Specific gravity PK I PK2

324.41 177°C - 1 l7”b

324.41 174-175°C + I92”<.

1/1900 110.8 114 (water saturated) 1/80 (slightly soluble)

I12000 1/36 1.56 - (soluble)

u1.2

lil.6

1.625 5.4 (20°C) 10.0

1.625 5.07 (18°C) 9.1

“Data from “CRC Handbook of Chemistry and Physics,” 53rd Ed. (1972-1973), Chem. Rubber Publ. Co., Cleveland, Ohio; “Martindale.” 26th Ed. (1973) Pharm. Press, London; and “The Merck Index,” 10th Ed.(1983), Merck, Rahway, New Jersey. h[a]l; chloroform, c = 1 5 g/cc. “[a]: chloroform. c = 1.5 gicc (calculated from data for c = 1.8 g/cc).

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Kenneth M. Willioms

The above discussion, while introducing some nomenclature and historical perspective, has aimed to emphasize that, while enantiomers are chemically equivalent, biochemically they are inequivalent. This inequivalence can be absolute. Such actions or metabolic disposition can then be termed stereospecijic or enantiospecijic. If not absolute, the differences are enantioselective or stereoselective. If, in the context of the discussion, one enantiomer makes a negligible contribution to the action or interaction, this will be taken to be enantiospecific.

11. Pharmacokinetic Consequences of Chirality A. Concepts A classification of potential chiral events in the disposition of a drug which helps to give an overview of the important issues is that formulated by Testa (1989). The three categories of interaction are penetration, recognition, and activation. The drug must first partition between body compartments and ultimately reach the relevant receptor. This process can be described as drug penetration. This distribution of drug will be controlled primarily by physicochemical characteristics of the molecule such as lipophilicity. As such, the effects of chirality on these processes generally are expected to be minor, although the molecules will partition across membranes which have an asymmetric component. Consistent with the ovemding achiral factors, there is no evidence for enantioselective absorption from the gastrointestinal tract for passively absorbed drugs, although this subject has received little attention. In contrast, active transport involves a recognition or affinity of a chiral carrier protein for the drug and it is expected that there may be enantioselective differences in processes such as active absorption, active cellular uptake and receptor storage, and renal secretion. There is even evidence of enantioselective secretion of tocainide into saliva (Pillai et al., 1984). The absolute penetration, reflected by the total amount of drug which partitions across the biological boundaries, may be influenced by additional factors. Thus, while there may be active absorption resulting in differences in the rate of transport of enantiomers across the gastrointestinal epithelium, the total absorption, a reflection of both active and passive components, may not be enantioselective. Furthermore, the absolute degree of partitioning of drug across membranes, such as the distribution of drug between blood and synovial fluid (Fig. 5 ) (Day et al., 1988a), will be limited by the relative albumin concentrations in the various Compartments for those drugs bound significantly to this protein. The enantioselectivity of the albumin binding to this protein will determine the pharmaco-

65

Molecular Asymmetry (8

5

-

. -

-

-----a

E

---__----_ 9

91

5

z 0 I-

d z g

1.8

-

8.5

-

I-

z

8

t 8.I

1

1

1

0

2

4

6

1

I

e

18

1

12

TIME (hr)

Fig. 5 The log concentration-time profiles of (S)-ibuprofen (m) and (R)-ibuprofen (0)in plasma, and the S (0)and R (0) enantiomen in synovial fluid. The patient was being treated with 400 mg (R,S)-ibuprofen 8 hourly and was given 800 mg as the test dose at time 0 (Day el a l . , 1988a).

logically more important unbound concentrations available for interaction at the target receptors. For the binding of a substrate to a metabolizing enzyme, chiral recognition can occur at the binding step and/or the catalytic step. This will be reflected by differences in the affinity between the substrate and the macromolecule and/or in the activation which results from the binding interaction. The interaction between the binding site of an enzyme or a neural receptor and the enantiomer will also involve recognition. The differential formation of the diastereomerically related complexes is reflected by differences in the K,s and Kds, respectively. Some Michaelis-Menten constants for enantiomeric pairs have been tabulated by Testa and Mayer (1988). A few more examples (Table 111) illustrate varying degrees of enantioselective recognition (K,s). However, even if there is equal recognition of the enantiomen by the macromolecule, as evidenced by complex formation (equal K,s or Kds), the interactions may not be equally productive, i.e., no activation may be elicited. Consequently, either the enzymatic transformation will proceed at a different rate for each of the enantiomers, as reflected by a difference in V,,, (Testa and Mayer, 1988) (Table 111). or there will be a difference in response at the neural receptor, as evidenced by a difference in potency. Many examples of both of these phe-

66

Kenneth M. Williams

Toble 111 Michaelis-Menten Parameters for Some Examples of Enantioselective Metabolism

K", (CWM)

Vmax

RatelK,,,

1 '-Hydroxylation of bufuralol (human liver microsomes) 12.0 2 4.8 I, 0.67 (+)-EMSO 17.9 ? 6.30 (-)-EMS 5.6 t 1.5 (+ )-PMs 118 -r- 85 7.5 t 2.0 0.06

Reference

Dayer er a / . (1987)

(-)-PMs 4.7 ? 0.9 7-Hydroxylation of warfarin (Sprague-Dawley rat liver microsomes) R 46 ? 24 1.65 ? 0.11' 0.035 Pohl er a / . (1976) 0.46 2 0.01 0.009 S 50 ? 10 Glucuronidation of propranolol (dog liver rnicrosornes) R 1000 t 100 6.4 2 0.4d 0.006 Wilson and Thompson (1984) 25 t I 0.015 S' 1700 ? 100 N-Demethylation of ketamine (rat liver microsomes) (+) 256 25 468 t 306 1.83 Marietta et a / . (1977) 292 2 12 1 .n5 (-) 158 L 25 I36 3 3 0 ? 10 (+Ye) 1772 20

*

OEMs, Extensive metabolizers; PMs, poor metabolizers bnrnol / mg/hr. cnrnol/mg/IO min. dnrnol/rng/min. '(S) was a noncompetitive inhibitor of the glucuronidation of ( R ) with a Ki of 1800 )LM.

nomena exist for enantiomenc pairs. The data also indicate that there may be interactions between the enantiomers, such as for the metabolism of ketamine and propranolol (Section V). The enantioselective recognition resulting in a differential rate of metabolism for an enantiomeric pair has been termed substrare enantioselectivity. It is this form of enantioselectivity which is the focus of this review because it deals with the disposition of chiral drugs. Substrate enantioselectivity is to be distinguished from product enantioselectivity and is the differential formation of enantiomeric metabolites from a common prochiral substrate. Very detailed discussions have been presented on the concepts of product and substrate enantioselectivity and the reader should refer to these for further discussion (Testa and Mayer, 1988; Jenner and Testa, 1973, 1980). After consideration of each of these steps, it is not surprising then that there are pharmacodynamic and pharmacokinetic differences between enantiomers. These considerations form the basis for the following discussion on the molecular asymmetry of drugs and its pharmacological consequences.

Molecular Asymmetry

67

B. Pharmacokinetics of Enantiomers The impact of asymmetry on the pharmacokinetics of drugs is most readily reviewed by considering the normal contributions to the overall pharmacokinetics by the processes of drug absorption, distribution, and elimination, including metabolic elimination and renal excretion.

1 . Drug Absorption Most drugs are absorbed across the intestinal epithelium entirely by passive mechanisms following oral drug ingestion. The primary determinants of drug absorption are the concentration gradient and the solubility of the drug in the lipid bilayer. As preempted under the discussion of the underlying concepts, this membrane barrier has, however, a chiral component, viz. the phospholipids. It is evident, however, from the limited data so far available on the enantioselective absorption of drugs, that the importance of the lipophilicity of a drug far exceeds the effect of chiral interactions. This is supported by recent data demonstrating the surprisingly little chiral discriminatory capacity of phospholipids (Arnett ef a / . , 1988). In contrast, for those drugs for which active or facilitated transport is a significant component of the absorptive process, chiral recognition is expected to be demonstrated by enantioselective differences in absorption. There appear to be only two drugs for which active transport is an important determinant of the absorptive process. These are dopa (Wade et al., 1973) and methotrexate (Hendel and Brodthagen, 1984). There are differences between these examples which are of interest. Dopa is both actively and passively absorbed, i.e., there are differences in the active component for the enantiomers but no difference in the passive component. The overall effect is that, while the rates of absorption of the enantiomers are different (Wade et al., 1973), the total absorption of the enantiomers is probabiy the same (Williams and Lee, 1985). The absorption of orally administered D-methotrexate has been reported to be less than 3% of the availability of the L-enantiomer, the form used clinically. This conclusion was based on data obtained by comparing the relative area under the concentration-time curves (AUCs) following oral administration of 10 mg/kg of the separate enantiomers to volunteers (Hendel and Brodthagen, 1984). In addition, the 24-hour urinary recovery of D-methotrexate was less than 3% of the dose. In contrast, there was 100% recovery of the L-enantiomer, whose absorption appears to be entirely dependent on active processes. It is interesting that there is so little passive absorption of the drug in this instance. Paradoxically, studies in a dog purport to demonstrate both rapid absorption and elimination of D-methotrexate in this species (Cramer e t a / ., 1984). However, the urinary recov-

68

Kenneth M. Williams

ery data suggest very poor absorption of the D-enantiomer in this species also. Indeed, even after intraperitoneal administration to mice in this same study, there appeared to be significantly reduced absorption of D-methotrexate compared with the L-enantiomer. D-Methotrexate, while not as potent as the L-enantiomer, has been reported to be a good inhibitor of dihydrofolate reductase (Cramer et al., 1984; Lee et al., 1974). The above discussion suggests, therefore, that the reason the D-enantiomer has been reported to be a poor inhibitor of cell growth (Cramer et al., 1984) is because of inadequate penetration through cellular boundaries. Additionally, it is appropriate to mention here that an important aspect of the prolonged inhibition of thymidylate by L-methotrexate is the ability of this enantiomer to form polyglutamates (McGuire and Bertino, 1981; Jolivet and Chabner, 1983). D-Methotrexate is not a substrate for folylpolyglutamate synthetase (McGuire and Bertino, 1981), another reason for its lesser in vivo activity. The very interesting literature on the stereoselective absorption of the leucovorin isomers should be read in the context of this discussion (Weir et al., 1973; Sirotnak et al., 1979; Straw et al., 1984; Bertrand and Jolivet, 1988). This subject is not discussed here because the isomers which have been studied are diastereomers rather than enantiomers.

2. Tissue and Protein Binding The binding of enantiomers to plasma proteins can occur enantioselectively. This is a reflection of binding to acceptor sites rather than receptor sites, i.e., binding which does not precipitate a pharmacological response (Laduron, 1984). However, such binding may determine the relative concentrations of the enantiomers which are available for interaction with receptors or the concentrations available for metabolic modification. Similar enantioselectivity may occur for tissue binding. Cellular uptake and storage may also demonstrate chiral discrimination, as these processes involve interactions with chiral transport proteins. a. Tissue Binding Tissue distribution of chiral drugs may be determined by enantioselectivity in uptake and storage mechanisms as it is with chiral neurotransmitters such as noradrenaline (Patil et al., 1975); atenolol, whose uptake and storage in neurosecretory cells, as well as its eventual secretion as a result of membrane depolarization occurred preferentially for the (-)-enantiomer (Bagwell et al., 1989); and amphetamine, for which there was enantioselective storage of the (+)-enantiomer and of the slowly eliminated metabolites (+)-a-methyl-p-tyramine and (+)-a-methyl-p-octopamine in striatal aminergic neurons in rats (Dougan et al., 1986). @-Blockerssuch as propranolol (Bai et al., 1983; Kawashima er al., 1976) and timolol (Tocco et al., 1976) were enantioselectively taken up into extravascular tissues, presumed to be a reflection of the enantioselective binding of the eutomers to the P-receptors. Interestingly, the

69

Molecular Asymmetry

binding of (S)-timolol to melanin in the iris is not affected by (S)-propranolol or by (R)-propranolol (Aula et al., 1988), suggesting that timolol is not bound to P,-receptors in this tissue. Unfortunately the binding of (R)-timolol was not studied. In addition to reversible distribution of drug into tissue compartments, whether by simple partitioning or by binding to specific receptors, there is also the interesting incorporation of drug into lipids, leading to long-lasting drug stores (Fig. 6). This can apparently occur for a broad spectrum of xenobiotics whose common structural feature is that they are carboxylic acids (Fears, 1985). The drug replaces one (or more) of the endogenous fatty acids to form a hybrid lipid. To date, this has only been demonstrated for triglycerides. The stereochemical focus has been on the 2-arylpropionates where the (R)-enantiomers are enantiospecifically incorporated into lipids (Williams et al., 1986; Sallustio et al., 1988). This subject is taken up in later discussion of chiral inversion and its consequences (Section II,B,3,c). b. Protein Binding The chiral interactions between a drug and the plasma proteins are of low energy and reversible (Alebic-Koibah et al., 1979). These associations are to be clearly distinguished from covalent binding via acylation reactions, which are discussed in detail in Section IV,C. Albumin and other plasma proteins were once considered to be nondiscriminatory for enantiomeric pairs. That this is not the case is evidenced by the now commercially available chiral columns whose stationary phases are the biomacromolecules albumin and

0

100

200

T i m (hrr) and adipose Fig. 6 Contrasting elimination rates for ['4C]-(RS)-ibuprofen from plasma (0) tissue ( 0 )in rats after cessation of chronic dosing (27 days, 20 mgikg, twice daily, i.p.). The slow elimination from fat is a reflection of covalent binding of drug as hybrid triglycerides. [Data adapted from Adam et nl. (1969) and reproduced from Williams (1990a). Reprinted with permission of the copyright holder, Akadkmiai Kiad8, Budapest.]

70

Kenneth M. Williams

,

a -acid glycoprotein. Many examples of enantioselective plasma protein binding have now been reported (Simonyi et al., 1986), although it is difficult to summarize and compare the quantitative data because of the diverse manner in which such data have been published (Table IV). As for most pharmacological events, species variability in binding is evident (Schmidt and Jahnchen, 1978). Interestingly, the individual plasma proteins may demonstrate reversed enantioselective binding for particular enantiomeric pairs. Thus, while (-)-propranolol binds more avidly to a ,-acid glycoprotein, the (+)-enantiomer prefers human serum albumin (Walle et al., 1983; Albani et al., 1984). In contrast, the (S)-enantiomer of disopyramide is bound more avidly by both a,-acid glycoprotein and human plasma (Lima et al., 1984). Although the plasma protein binding is enantioselective, it does not necessarily follow that binding to all tissues will also be enantioselective. Thus, while the interactions of propranolol with plasma proteins and cardiac tissue are enantioselective, accumulation of propranolol in red blood cells is not enantioselective, at least in the overall sense (Walle et a l ., 1983). It must be concluded that, while there may be interesting similarities between the binding of, for example, P-antagonists and agonists to a,-acid glycoprotein and to the P-adrenergic receptor (Sager et al., 1985), in general, plasma binding cannot be used as a model to predict the enantioselectivity of interactions with other macromolecules or specific receptors. The enantioselectivity of binding is not an invariant factor and the ratio of binding may change depending on the relative enantiomer concentrations or on factors such as pH. While there are clearly and not unexpectedly enantioselective preferences for enantiomers to bind to plasma proteins, which may be considered to be “pharmacologically inert” sites (Alebic-Kolbah et al., 1979), the clinical consequences of this enantioselectivity are not so evident. Factors such as age and renal or hepatic dysfunction or disease states such as rheumatoid arthritis may lead to changes in the protein binding of drugs. Although this area has received little attention before now, it has recently been reported that there is

Table IV Examples of Enantioselective Binding to Human Plasma Proteins Ibuprofen Flurbiprofen Tocainide Ketoprofen Lorazepam Oxazepam Gliflumide Chloroquine Phenprocoumon

Evans et al. (1989) Knadler et al. (1989) Sedman ef al. (1982) Rendic et a / . (1979) Fitos et nl. (1986) Muller and Woliert (1975) Schillinger er al. (1978) Ofori-Adjei er al. (1986) Brown et al. (1977)

Verapamil Propranolol

Disopyramide Warfarin Alprenolol

Gross et a / . (1988) Walk et al. (1983) Colangelo rt al. ( 1989) Albani et a / . (1984) Oravcova ef a / . (1989) Lima et at. ( 1984) Sellers and Koch-Weser (1975) Yacobi and Levi ( I 977) Sager et a / . (1985)

Molecular Asymmetry

71

decreased binding of (R)-flurbiprofen in uremics, an effect not observed for the eutomer, (S)-flurbiprofen (Knadler er a/. , 1989). The only study yet to consider the effect of age found no difference in the enantioselectivity of plasma protein binding for the enantiomers of propranolol in the elderly (Colangelo er al., 1989). Differences in protein binding between pairs of enantiomers will be manifested most clearly for drugs which have a low intrinsic clearance, such as the coumarin anticoagulants and the nonsteroidal anti-inflammatory drugs, because, in these cases, glomerular filtration and the total clearance are directly proportional to the unbound fraction of the enantiomer. For the same reason, interactions with other drugs are more likely to be recognized for low-clearance drugs. Some very interesting data relate to allosteric interactions with the warfarin enantiomers in their binding to albumin. This subject is taken up in later discussion (Section VI). For drugs with a high intrinsic clearance, such as propranolol and verapamil, which also are often only moderately bound to plasma proteins (Gross er al., 1988), enantioselective effects on total pharmacokinetics may be less evident. With no effect on total clearance being expected, but with potential differences in the volume of distribution, enantioselective binding may be demonstrated by differences in half-life, and therefore, time to steady state. Pharmacodynamic responses may be affected, however, because there will be differences in the free concentrations of the enantiomers. While not wishing to discount the importance of such issues to the basic understanding of the mechanisms of drug action and disposition, the real therapeutic consequences of these factors are unknown. Interestingly, the binding of the enantiomers of verapamil are dependent on the route of drug administration. The free fractions were doubled after oral administration as compared to intravenous administration (Gross et al., 1988). This was attributed to displacement of the enantiomers from plasma protein sites by metabolites, a hypothesis which deserves further investigation. The unbound concentrations of the enantiomers ought to be considered when estimating the relative potencies of enantiomers from concentration-response data. Thus, the intrinsic activity of (S)-warfarin was estimated to be 3-4 times that of (/?)-warfarin (O’Reilly, 1974; Wingard and Levy, 1977), but when one considers the unbound drug, the (S)-enantiomer is approximately 8 times more active than (R)-warfarin (Toon and Trager, 1984). An interesting feature of the enantioselective binding of the enantiomers of propranolol is that the (-)-enantiomer had saturable binding, but the ( +)-enantiomer was not saturable even at molar drug/protein ratios of up to 50 (Oravcova el al., 1989). These data are of interest because they suggested that there was a nonspecific binding component for the ( +)-enantiomer but not the (-)-enantiomer. Additionally, there was the very interesting observation based on the circular dichroism spectra that there were different conformational states of the protein depending on which enantiomer was binding to it. While not studied, one

Kenneth M. Williams

72

MeEt

-

:-o elut M e 0 Me0

Conformatlon A

MMe0 OMe

e

O

-

Me0

Vt i OMe

Conformation B

Fig. 7 (S)-Tofisopam adopts two conformations in solution, the major conformation (A) [(-)(S)-tofisopam] and a lesser conformation (B) [( +)-(S)-tofisopam]. Interestingly, these conformations have rotations of opposite signs under the experimental conditions. The conformer A is much more avidly bound to human semm albumin and is the major conformer in the solid state. (From Simonyi and Fitos, 1983. Reprinted with permission of the authors and the copyright holder, Pergamon Press, Oxford.)

could surmise on this basis that there may be an enantiomer-enantiomer protein binding interaction for the propranolol enantiomers. Examples of such interactions are taken up in later discussion. Finally, it is not only the conformation of the protein which may be important for the chiral interaction but also the conformation of the individual enantiomers. Thus, studies with tofisopam have demonstrated there is approximately a 7-fold difference between the association constants for the major and minor conformers of the (S)-enantiomer (Fig. 7) (Simonyi and Fitos, 1983; an interesting observation was the change in rotation associated with the change in conformation).

3 . Clearance Enantioselectivity may be displayed in the clearance of drugs, whether this is by direct renal excretion or secretion or whether by metabolism. The following discussion deals with enantioselectivity as displayed by the pharmacokinetic parameters of drug disposition. a. Total Body Clearance Total body clearance is a gross and, therefore, not necessarily a sensitive measure of enantioselective drug disposition. Consequently, while enantioselective disposition may be evident in the pharmacokinetic parameters determined from plasma data, it cannot be inferred that the absence of a difference means that there is not enantioselective disposition. Thus, the kinetic parameters for the enantiomers of ibuprofen are very similar, as are those for tiaprofenic acid enantiomers (Table V). However, a consideration of specific metabolic pathways has shown that there is highly enantioselective chiral inversion of @)-ibuprofen but not of (S)-ibuprofen nor of ( R ) - or (S)-tiaprofenic acid (Section II,B ,3,c). In contrast, there are large differences between the clearances of other enantiomeric pairs such as verapamil and mephenytoin. Large differences in clearance will account for important differences in in vivo response. Consequently, (S)-nicoumalone has a more potent effect on blood

73

Moleculor Asymmetry

Table V Pharmacokinetic Parameters for Some Representative Drug Enantiomers in Humans

Drug Anti-inflammatory Ibuprofen

Chirality

R S

Tiaprofenic acid'

Rb Sb R S

lndoprofen

R S

R SC

Cardiac Disopyramide (i.v.)

S

Ill

Ill 83 127 4085 2456 438 486 1210 1030 990 850 I000 2500 490 650 104 185 1720 7460

Rc R em S

R Pm S Propran~lol~ (i .v. )

Perhexiline Mexiletine' TocainideC (i.v.1 Verapamil' Anticoagulants Acenocoumarol Nicoumalone' Phenprocoumon Warfarin

68 74 16.2 11.5 45 47 72 41 41 49 32

R S C

Metoprololc

CLU (mllmin)

(+) (-)

35 496 24 225 0.62 0.46 3.5 4.9 2.6

V

1112

(liter)

(hr)

9.9 10.5 -

2.0 1.7 2.7 3. I 2.3c 2.8< 2.9 3. I 3.4

-

10.5 10.7 12.6 9.0 48 50 33 60 4.8" 4. I d 422 358 134 I36 32 38 14.8 30.5 0. I54 0.160 0.14

5.2 5.5 5.6 4.7 2.8 2.9 7.7 1.2 3.6 3.5 4.8 4.5 11.7 18.9 12.4 11.1 17.1 9.3 4.0 5.4

11.5 1.2 7.1 1.6 Ill 144 35 24 37

Reference

Lee

el

al. (1985)

Evans ef al. ( 1989) Singh e l a/. (1986) Tasmassia ef ul. (1984)

Bjorkman ( I 985)

Giacomini et a/. (1986)

Lennard el a/. (1983)

Olanoff et a / . (1984) Jackman et a/. (1981) Gould ef al. (1986) Grech-Belanger et a / . (1986) Thomson et a/. (1986) Vogelgesang ef a/. (1 984)

Thijssen er al. (1986) Gill et a / . (1989) Hewick and Shepherd (1976) Breckenridge er a / . ( 1 974) Banfield er a/. (1983) (continued)

74

Kenneth M. Williams

Table V (Continued)

Drug

Chirality Sd

R' SP

Anticonvulsants Mephenytoin'

Miscellaneous Fenfluramine

4.0 480 940

0.14 25.0d

21 4700 20 29

(+F

750 760 750 22 I06 59 52

(-)' (+)

(-1

Misonidazole

V (liter)

R em S em R Pm s Pm

(+)

Gossypol

CLU (ml/min)

(+) (-)

33d

1.9d I.5d

1.7d -

254 42 53 51

1112

(hr)

Reference

25 -

76 2. I 77 63 17.8 18.3 18.4 133 5 10.2 11.3

Wedlund et

a/.

(1985)

Caccia et a / . (1982)

Wu et

a/.

(1986)

Williams (1984)

aApparent oral clearance (CL). bunbound CL = literlmin; estimated from data from Lee er al. (1985)and the average unbound concentrations reported by Evans et a / . ( 1989). rRacemic drug administered. em, Extensive metabolizers; pm. poor metabolizers. dCL = ml/hr/kg, V = literikg.

coagulation, although in practice it is the less active (R)-enantiomer which will determine the bleeding times because it is so much more slowly eliminated (Godbillon et al., 1981) (Fig. 8). This contrasts with warfarin, where the (S)-enantiomer has the greater potency in vivo (O'Reilly, 1974). Interestingly, in the case of the male antifertility drug, gossypol, the (+)-enantiomer was reported to have similar activity to its antipode in vitro, but in vivo only (-)-gossypol exhibited efficacy (and toxicity), i.e., the enantiomer which was more rapidly eliminated was more active in vivo, suggesting perhaps the involvement of an active metabolite. An interesting feature of gossypol is that asymmetry is not due to a chiral carbon but to restricted rotation around the bond joining the two biphenyl rings (Fig. 9). This form of stereoisomerism is termed atropisomerism. Several other important observations should be made concerning the examples of pharmacokinetic data shown in Table V. Clearance may be the same when the enantiomer is administered as part of the racemate as compared to alone, e.g., ( +)-fenfluramhe and (S)-indoprofen, but interactions may also be evident. For example, the clearances of the enantiomers of disopyramide were enhanced

Molecular Asymmetry

75 4000 3000

2000

1000

500

1

2

3

4

Time (h) Fig. 8 Plasma concentrations following intravenous administration of 25 mg each of (RS)the (R)-enantiomer (A),and the (S)-enantiomer ( 0 )to a acenocoumarol (Nicoumalone; subject. The inset shows the time course in expanded view over the first 4 hours. (From Godbillon et al.. 1981. Reprinted with permission of the authors and the copyright holder, Macniillan Publishers. Hampshire.)

m),

CHO

CHO

OH

Fig. 9 The male antifertility drug gossypol. Asymmetry is not due to a chiral carbon, but to restricted rotation around the bond joining the biphenyl rings.

76

Kenneth M. Williams

when administered as the racemate (Section V). Unbound or intrinsic clearances are a better indication of the enantioselectivity of the metabolic enzymes than total clearances. For example, in the case of ibuprofen (Table V), although the clearances for total drug enantiomers are very similar [(S)I(R) = 1.1)], the unbound clearances of the enantiomers are dissimilar [ ( S ) / ( R )= 0.71)]. One final point of importance is the effect of enantioselective clearance on the disposition of highly extracted drugs. This is illustrated by the data for metoprolol, where the effects of polymorphic drug metabolism are also demonstrated (Lennard et al., 1986; see also Section 111). In this instance, the less active (R)-enantiomer is eliminated more rapidly in extensive metabolizers. In contrast, the clearance of the enantiomers are approximately equal for poor metabolizers, a situation also observed in extensive metabolizers treated with quinidine (Leemann et al., 1986; Dayer et al., 1987). Clinically, this means that, at equal plasma metoprolol concentrations, there will be a greater response in extensive metabolizers but that the duration of P-blockade will be longer in the poor metabolizer phenotype. Similar effects have been observed for verapamil (Vogelgesang et al., 1984; Eichelbaum et al., 1984). These are insights which could only be obtained by an understanding of the disposition of the enantiomers. Interestingly, an enantioselective interaction can be elicited in vivo using an achiral ligand (Huang, 1988). Oral activated charcoal was shown to increase the clearance of (R)-disopyramide by 58%, but had no significant effect on the (S)enantiomer. This paradoxical result occurred because, in the species studied (rabbit), (R)-disopyramide has an intermediate extraction ratio and its antipode a high extraction ratio. Under these circumstances, the introduction of another clearance pathway can significantly alter the total clearance. In contrast, the proportional effect on the (S)-enantiomer, whose clearance is already high, is not as significant. Incidentally, the binding to charcoal was shown in this study to be nonenantioselective, as expected. It might reasonably be expected that similar enantioselective effects would be demonstrated for other enantiomeric pairs for which one enantiomer has a high clearance and the other a low clearance, such as mephenytoin (Table V) in extensive metabolizers. b. Renal Clearance Enantioselective renal clearance (Table VI) will be the consequence either of enantioselective protein binding and, therefore, selective filtration by the glomerulus, or of differential secretion or reuptake by the renal tubules. Thus, the enantiomers of pindolol had a small but significant difference in their renal clearances. Because the protein binding of the enantiomers was the same, this difference was attributed to either enantioselective renal transport or enantioselective renal metabolism (Hsyu and Giacomini, 1985). Similarly, there appeared to be enantioselective secretion and/or reabsorption of the enantiomers of terbutaline (Borgstrom ef al., 1989), chloroquine (Ofori-Adjei et al., 1986), and N-dealkyldisopyramide (Le Come et al., 1988; Lima et al., 1985). In contrast, the enantioselective renal excretion observed when the enantiomers of

Molecular Asymmetry

77

Table Vl Renal Clearance of Enantiomers

Chirality Disopyramide

S R

N-Dealky ldisopyramide Mexiletine

S R S R

Chloroquine

(+) (-)

Metoprolol

S

R Pindolol Terbutaline (i.v.) Terbutaline (oral)

CLre,,, (rnllrnin) 56 55 345 170 0.33 0.53 315 268 70 em“ 57 prna 75 ern0 62 pma 200 240 2.65h 1.47” 2.47”

Reference Giacomini ef nl. (1986) Lima et a!. (1985) Grech-Belanger et al. (1986) Ofori-Adjei er al. (1986) Lennard er nl. ( 1983)

Hsyu and Giacomini, (1985)

Borgstrorn ef al. (1989)

1.87”

aem, Extensive metabolizers; pm, poor metabolizers of metoprolol bmllminlkg.

disopyramide were administered as the racemate (Giacomini et al., 1986) probably reflects an effect of protein binding differences on renal excretion. D-Methotrexate presumably is excreted by glomerular filtration because it appears to be unable to passively traverse cellular boundaries and is not a substrate for camer proteins (Section II,B, 1). Consequently, active transport m2y play little role in L-methotrexate excretion, the renal elimination rates being similar (Hendel and Brodthagen, 1984). Renal dysfunction might be envisaged to change the enantioselectivity of renal secretion, but no studies have addressed this issue. Interestingly, renal dysfunction might affect the chiral inversion of 2-arylpropionates, even though the parent enantiomers themselves are not renally excreted (see following discussion). c. Chiral Inversion The subject of chiral inversion has been reviewed several times recently. Therefore, while it is a subject of current interest and has potentially important ramifications, the following is an attenuated discussion that aims to highlight the issues. For more detailed discussion on the subject, the interested reader should refer to other reviews (Williams, 1987, 1990a,b; Hutt and Caldwell, 1983, 1984; Caldwell et al., 1988).

78

Kenneth M. Williams

Some enantiomers such as oxazepam and the 5-aryl-substituted anticonvulsant hydantoins and succinimides are relatively labile with respect to racemization when placed in solution (Table VII). Interestingly, the natural product alkaloid, (S)-hyoscamine, also readily racemizes. Its racemate is the clinically administered atropine (Ariens, 1988). Oxyphenbutazone, a minor metabolite of the achiral anti-inflammatory drug phenylbutazone, is also an interesting example. In keeping with other aromatic hydroxylations, it is likely that there is enantioselective product formation of oxyphenbutazone from phenylbutazone in vivo, but ready racemization of the product will ensure that it is essentially racemic. These examples represent a relatively limited number of drugs for which there is spontaneous equilibrium between the enantiomers. Normally, however, the barriers to racemization are too great for this to occur “spontaneously,” particularly within a biologically significant time frame. Thus, racemization is not a common phenomenon in the overall scheme of things. The more interesting chiral interconversion is that which has been reported to occur enantiospecifically, that is, unidirectionally, for drugs belonging to the chemical class of 2-arylpropionates. In this instance, inversion requires interaction with a chiral force. The initial evidence that such a chiral inversion was occurring was the comparison between the in vivo and in vitro activities of the ibuprofen enantiomers. While there was a large difference in their abilities to inhibit prostaglandin synthetase in vitro, their in vivo potencies were similar (Adams et al., 1976). In summary, the key to inversion is the activation of the 2arylpropionate to its CoA thioester (Nakamura el al., 1981; Knights et al., 1988; Knihinicki et al., 1989) (Fig. 10). The following racemization of the CoA thioester is enzymatically mediated (Williams, 1987; Mayer et al., 1989) and occurs with retention of the a-methyl hydrogens (Baillie et al., 1989). The enantioselectivity of the subsequent hydrolysis of the 2-arylpropionate-CoAs to release the parent drugs is unknown (Williams, 1987). Inversion is substrate and species dependent (Caldwell and Marsh, 1983).

Table VII Examples of Drugs which Racemise “Spontaneously” in Solution Drug 5-Arylhydantoins 5-Arylsuccinimides Oxazepam 15-R-15-Methyl PGE? Thalidomide analog (EM- 12) Ox yphenbutazone Atropine

Reference Dudley and Bius (1976) Dudley et a / . (1972) Blaschke and Markgraf (1980) Cox ct af. (1986) Schmahl et a / . (1988) Wiley and Wiley (1964) Ariens (1988)

Molecular Asymmetry

79

/

W

acyl-CoA

C

H

\

3

-

synthetase

-CH3

\

COOH

(R)-ibuprofen

,C-SCoA 0'

(R)-ibuprofen-CoA

I,

ibuprofen-CoA racernase

hydrolase

,c-sc (Spibuprofen

0A

0' (S)-ibuprofen-CoA

Fig. 10 Illustrating the proposed mechanism for the inversion of ibuprofen and related 2arylpropionic acids. The enantioselectivity of inversion is determined by the acyl-CoA synthetase. The racemase (more correctly an epirneraae) interconverts the CoA thioesters. Hydrolase(s) of unknown enantioselectivity releases the 2-arylpropionates. (After Nakamura el a / ., 198 I .)

Chiral inversion in man has only been demonstrated for a limited number of 2arylpropionates, notably ibuprofen (Adams et a/., 1967; Kaiser ef a/., 1976; Lee et ul., 1985), benoxaprofen (Bopp ~t a / . , 1979; Simmonds et al., 1980), fenoprofen (Rubin et a l . , 1985), and flunoxaprofen (Palatini et al., 1988). Apart from the (R)-configuration, the structural requirements for inversion are unknown. The study of chiral inversion has, however, raised some very interesting issues. In particular, the relationship between inversion and lipid biochemistries. As noted in earlier discussion, 2-arylpropionates and other carboxylic acidderived xenobiotics may be incorporated into triglycerides (Fears, 1985; Fears et a / ., 1978). The stereochemical selectivity of CoA formation evident in the studies of inversion was the basis for the hypothesis that, similarly, since the CoA thioester intermediates are obligatory for the uptake of 2-arylpropionates into these hybrid triglycerides, incorporation would also be enantiospecific for the (R)-enantiomers (Williams and Day, 1985). Both in vivo data for ibuprofen (Williams er al., 1986) and in vitro data for fenoprofen (Sallustio et a / . , 1988) in rats confirmed the hypothesis. While there are only preliminary data (Table VIII), they do suggest that this is a relevant consideration for the disposition of this class of drugs in man. Importantly, it IS likely that there is a correlation betwcen inversion and lipid incorporation (Williams and Day, 1988) (Fig. 11). One can only speculate about the possible clinical consequences of such

Kenneth M. Williams

80 Table Vlll Accumulation and Excretion of Ibuprofen Enantiomers in Man after Chronic Dosing (7 days) ~~

~

Concentration in fat (pgLg/g)

24 h p Subject 1

2 3

96 hr

Dose (mg)

R

S

800 tds 800 bd 800 tds

11.4 0.V -

2.4 0.4b -

R

S

8.5

2.1 0.8 9.5

1.8 19.3

OTime after last dose. bSamples collected at 4 hours after the last dose

hybrid lipid formation (Fig. 11). It has been suggested that there are potential toxicological (Caldwell and Marsh, 1983) effects mediated by this mechanism. There is evidence that some CoA thioesters are themselves toxic. Clearly, use of the (S)-enantiomers alone would circumvent this issue if it were demonstrated to be a toxicological problem. However, there is no clear clinical evidence at this stage that ibuprofen, a racemic 2-arylpropionate, is less or more toxic than naproxen, an (S)-enantiomer. Disposition

/ z

Metabolic

Pathways

Consequences

lipids

Lipid incorporation

Perturbation of lipid biochemistries (?)

S-CoA

R-CoA inversion

11,

oxidation

R

z

n

S

11 S-gluc \C /

R-gluc Renal clearance (futile cycle)

1

Direct toxicity

-====b

I?)

P G synthetase inhibition Drug-protein adducts Immunological effects

Lrcnr 1

Fig. 11 Schema (Williams, 1990a) illustrating the potential interrelationships and consequences of the inversion of the (R)-2-arylpropionates (R). Both inversion and lipid incorporation are controlled by formation of the CoA thioesters (R-CoA, S-CoA). If renal clearance of the glucuronides (R-gluc, S-gluc) is reduced, hydrolysis of the glucuronides back to the parent drug may occur, making more (R)-enantiomer available for inversion and lipid incorporation. The toxicological consequences are uncertain. (Reprinted with permission of the copyright holder, Pergamon Press, Oxford.)

Molecular Asymmetry

81

The schema (Fig. 11) also indicates the potential interrelationship between the effects of renal dysfunction and the chiral disposition of the 2-arylpropionates. Reduced renal function may cause an accumulation of the glucuronides which in turn can then be hydrolyzed back to the parent enantiomers. This “futile cycle” (Meffin, 1985) means that, although there is no direct effect of renal dysfunction on the disposition of the 2-arylpropionates, there may be secondary effects resulting in more (R)-enantiomer being available for chiral inversion or lipid incorporation. Additionally, the glucuronides are activated species with the potential for formation of drug-protein adducts (Section IV,C). There is still much to be learned concerning these issues. In concluding this section on chirality and its pharmacokinetic consequences, brief attention should be drawn to the problems associated with pharmacokinetic analysis of racemic drugs based on nonenantioselective assays. A recent assessment of the pitfalls of achiral analysis using a model racemic drug (Evans et al., 1988) illustrated, for example, that even if the individual enantiomers follow linear kinetics, concentration and/or time dependence may be falsely indicated by the pharmacokinetics of the total racemic drug. Similar problems also arise for the interpretation of unbound concentrations based on achiral analysis, with the possibility of concentration dependence of binding to plasma proteins being falsely indicated (Evans et af., 1988). Chiral drugs require chiral analysis for a meaningful interpretation of the data (Ariens, 1984).

111. Chirality and Polymorphic Drug Disposition There is a large body of literature dealing with the many examples of differential rates of metabolism between enantiomerically related drugs (substrate enantioselectivity). The following discussion reviews some of the literature dealing with specific genetic defects in metabolism which present themselves by significant intersubject variability in substrate enantioselectivity. For more detailed discussion concerning enantioselective metabolism, the reader should refer to other literature (see, e.g., Jenner and Testa, 1973; Trager and Testa, 1985; Testa, 1986; Testa and Mayer, 1988).

A. The Sparteine-Debrisoquin Phenotype One area of current research receiving considerable attention is that of genetically determined variability in disposition of drugs. Polymorphic drug oxidation has been characterized by determining the ability of subjects to oxidize debrisoquin or sparteine. These studies have identified subgroups of the population with reduced ability to carry out these oxidative processes. The “poor metabolizer” phenotype is inherited as an autosomally recessive trait and presents itself in up to 10% of the Caucasian population. Debrisoquin polymorphism cosegregates

82

Kenneth M. Williams

with the polymorphic oxidation of sparteine. Other examples of drugs whose metabolism is controlled by the phenotype are bufuralol, metoprolol, nortriptyline, desipramine, and encainide. The decreased capacity to eliminate these drugs puts the poor metabolizer at increased risk of adverse effects due to accumulation of supraoptimal concentrations of drug. The interest for the present discussion is the enantioselectivity exhibited by the isozymes of the hepatic cytochrome P-450 system responsible for the oxidation of debrisoquin and other related racemic drugs. I n vitro studies with microsomes (Dayer et af., 1985) demonstrated the enantioselectivity of the 1'-hydroxylation of bufuralol in extensive metabolizers [(-)/(+) ratio range 0.3-0.6; Fig. 12), whereas poor metabolizers had a much reduced enantioselectivity [ ( - ) I ( +) ratio 0.7-0.91. A preparation of purified isozyme was more enantioselective, with the mean (-)/(+) ratio being 0.13 (0.11-0.15). In vitro studies have been useful in determining potential candidates for the debrisoquin phenotype and in predicting drugs which might inhibit drug metabolism (Inaba et al., 1985). The enantioselective disposition of metoprolol is illustrative of this discussion. Metoprolol is a racemic P-antagonist whose metabolism to a-hydroxymetoprolol and 0-demethylation are significantly controlled by the debrisoquin phenotype (Lennard et al., 1986). Plasma clearance data (Table V) indicated that metoprolol is a medium- to high-clearance drug in extensive metabolizers and a low-clearance drug in the poor metabolizer phenotype. Consistent with these data, there

7 4

-

2

4

E

0

40

80

120

160

200

Bufuralol C p I o l / i )

Fig, 12 The 1'-hydroxylation of (+)-bufuralol (A:K, = 41, V,,, = 8.6) and (-)-bufuralol = 2.7) by microsomes from an extensive rnetabolizer [(-)/(+) ratio = 3.21. (From Dayer et a!.. 1985. Reprinted with permission of the authors and the copyright holder, Pergamon Press, Oxford.)

(V;K,,,= 22, V,,,,

83 0

300-

Plasma concentration 100. (nglmll

(Sl-M

10.

(Rl-M

1.

0

4

8

1

2

0

6

i2

24

Time Ih) Fig. 13 Log plasma concentration-time profiles for (Shnetoprolol and (R)-metoprolol after oral ingestion of (RS)-metoprolol (200 mg) in ( A ) an extensive and (B) a poor metabolizer of debrisoquin. (From Lennard el a/.. 1983. Reprinted with permission of the authors and the copyright holder, C. V. Mosby Company, St. Louis.)

was an enantioselective oral availability such that the AUC for (S)-metoprolol was on average 35% greater than for the (R)-enantiomer in extensive metabolizers and the half-lives of the enantioniers were similar. In contrast, the oral availability of the enantiomers was similar, while the half-life of the (R)-enantiomer was greater than for its antipode, in poor metabolizers (Fig. 13) (Lennard er a l . , 1983). Because the P-antagonist activity of metoprolol resides in the (S)-enantiomer only, there are interesting and perhaps clinically significant consequences of this enantioselective metabolism. Thus, for the same total concentrations of metoprolol, a greater effect would be anticipated in extensive metabolizers since the contribution of the active (S)-enantiomer to the total drug concentration will be greater in this group, i.e., there will be a shift in the concentration response curve to the right in poor metabolizers compared with extensive metabolizers (Lennard et af., 1983) when total drug concentrations are considered. Similar data have been reported for verapamil, as discussed previously.

B. The Mephenytoin Phenotype Another genetic polymorphism of drug oxidation which does not cosegregate with debrisoquin is that characterized by a deficiency in the ability to metabolize

84

Kenneth M. Williams

mephenytoin (Kupfer et al., 1984a; Wedlund et al., 1985), a racemic anticonvulsant. The relative anticonvulsant activities of the enantiomers apparently are unknown. The metabolism of hexobarbital (Knodell et al., 1988), 5-phenyl-5-ethylhydantoin (Kupfer et al., 1984b), and methylphenobarbitone (Kupfer and Branch, 1985; Jacqz et al., 1986) also cosegregates with the mephenytoin phenotype which presents itself in 3-5% of the Caucasian population (Wedlund et al., 1985) and in up to 25% of the Japanese population (Nakamura et al., 1985; Jurima et al., 1985). In v i m data suggest that other drugs which cosegregate with this deficiency are tranylcypromine, nialamide, and papaverine (Inaba et al., 1985). [Tranylcyprornine, (+)-trans-2-phenylcyclopropylamine,is a chiral monoamine oxidase inhibitor whose monoamine oxidase potency is primarily associated with the ( lS,2R)-stereoisomer. In contrast, the eutomer for inhibition of catecholamine uptake is the (lR,2S)-enantiomer (Nickolson and Pinder, 1984).] Again one of the interesting features of this polymorphism is the enantioselectivity of the isozyme associated with this deficiency. The isozyme catalyzes the aromatic 4-hydroxylation of (S)-mephenytoin, which is the primary mode of elimination of this enantiomer (Fig. 14). In the deficient state there is a substantially reduced rate of elimination of (S)-rnephenytoin by hydroxylation, and the

PMs 0 (R,S)-rnephenytoin

(R,S)-phenylethylhydantoin

HO

0

(S)-4-hydroxymephenytoln

0

(S)-4-hydroxyphenylethyl hydantoln

Fig. 14 The metabolic fate of (RS)-mephenytoin in extensive (EM) and poor metabolizers (PM). Mephenytoin is primarily cleared by aromatic 4-hydroxylation in EMS. but by N-demethylation in PMs. EMS also clear phenylethylhydantoin by aromatic 4-hydroxylation of the (S)-configuration.

85

Molecular Asymmetry

previously minor demethylation pathway becomes important for the clearance of this enantiomer. In contrast, (R)-mephenytoin is primarily cleared by N-demethylation to phenylethyIhydantoin and its metabolism is unaffected by the deficiency. Moreover, the 4-hydroxylation of (R)-mephenytoin is also unaffected by the polymorphism. The differences in the plasma concentrations of the respective enantiomers and metabolites are very large (Fig. 15) (Wedlund er a l . , 1985). While the oral clearances in the poor metabolizer are similar for the enantiomers, this contrasts with the highly enantioselective oral clearance of the (S)-enantiomer in the extensive metabolizer phenotype (Table V). The cumulative urinary excretion data for the parent drug and the metabolites further complete the picture (Fig. 15).

-n A

k

Y

500

\ .

‘OJ,.

0

, . , 2

4

.

, 6

,

, . , S

TIME (DAYS)

,

,

1 0 1 2

,

, 14

lo 2

4

6

8

10

12

TIME (DAYS)

Fig. 15 Contrasting profiles after oral administration of (RS)-mephenytoin to an extensive metabolizer (EM) (300 mg) and a poor metabolizer (PM) (200 mg; Wedlund el a l., 1985). (A) Plasma concentrations of the enantiomers of mephenytoin and phenylethylhydantoin (PEH) in an EM. (B) Plasma concentrations of the enantiomers of mephenytoin and PEH in a PM. (C) Cumulative urinary excretion of mephenytoin and its 4-hydroxy and N-demethylated (PEH) metabolites in an EM and a PM. (Reprinted with permission of the authors and the copyright holder, The American Society for Pharmacology and Experimental Therapeutics, Baltimore.)

14

J

TIME (DAYS)

POOR METABOLIZER

EXTENSIVE METABOLIZER w

1

4-

.-& *-.. 4 4-

4-C.

-A.

57

4-0H MEPHENMOIN

i

I

I I MEPHENYTOlN

"

TIME (DAYS)

TIME (DAYS)

Fig. 15 (conrinued)

Molecular Asymmetry

87

Phenylethylhydantoin is an active metabolite which contributes significantly to the anticonvulsant activity of mephenytoin during chronic administration (Kupferberg and Yonekawa, 1975). In extensive metabolizers the primary anticonvulsant activity is presumably mediated through (R)-mephenytoin and (R)-phenylethylhydantoin. However, because the relative activities of the enantiomers of phenylethylhydantoin are unknown, it is not clear as to the clinical consequences of the accumulation of (S)-mephenytoin and (S)-phenylethylhydantoin for the poor metabolizer phenotype. Indeed, it may be the extensive metabolizer who is at greater risk of adverse effects by the formation of potentially toxic oxidative metabolites of (S)-mephenytoin (Kupfer et al., 198I). Phenylethylhydantoin is also enantioselectively hydroxylated to give (S)-4-hydroxyphenylethylhydantoin in extensive metabolizers (Fig. 141, presumably by the same enzymes involved in the hydroxylation of mephenytoin (Kupfer et al., 1984b). Phenytoin (which is achiral) is primarily oxidized to the (S)-4-hydroxylated derivative, p-hydroxyphenylphenylhydantoin (HPPH) (Poupaert et al., 1975; example of product enantioselective metabolism, Fig. 16). The topographical equivalence of the aromatic rings, which are hydroxylated in phenytoin and mephenytoin (Fig. 16), suggests that the poor metabolizer phenotype for mephenytoin would also be a poor metabolizer of phenytoin. Surprisingly, this is not the case. (Debrisoquin also does not predict the metabolism of phenytoin; Steiner et al., 1987.) Phenytoin does not competitively inhibit the metabolism of mephenytoin in vitro (Hall et ul.. 1987) and was not found to cosegregate with the mephenytoin deficiency phenotype (Fritz et al., 1987). Indeed, it was the minor metabolic pathway of hydroxylation of the pro-(R)-phenytoin which cosegregates with genetic (S)-mephenytoin hydroxylation deficiency. An explanation for this apparent paradox (Fritz et al., 1987) is that there are two isozymes responsible for the hydroxylation of (SI-mephenytoin. Evidence supporting this hypothesis is that autoinduction results in a significant change in the relative excretion of the enantiomers of HPPH. Presumably then, the mephenytoin phenotype is associated with a genetic deficiency in two isozymes for which (S)mephenytoin is enantiospecifically the substrate. Phenytoin is only a substrate for one of these isozymes and, in this instance, it is the pro-(R)-form which is the substrate. It was thus suggested that monitoring (R)-HPPH formation from phenytoin could be used to identify poor mephenytoin metabolizer phenotypes (Fritz et al., 1987). Clearly, these data have therapeutic implications. In fact, in the original report on the genetic mephenytoin disability, the slow metabolizer experienced adverse effects due to accumulation of phenylethylhydantoin, despite “therapeutic” concentrations of the parent anticonvulsant (Kupfer et a l . , 1984a). Future therapeutic drug monitoring may see patients screened with a series of marker compounds to determine a genetic fingerprint for drug metabolism prior to drug therapy. In addition, as suggested by Meyer et ul. (1986), an increased understanding of polymorphism as exhibited in the metabolism of xenobiotics may also lead to an

88

Kenneth M. Williams

OH

I

A

CH3

(S)-mephenytoin OH I

0

pro-S

/ pro47

H

\

phenytoin

Fig. 16 (A) (S)-Mephenytoin is primarily oxidized to (S)-4-hydroxymephenytoin. (8)Phenytoin, which is achiral, is also metabolized to the 4-hydroxylated metabolite and is also of the topographically equivalent (S)-configuration. Surprisingly, it is the pro-(RFphenytoin which cosegregates with the genetic (S)-mephenytoin hydroxylation deficiency.

understanding of interindividual variability in the metabolism of endogenous substrates of the cytochrome P-450 isozymes.

IV. Pharmacodynamic Consequences of Chirality A very comprehensive collection of pharmacodynamic data for enantiomeric pairs has been published in the excellent CRC Handbooks edited by Donald Smith (1984, 1989a). In addition, the relative pharmacodynamic potencies of a large number of enantiomers have been tabulated (Jamali et al., 1989), and there

Molecular Asymmetry

89

are excellent reviews dealing with stereoselectivity of transmitter storage vesicles and the processes at neuronal junctions (Patil et a / . , 1975). Consequently, a limited number of examples which illustrate some chiral pharmacodynamic concepts are reviewed. Our sensory functions of taste and smell and immunological responses to drug-related haptens may occur enantioselectively. Receptor binding studies require careful attention to chirality, and enantioselectivity may occur at acceptor as well as at receptor sites. Finally, data on the relative activities of the enantiomers of timolol and the potential clinical and mechanistic consequences complete this brief overview of the pharmacodynamic consequences of chirality.

A. Our Asymmetric Senses National Geographic conducted a smell survey in association with an excellent article on “The Intimate Sense of Smell” (September 1986 edition, B. Gibbons and L. Psihoyos) which demonstrated the large intersubject variability in the perception of odors (October 1987 edition; N . Gilbert and C. J. Wysocki) among the 1.5 million participants. Unfortunately, they did not include any enantiomeric pairs in the survey, but it is known that the receptors in the olfactory epithelium do have a discriminatory capacity for some enantiomers (Polak et al., 1989). For example, (+)-carvone is the smell of caraway, while the (-)-enantiomer is the clearly different odor of spearmint (Leitereg et al., 1971; Murov and Pickering, 1973). The chiral discrimination demonstrated by our senses is further illustrated by studies on the enantiomers of menthol (Eccles et a l ., 1988a,b). Qualitatively, the enantiomers have similar odors-that of menthol, the active component of some nasal decongestants commonly applied topically as a gel to the skin and subsequently inhaled. While the enantiomers of menthol are quantitatively different, the (-)-enantiomer having the greater odor, Eccles et al. have looked at a more subtle and interesting qualitative difference in activity, that of the differences in the perception of nasal airflow induced by smelling the enantiomers of menthol. It was found that the (-)-enantiomer elicited a “decongestant” action while the (+)-enantiomer was inactive in this respect. The subtlety and insight, however, is that there was no change in measured nasal airflow (Fig. 17). There was only a perception of nasal decongestant activity. There are interesting implications of these data for the study of conditions in which there is believed to be a failure of upper airways stability, e.g., snoring and sleep apnea (Eccles el a/., 1988b). These data are sufficient to illustrate in a simple but convincing manner that there may be pharmacodynamic differences between enantiomers, differences which may be either quantitative and/or qualitative.

Kenneth M. Williams

90 A

nasal 50 sensat ion

lB

T

score (mm)

(-)-men t hot

(+)-men t hot

0

vanilla

(+)

(-1

(menthol,

Fig. 17 (A) Structures of the enantiornerically related (-)-(lR,2S,SR)-menthol and ( + ) - ( 2 S , 2 R , 5s)-menthol. (B) The effects of the inhalation of vanilla (control) and of the enantiorners of menthol on nasal sensation of airflow (Eccles et a / . , 1988b). The histogram illustrates the mean scores (rnrn) on a visual analog scale (&SEM, n = 20) where a score of 50 rnrn indicates the maximum sensation of nasal airflow. (Reprinted with permission of the authors and the copyright holder, lnvicta Press, Kent.)

B. Receptors, Acceptors, and Chiral Discrimination 1. Chiral Discrimination The concept of a three-point interaction for chiral discrimination (Easson and Stedman, 1933) at receptors has been discussed previously. This has been a useful and practical approach to understand and characterize enantioselectivity demonstrated by receptors (Fig. 18) and helps understanding of the examples of enantiomer-enantiomer interactions in later discussion. However, this concept represents a limited and perhaps naive approach to understanding chiral recognition since there is evidence of apparent one- and two-point interactions resulting in chiral discrimination. These data cannot be explained by the simple EassonStedman model. Thus, while a molecule can be viewed in terms of separate distinct entities interacting with discrete loci on a receptor, much in keeping with the lock-and-key concept of substrate-receptor interaction, it is more realistic to view the molecules as a whole. Thus a “one-point model” is really an interaction between all the nuclei and electrons of each of the interacting species (Topiol, 1989). This is an interesting concept to bear in mind when considering chiral interactions. The degree of enantioselectivity can be quantitated. The original basis for a quantitative approach to chiral interactions was Pfieffer’s Rule (1956), which states that the enantiomeric potency ratio increases with the potency, i.e., the more potent the eutomer the less potent the distomer. A useful analogy is the hand-glove or feet-shoes relationship. Thus, the better the fit of a shoe for one foot, the more difficult it becomes to fit that foot into the “enantiomeric” shoe (Lehmann, 1982).

91

Molecular Asymmetry

H H

w-1

Desoxy

Fig. 18 Schematic representation of the Easson-Stedman hypothesis for interaction of ( - ) - ( R ) and (+)-(S)-noradrenaline, and [he corresponding p-desoxy derivative, dopamine, with adrenergic receptors (Ruffolo, 1983). P, H, and A represent three hypothetical binding sites to which attach the phenyl, hydroxyl, and amino functional groups of phenylethylamines, respectively. (Reprinted with permission of the author and the copyright holder. Blackwell Scientific Publications, Oxford.)

The concept of the eudismic index, which is the logarithm of the ratio of the affinities or the potencies of the eutomer and the distomer, has been developed to quantify these relationships (Lehmann, 1982). Among many other examples, the correlation between sweetness and bitterness of several amino acids (log sweetness = 0.084 + 0.702 bitterness; R2 = 0.759) was established by this means (Lehmann, 1978). An interesting additional result of this study was an hypothesized three-point association with the receptors for bitterness and taste which incorporated sites that were essentially mirror images of each other. It can thus be surmised that, when Alice went through the looking glass (Lewis Carroll, Through the Looking Glass and What Alice Found There, 1872), those chiral substances which normally were sweet, would to her have been bitter, and vice versa.

2. Receptors and Acceptors Enantiomers are useful probes to characterize receptors because their physicochemical properties are identical. Indeed, one of the key criteria or requirements necessary to define a receptor is that of enantioselective discrimination. However, while this is a useful criterion, it is on its own insufficient to prove the presence of receptors (Laduron, 1984, 1988). Thus, enantioselective protein binding, active transport, enzymatic reactions, and nonspecific tissue binding may all demonstrate enantioselectivity but are all examples of acceptors since they occur without eliciting a pharmacological response (Fig. 19). Indeed, the enantioselectivity of both the acceptor and the receptor may be similar, as illus-

92

RECEPTOR

1

Kenneth M. Williams

ACCEPTOR

U P T A K E PROCESS RESPONSE

A C T I V E TRANSPORT E N Z Y M A T I C R E AC T I O N NON S P E C I F I C BINDING

Fig. 19 Schematic representation of a receptor site and an acceptor site. (From Laduron, 1984. Reprinted with permission of the author and the copyright holder, Pergarnon Press, Oxford.)

trated for the neurotensin acceptor and the histamine H,-receptor (Schotte et al., 1986). Very high enantioselective binding will, however, generally be discriminatory for a receptor, especially when displacement from the receptor also occurs with another drug belonging to another chemical class, but eliciting the same pharmacological response (Laduron, 1988) (Fig. 20). In contrast, nonspecific binding is likely to demonstrate much less enantioselectivity, and displacement may often occur over a wide range of concentrations (Laduron, 1988) (Fig. 20). One of the consequences of enantioselectivity of receptor binding is that there are potential pitfalls in the common practice of using racemic ligands for radioreceptor binding studies. This subject has been reviewed previously (Hoyer, 1986). In brief, both enantiomers may contribute to total binding, although only one enantiomer may be binding to the relevant receptor, i.e., there may be high affinity for the distomer at acceptor sites. Acceptor binding of the distomer will also lead to unnecessarily high nonspecific binding. Alternatively, the distomer may bind at the receptor, leading to competitive binding effects between the enantiomers. Finally, the distomer may bind to a different class of receptor such that data using racemic radiolabeled ligand will be confused by the simultaneous binding at more than one class of receptor. Clearly, these factors may lead to

93

Molecular Asymmetry

B

d - BENZETlMlDE

1

I

11

10

I

9

8

7

6

I

5

-log C (MI Fig. 20 (A) Inhibition of (3H]-(+)-henzetimide (dexetimide) binding in a total homogenate of dog splenic nerves by (RS)-atropine, dexetimide, and ( - l-benzetimide (levetimide). (B) Doseresponse inhibition of (t)-benzetimide and (-kbenzetimide in the ['Hllevetimide assay using a total homogenate from rat striatum. (From Laduron, 1988. Reprinted with permission of the author and the copyright holder, Pergamon Press, Oxford.)

94

Kenneth M. Williams

errors in the estimation of the affinity and the number of binding sites and, as is the case for nonenantioselective assays in pharmacokinetic studies, misinterpretation of the data is likely.

C. Immunological Enantioselectivity Further evidence for the chiral nature of biological mechanisms is the enantioselective formation of antibodies with enantiomerically pure haptens. This applies to antisera formed by immunization with drug enantiomers. In fact, enantioselective assays have been developed for drugs such as propranolol where cross-reactivities of the (+)-enantiomer following preparation of antibodies using the (-)-enantiomer were less than 10% (Kawashima et al., 1976; Bilezikian et al., 1979). Similarly, cross-reactivity was only 1-2% in a study of antibodies generated to the enantiomers of pentobarbital (Cook er al., 1987). By contrast, when antiserum was prepared by immunization with, for example, racemic propranolol (Kawashima et al., 1976) or racemic warfarin (Satoh et al., 1982) enantioselectivity of the antibodies was lost. Presumably, under these conditions a mixed (and equal?) population of antibodies is formed, one enantioselective for each enantiomer. It should not be assumed on the basis of these data, however, that immunization with a racemic hapten will lead to formation of nonenantioselective antisera. Even assuming nonenantioselective reaction between the drug-protein conjugates, the “immune system could respond by producing antibodies to only one hapten, equal amounts of antibodies to each hapten or some intermediate situation” (Cook et al., 1982), as illustrated for the antimalarial drug WR 171,669 (Cook et al., 1982). This has clear implications for drug analysis. While we normally think of formation of acyl glucuronides to be a means of facilitating drug excretion, it has also been demonstrated that these metabolites are highly reactive species, capable of acylating biopolymers (Van Breemen and Fenselau, 1985; Hyneck et al., 1988). In the context of the present discussion, the reader will recognize that this is analogous to the procedures for hapten formation used to generate antibodies suitable for drug analysis. Furthermore, glucuronide formation may occur enantioselectively (Koster et al., 1986; Spahn et al., 1989) and the clearance of acyl glucuronides may be affected in an enantioselective manner by concurrent drug administration (e.g., probenicid; Spahn et al., 1987, 1989) or in renal failure (e.g., ketoprofen; Verbeeck et al., 1984). Thus, it would not be surprising to find mixed populations of antibodies generated by the drug enantiomers, and that there would be an increased potential for antibody formation in renal failure and during concurrent administration of drugs affecting the renal clearance of glucuronides. It should be a consideration, therefore, that the potential for adverse immunological sequelae is increased by using racemic drugs which are cleared by conjugation with glucuronic

95

Molecular Asymmetry

acid. Thus, “inactive” enantiomers may still generate antibodies which could increase the risk of hypersensitivity reactions to drugs. The practolol syndrome, a serious adverse hypersensitivity reaction to treatment with practolol, but not other P-blockers, has been attributed to antibody formation to metabolites of practolol containing the acetylanilide function (Amos et a l . , 1978). It has been proposed that antibody formation may only be associated with metabolites of the inactive (R)-practolol, a situation which could be avoided by use of the (S)-enantiomer alone (Simonyi, 1984). This suggestion is speculative, but it is a concept which ought to be considered when comparing the risk-benefit ratios for the use of enantiomeric versus racemic drugs.

D. Timolol and the p-Receptors Timolol is used as the (S)-enantiomer in the management of hypertension and is indicated for the reduction of mortality and reinfarction after a heart attack. Presumably, the (S)-enantiomer was developed on the basis of the observation that the (S)-enantiomer was 50-90 times more active than the (R)-enantiomer as a P-antagonist in preparations of heart tissue, an enantioselectivity common for the P-antagonists (Table IX). Control of ocular pressure is a complex process as evidenced by the hypotensive action of both P-agonists and P-antagonists. Presumably, on the basis, therefore, that P-antagonists are useful in the control of intraocular pressure, the (S)-enantiomer of timolol was marketed for topical application in the treatment of glaucoma. However, because of systemic adverse effects mediated by antag-

Table IX Extraocular P-Antagonist Activities of the Enantiomers of Timolol“ PA2 Test tissue Human lung Carbachol contracted PGF2” Guinea pig Trachea (PGF?) Atria

S

R

SIR

10.7 10.8

8.8 9.1

1\79 1/49

9.8 9.S

7.9 7.6

1191 1/91

“From Share et a / . (1984. Reprinted with permission of the authors and the copyright holder, SpringerVerlag, New York). hPGF,, Prostaglandin FZu.

96

Kenneth M. Williams

onism at extraocular @-receptors,it is contraindicated for patients with asthma or patients with cardiac failure. Indeed, this drug has been associated with significant morbidity and mortality when used in these groups of patients. However, a comparison of data for the activities of the enantiomers in preparations of ciliary process 6-receptors from human eyes (Nathanson, 1988) revealed a very interesting relative shift in the dose-response curve for the (R)-enantiomer for @-receptorsin the eye as compared to those in heart or lung such that the potency of the (R)-enantiomer approximated that of its antipode (Fig. 21). Other in vitro data (Share ef al., 1984) suggest a larger eudismic ratio [ ( S ) / ( R )= 3-41, but this still contrasts with the 90-fold difference in @-antagonismin heart and lung preparations (Table IX). Similarly, few enantioselectivities have been reported for the action of adrenergic agonists on intraocular pressure. Thus, in contrast to the midriatic action of the noradrenaline enantiomers in rabbits, where the (+)/(-) ratio of ED,,s (pupillary dilation) was 0.5, the ratio for the hypotensive action on intraocular pressure was 1 . 1 (ED for a decrease in intraocular pressure of 3 mmHg; Green et al., 1986). Clinical studies demonstrated the effectiveness of reducing intraocular pres-

c

"

.I

I

10

DRUG CONCENTRATION ( p M ) Fig. 21 Effects of (S)-timolol and (R)-timolol on human ciliary process isoprenaline-stimulated adenylate cyclase activity (From Nathanson, 1988. Reprinted with permission of the author and the copyright holder, The American Society for Pharmacology and Experimental Therapeutics, Baltimore.)

Molecular Asymmetry

97

sure with (R)-timolol (Richards and Tattersfield, 1985; Mills et al., 1988), and it was generally thought that (R)-timolol would have a better therapeutic index for the management of glaucoma than the (S)-enantiomer, being much less likely to elicit adverse systemic effects (Keates and Stone, 1984). Unfortunately, recent data in asthmatics revealed that at doses effective in lowering intraocular pressure, (R)-timolol did have some adverse effects on respiratory function such that it was concluded that there would not be a greater margin of safety with (R)timolol (Richards and Tattersfield, 1987). There is an additional consideration, that of the increased risk for adverse effects in the poor rnetabolizer phenotype (Lennard ef al., 1986, 1989), which has not been addressed, although peak concentrations are not likely to be greatly different between the two groups because first pass metabolism would be circumvented by ocular administration (McGourty et al., 1985). It should not be overlooked, however, that even if there is not a great improvement in therapeutic index with the (R)-enantiomer, the data still may give important insights into the characteristics of the ocular f3-receptors. The data suggest that there may be a distinct subset of P-receptors in the eye, although the data for the adrenergic agonists discussed above were interpreted as indicating a difference in the disposition of the enantiomers, i.e., that there was limited uptake of (+)-noradrenaline into noradrenergic stores and a relative resistance to metabolism of the (+)-enantiomer (Gherezghiher and Koss, 1985). There are clearly further interesting insights to be gained into the factors controlling intraocular pressure by the use of chiral probes.

V. Enantiomer-Enantiomer Interactions According to the Easson-Stedman hypothesis ( 1933), the ability to differentiate between enantiomers is thought to require at least a three-point interaction between the macromolecule and the enantiomers, as discussed earlier. Thus, the distomer may still interact with the receptor at two points and have a significant affinity for the receptor (K,s similar). However, the binding may be nonproductive and thus antagonize the action of its enantiomeric pair (Fig. 18). Precedents for such interactions have been reviewed for the modulation of various enzymes by their unnatural enantiomeric substrates. Thus, (+)-carnithe and (+ )-acetylcarnitine are competitive inhibitors of carnitine acetyltransferase, and proteolytic enzymes are often inhibited by D-amino acid-containing peptides (Grafstein, 1985). Therefore, although the potential for interactions between enantiomers of drugs has received little attention, it should not be surprising to find both pharmacokinetic and pharmacodynamic enantiomer-enantiomer interactions.

98

Kenneth M. Williams

A. Protein and Tissue Binding Enantiomers may compete with each other for plasma protein binding sites, with consequent increases in blood clearance for low-clearance drugs, although the clinical consequences of such interactions may not be significant. The data for ibuprofen in man showed that the clearance of the (R)-enantiomer was increased when administered as part of the racemate, in contrast to when it was administered alone, a finding which suggested that the (S)-enantiomer displaced the (R)-enantiomer from plasma binding sites (Lee et af., 1985). Area data also suggested a mutual effect of the (R)-enantiomer on the binding of the (S)-enantiomer. A recent study has confirmed this hypothesis, with (R)-ibuprofen displacing (S)-ibuprofen to a greater extent than vice versa (Fig. 22) (Evans et al., 1989). However, the very small displacement of @)-ibuprofen by (S)-ibuprofen observed over the normal range of plasma concentrations (up to 30 mglliter) suggests that other unknown factors contributed to the altered clearance in the study of Lee et al. (1985). Mutual protein binding displacement by the enantiomers of flurbiprofen has also been described, but interestingly, in this case, the (S)-enantiomer exhibited the greater displacing capacity, consistent with its greater affinity for binding sites (Knadler et al., 1989). [While there is a reversed

h

1.4-

0 0

-

Y-

X

-0 C 3

2

1.2-

1.0-

C

3

.-c

0.8-

0

m

0.60.4 -

0.2 ! 0

10

20

30

40 50 Concentration (rngil)

Fig. 22 Plots of the fraction unbound versus the plasma concentration of (!?)-ibuprofen both alone (0) and in the presence of an equal concentration of (S)-ibuprofen (W), and of (S)ibuprofen both alone (0) and in the presence of an equal concentration of (R)-ibuprofen (0) (From Evans el al., 1989. Reprinted with permission of the authors and the copyright holder, Springer-Verlag, New York.)

99

Molecular Asymmetry

selectivity for binding of ibuprofen enantiomers and flurbiprofen enantiomers, in both instances the (S)-enantiomers are still the inhibitors of prostaglandin synthesis.] However, these interactions were only observed at concentrations above those expected in normal therapeutic practice. Mutual displacement interactions between the enantiomers of phenylpropionic acid in rabbits are also of interest within this context (Jones et al., 1986). Jones et af. noted the problems that such interactions pose for the interpretation of drug disposition data when the relative concentrations of the enantiomers are changing with time, such as occurs for ibuprofen. Finally, it should not be assumed that the enantiomers will always interact as illustrated by data for verapamil, where the binding of each enantiomer is independent of the other (Gross et al., 1988).

B. Metabolic Interactions There may be competition between enantiomers for metabolism. Thus, (+)-ketamine has a higher affinity (recognition) for the N-demethylase than dose (-)-ketamine but also has a lower activation, as reflected by a lower V,,, (Table 111). This leads to a competitive antagonism between the enantiomers such that the racemic drug has a lower rate of demethylation than predicted (Marietta et af., 1977). (S)-Propranolol was also found to be a competitive antagonist of the glucuronidation of (R)-propranolol (Table 111). Similarly, data for the metabolism of p-chloroamphetamine by a rabbit liver microsomal preparation (Table X) (Ames and Frank, 1982) demonstrated a reversal in the rates of metabolism of the enantiomers when the enantiomers were incubated separately as compared to the racemate. Analogous data have also been reported for metabolism of the enantiomers of amphetamine (Gal et al., 1976).

Tuble X Metabolism of p-Chloroamphetamine Enantiomers by Rabbit Liver Microsomes following Incubation of ( R S ) - , ( R b , and (S)-PCAU Rate of disappearanceh Concentration Substrate

RS R S

(I-LM)

R

S

50 25 25

0.037 2 0.008 0.329 t 0.024

0.145 t 0.020 0.258 2 0.029

"From Ames and Frank (1982. Reproduced with permission of the authors and the copyright holder, Pergamon Press, Oxford). hRates of disappearance (nmolimglmin; mean 5 SEM; n > 3) over 15 minute incubations with 3 mg of microsomal protein.

100

Kenneth M. Williams

C. Pharmacodynamic Interactions As well as potential interactions at protein binding and metabolizing enzyme sites, similar enantiomer-enantiomer interactions are likely to occur at receptors. Again, such interactions have received little attention to this time but there are a limited number of examples which can be cited. Thus, (S)-flurbiprofen is eight times more active than the racemate (antagonism of the effects of SRS-A on guinea pig tracheal chain; Greig and Griffin, 1975). Similarly, the antihelminthic drug levamisole potentiates the chemotactic response of human monocytes to a number of stimuli. Its enantiomer, dextramisole, was found to inhibit this action in a manner indicative of competitive binding to the monocyte (Pike and Snyderman, 1976). The a-adrenergic action of (-)-isoprenaline is antagonized by its (+)-enantiomer, which is devoid of a-activity, as evidenced by the much reduced activity of the racemate (Fig. 23) (Ariens, 1963). Another interesting pharmacodynamic interaction is that demonstrated between the enantiomers of propranolol. (S)-Propranolol causes a decrease in liver

\

dl-isopropylart.

I-artereno1

1-isoprop ylart .

xio-*

*lo-*

I

d-isopropyla rt. *i0-2

x10-*

mM

Fig. 23 Cumulative log dose-response curves for the optical isomers and the racemic mixture of isopropylnoradrenaline (isoprenaline or isopropylarterenol) tested on the vas deferans of the rat (Ariens et al., 1983). Although the (+)-enantiomer was apparently inactive, the a-adrenergic activity of the racemate was less than expected, because (+)-isoprenaline was in fact a competitive antagonist. (Reprinted with permission of the author and the copyright holder, Blackwell Scientific Publications, Oxford.)

Molecular Asymmetry

101

blood flow as a result of P-blockade. Consequently, as the propranolol enantiomers are high-clearance drugs, the clearance of the (R)-enantiomer is decreased when administered as the racemate (Nies et al., 1973, 1976; Branch ef a l . , 1973). Presumably, this interaction also occurs for other racemic highclearance P-antagonists. Finally, as discussed previously, enantiomers may compete for any active transport process, such as renal secretion. Terbutaline appears to be an example of this situation (Borgstrom et al., 1989). The inevitable conclusion is that the racemic drug is not necessarily the simple sum of the activities of the component enantiomers. The relationships are even more complex when, as indicated by the above pharmacokinetic data, the relative proportion of the enantiomers available for interaction at the relevant receptors is changing with time.

VI. Enantioselective Drug-Enantiomer Interactions It should not be surprising that, besides the potential for enantiomers to interact with each other, a drug may interact selectively with a pair of enantiomers, given that enantiomers are distinct pharmacological entities. The enantioselective drug interactions with warfarin are numerous (Table XI) and are an important illustration of the need to understand the enantioselective disposition of a drug to appreciate the subtle interplay of forces which may occur. (S)- and (R)-warfarin appear to differ pharmacodynamically only in the degree of inhibition of vitamin K , -2,3-epoxide reductase (Choonara ef ul., 1986b). When relative concentrations of the unbound enantiomers are taken into account, the (S)-enantiomer is about eight times more active than the (RFenantiomer, as discussed previously (Toon and Trager, 1984). The metabolism of the (S)-enantiomer is primarily oxidative, while the (R)-enantiomer is both reduced and, to a lesser extent, oxidized. The most recent data do not support the view (occasionally still being cited) that the interaction between warfarin and drugs such as phenylbutazone and sulfinpyrazone, leading to prolongation of bleeding times with no change in the plasma kinetics of warfarin, involves inhibition of the metabolism of the more active (S)-enantiomer and induction of the metabolism of the less active (R)enantiomer, or is simply a protein binding displacement interaction. Rather, it is a combination of these phenomena. That is, there is inhibition of the oxidation of (S)-warfarin and also of the (R)-enantiomer. However, the effect is not as pronounced for (/?)-warfarin as oxidative metabolism accounts for a smaller proportion of its overall clearance. Additionally, an enantioselective protein binding interaction increases the clearance of (R)-warfarin, more than offsetting the decreased clearance due to metabolic inhibition (Fig. 24) (Banfield er al.. 1983;

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Kenneth M. Williams

Table XI Studies of Enantloselective Interactions with Warfarin

in

Humans

Metabolic inhibition

D w

S

R

Protein binding displacement

Reference

-

Omeprazole Cimetidine

Yes Yes

No No

Unknown Unknown

Surfin er ul. (1989) Choonara et ul.

Sulfinpyrazone

Yea

Yes

R >> S

O’Reilly (1982a), Toon er a / .

Enoxacinu Amiodarone‘

Yes Yes

No Yes

No effecth Unknown

Toon p t ul. (1987) O’Reilly er a / .

Unknown

O’Reilly er a/.

R and S

Banfield et a / . (1983). Lewis et ul. (1974). O’Reilly er ul.

( I986a)

(1986)

(1987)

Secobarbital

Induction

( 1 98Oa)

Phenylbutazone

Yes

Yes

(1980b)

Aminoglutethimide‘

Induction

Roxithromycin

No effect

Unknown

Lonning er ul.

’? induction

Yes No

Unknown Unknown Unknown

Paulsen et ul. ( I 988) O’Reilly ( 198 I) O’Reilly (1982b) Bjornsson et ul.

No No

Yes Yes

Unknown Unknown

O’Reilly (1976) O’Reilly (1980)

( 1986)

Disulfiramd Ticrynafen Clofibrated

No effect NO

(1977)

Metronidazolep TrimethoprimiSulfamethoxazole

“No effect on prothrombin time. hRacemate. “Nonenantioselective. dEnantioselective pharmacodynamic effect on (.%warfarin. PRat studies indicate possible effects on protein binding and a pharmacodynamic effect as well as enantioselective inhibition of (,.?)-warfarin metabolism (Yacobi er a/., 1984).

Toon et al., 1986). That the effects so neatly cancel each other so that there is no change in the plasma kinetics of the racemic drug appears to be a purely random and probably unusual event. Interactions with warfarin are numerous and are often, but not universally, enantioselective. In vitro studies have demonstrated that both meclofenamate and phenylbutazone displace the enantiomers of warfarin from human serum albumin (Li et d . , 1988). The interesting feature of

Molecular Asymmetry

Lk

103

A

Total plasma

.s-

1 9

0.05

Unbound drug

0.01

1

0.5

0.005

0.001

0.1

c

B

0.005

-

10

Time (days)

0.001 0 2 4 6 Time (days)

810

Fig. 24 The total and estimated unbound concentrations of ( A ) (Rbwarfarin and ( B ) (S)warfarin following oral administration of (RS)-warfarin ( I .5 mgikg) to a subject, before ( 0 )and 4 days after (I a) regimen of phenylbutazone (100 mg, 3 times daily). (From Banfield e t a / . . 1983. Reprinted with permission of the author and the copyright holder, Macmillan Publishers, Hampshire. )

these data was that there was opposite displacement enantioselectivity, with meclofenamate displacing (R)-warfarin to a greater extent and phenylbutazone displacing (S)-warfarin more effectively. While there are thus a large number of enantioselective interactions with warfarin, interestingly there appears to be no significant pharmacokinetic or pharmacodynamic enantiomer-enantiomer interaction for this drug (Levy el al., 1978; Hignite et al., 1980). That is to say, the pharmacology of the racemate Is the sum of the individual enantiomers. Interestingly, the greater clearance of ( S ) warfarin is compensated by its more shallow concentration-effect curves, such that there was found to be no significant difference between the enantiomers in their maintenance of prothrombin complex activity over a dosage interval (Wingard and Levy, 1977). It has been suggested, therefore, that the only advantage in the use of the (R)-enandomer alone might be to lessen the frequency of drug interactions (O'Reilly, 1976; Wingard and Levy, 1977). However, in the light of

104

Kenneth M. Williams

more recent data (Table XI), it appears that interactions with the enantiomers are unpredictable and approximately evenly distributed between both eutomer and distomer. Indeed, even for those drugs where the interaction is primarily with the (S)-enantiomer, an occasional patient may have potentiation of the hypoprothrombinemia with the (R)-enantiomer (O’Reilly, 1976). It is clear, however, that interactions with racemic drugs ought to be treated as an interaction with two drugs. The additional insight to be obtained by attention to the effects on the enantiomers is not the least of the benefits from a stereospecific approach. Not surprisingly, similar interactions have also been reported for other coumarin anticoagulants. Cimetidine enantioselectively inhibits the metabolism of (R)-acenocoumarol, as observed also for warfarin (Gill et al., 1989). Cimetidine also enantioselectively inhibits the enzymes responsible for the first-pass metabolism of metoprolol, the effect being significantly greater for the inactive (R)enantiomer (Toon et al., 1988). As noted by Gill et al. (1989), the enantioselective inhibition elicited by cimetidine is an interesting finding and illustrates the selective action of cimetidine, which is frequently considered to be a relatively nonselective inhibitor of drug oxidation. Another potential enantioselective metabolic interaction is that of enzyme induction. An interesting illustration of this phenomenon is that displayed by phenylpropionic acid. (S)-2-Phenylpropionic acid induced the rat liver enzyme bilirubin UDPglucuronosyltransferase and the enzyme(s) responsible for the 12hydroxylation of lauric acid. The lesser degree of induction by the (R)-enantiomer is almost certainly attributable to (S)-2-arylpropionic acid formed by inversion of the (R)-enantiomer in vivo (Fournel er al., 1986). It would be interesting to investigate this phenomenon further and compare the effects of other inverted and noninverted 2-arylpropionate enantiomers on these enzymes. These data are also further evidence of the interesting relationships between lipid metabolism and the metabolism of the 2-arylpropionates. Warfarin has also been reported to interact enantioselectively with the binding of benzodiazepines at albumin binding sites (Fitos et al., 1986). These interesting allosteric interactions result in both increased (Fig. 25) and decreased binding of the (S)-benzodiazepines, a greater change being elicited by (S)-warfarin than by (R)-warfarin. The greatest interaction was observed between (S)-warfarin and (S)-benzodiazepines, notably with (S)-lorazepam acetate. An added complexity was that the increased binding of (S)-lorazepam acetate induced by (S)-warfarin was inhibited by (R)-warfarin. Furthermore, there was a mutual effect of the benzodiazepines on the binding of the warfarin enantiomers. While these are a complex set of data, they clearly illustrate the need to address the disposition of the enantiomers in order to interpret the interactions correctly at the molecular level (Fitos et af., 1986). They also suggest that, while drug-protein interactions may not be associated with definitive structural changes of either partner (AlebicKolbah et al., 1979), this idea should not be too generalized. These allosteric interactions support the view that there may be definitive alterations in the

Molecular Asymmetry

I05

Fig. 25 Displacement of [14C]-(S)-lorazepamacetate (0, 11.6 pM) and [ i4C]-(R)-lorazepam 11.6 (LM) from human serum albumin microparticles (15.5 p W ) by (RS)-warfarin; acetate (0, 27% of (S)-lorazepam acetate and l4%, of (R)-lorazepam acetate were bound in the controls. (From Fitos rt ul.. 1986. Reprinted with permission of the authors and the copyright holder, Pergamon Press, Oxford.)

protein structure and it is reasonable to assume that binding will be associated with specific drug conformations. The observation that quinidine was a potent inhibitor of sparteine metabolism in vitro (Otton er al., 1984) led to investigations showing that extensive metabolizers were converted to poor metabolizers of sparteine (Brinn et al., 1986) and debrisoquin (Brosen et al., 1987). In the light of earlier discussion, it is not surprising that there was enantioselective inhibition, which was particularly evident in extensive metabolizers treated with quinidine (Leemann et al., 1986) (Fig. 26). An interesting illustration to complete this discussion on drug-enantiomer interactions is that reported by Smith (1989b). Lithium was found to potentiate the inhibitory effect of (- )-tranylcypromine on monoamine oxidase. This effect was apparently mediated by a conformational change in the enzyme induced by the lithium.

VII. Enantiomeric Impurity and Potential Problems The subject of enantiomeric purity is one which has often been ignored when data are presented comparing the differences in the pharmacokinetics and pharmacodynamics of a pair of enantiomers. Particularly prior to the advent of sensitive and enantiospecific chromatographic techniques, optical purity was assessed by determining the optical rotation, an inadequate approach to guaran-

106

Kenneth M. Wil/iams

EM

Qd

metoprolol ‘ng’m‘

’I

Fig. 2 Plasma concentrations of (+)-metoprolo1 and (-)-metoprolo1 measure in five extensive metabolizers (EM) 3 hours after an oral dose of (RS)-metoprolol (100 mg. oral), and in the same volunteers after ingestion of quinidine (+Qd; 50 mg quinidine sulfate capsule). (From Leemann er al.. 1986. Reprinted with permission of the authors and the copyright holder, Springer-Verlag, New York.)

tee sufficient purity to give confidence in reported eudismic ratios (Barlow et al., 1972). It was thus suggested that purification of the distomer to minimum biological potency was a more reliable index of optical purity (Lands et al., 1954). Using this approach it was demonstrated that (+)-isoprenaline, after initial purification [[all,= +32.8; estimated to contain 10% (-)-isoprenaline impurity], had a eudismic ratio [(-)/(+); vasodepression, cats] of 3-6, which was of the same order of magnitude as previously reported. In contrast, the “pure” preparation (i.e., recrystallized to constant biological potency; [aID= +39.3) had a eudismic ratio of 1000 (Lands et al., 1954). Similarly, it was concluded that

Molecular Asymmetry

107

sulindac enantiomers had the same potencies in animal models of inflammation (Shen and Winter, 1977). However, the specific rotation reported for (+)-sulindac ([a],= +22.6) in this study was considerably lower than that more recently reported ([&ID = +61.6; Light et al., 1982). Finally, the interplay between enantiomeric impurity and the contribution of enantioselective pharmacokinetics can be very important, as now illustrated for the case of thyroxine. Thyroxine is used according to the therapeutic indication as either the Lenantiomer for thyroid hormone replacement therapy or the D-enantiomer, which has found some use as a hypolipidemic drug. The physiological potency of natural L-thyroxine was said to be 15-40 times that of the D-enantiomer, and Lthyroxine is used as hormone replacement therapy for athyreotic patients. The Denantiomer was found to have an hypolipidemic action without the associated hypermetabolic activity of the natural enantiorner. Subsequently, an assay based on the use of D-amino acid oxidase was developed and it was shown that the commercial D-enantiomer was contaminated with up to 15% of the natural enantiomer (Mather and Carroll, 1963). This was then rectified and current preparations of choloxin, the hypolipidemic D-enantiomer, contain between 0.5 and 2% of the L-hormone. These data raise the general question as to what level of enantiomeric “contamination” is acceptable when we choose to use an enantiomerically “pure” drug. It may appear that less than 2% is quite acceptable, after all some enantiomeric drugs would have this order of contamination, e.g., commercial samples of L-methotrexate were reported to be contaminated by 0.6 to 7.1% of the Denantiomer (Cramer et al., 1984). At this point, however, differences in the pharmacokinetics of the enantiomers may become significant. Thus, L-thyroxine is eliminated much more slowly than the D-enantiomer, as reflected by data for the enantiomer concentrations attained following chronic dosing with choloxin containing 1.9% of the L-enantiomer (Fig. 27) (Young et al., 1984). Under these circumstances, the concentrations of the L-enantiomer are about the same as those achieved by dosing with Synthroid, the L-enantiomer, when using this drug for hormone replacement therapy. Even when the choloxin formulation contained as little as 0.5% contamination, it was sufficient to generate therapeutically active plasma concentrations of the L-enantiomer. While it is true that this may be a somewhat unusual situation, these data not only illustrate the potential magnitude of the problem but also throw into question all previous data reported on the relative activities of the enantiomers of this drug. Thus, when there is not a concordance in data from different studies comparing the activities of enantiomers, enantiomeric contamination should always be considered. Purification to constant biological activity is a better criterion for optical purity than is optical rotation (Patil er al., 1970). However, optical purity should now generally be confirmed by stereospecific assay.

108

Kenneth M. Williams

P r e t r e a t m e n t Choloxin

Lot A Choloxin Lot B

Synthroid

Fig. 27 Mean ( ? SEM) serum TT4 levels measured by radioimmunoassay and L-T4 and D-T4 measured enantiospecifically in nine subjects with hypothyroidism before treatment and after three , D - T (8 ~ periods of thyroxine therapy (Young et al., 1984): Choloxin, Lot A: I .9% L - T ~98.1% mg/day, 5 months); choloxin, Lot B: 0.5% L - T ~99.5% , D - T ~Synthroid: ; 98.1% L - T ~1.9% , DT4. (Reprinted with permission of the authors and the copyright holder, C. V. Mosby Company, St. Louis.)

VIII. Enantiomers as Biochemical Probes The property of enantiomers that they have identical physical characteristics and often have distinct actions at receptors makes them ideal probes for understanding biological systems and understanding the mechanisms of drug action. Thus, there have been an increasing number of instances where advantage has been taken of these features to use enantiomers as biochemical probes (Table XII). The comments of Pasteur on the difference in tastes of the asparagines have been discussed previously. In particular, Pasteur concluded that the taste “nerve” must be asymmetric. Clearly, enantiomers are excellent probes for determining structure-activity relationships. The important contribution of Easson and Stedman ( 1933) to understanding the structural requirements of receptors has received previous attention and studies have continued to address the mechanisms of taste and smell. An interesting use of enantiomers to understand structure-activity is that of Brunner and Muller (1987), who have characterized the binding site on a,-acid glycoprotein. They adopted a method commonly used in receptor-radioligand binding studies, i.e., to label the binding site with two specific radiolabeled ligands. They then studied the ability of various enantiomers to displace the ligands, concluding that there is a single binding site on &,-acid glycoprotein and reaffirming the enantioselectivity of this binding site.

109

Molecular Asymmetry

Table XI1 Some Examples of the Use of Enantiomers as Biochemical Probes General area

Specific application

Reference ~

SARsa

Gustatory nerve Asparagines Amino acids Adrenergic receptors Phenethylamines Olfactory receptors Carvone

DNA

Mechanisms of drug action

Drug disposition

Menthol DNA mapping Organometal complexes Site specific cleavage Antitumor drug design actinomycin analog Cerebral ischemia Propranolol Anticonvulsants Propranolol Metabolism versus blood flow Propranolol Mephenytoin Polymorphic metabolism Bufuralol Debrisoquin Protein binding a , Acid glycoprotein Methotrexate

Pasteur ( 1886) Solms et a / . (1965). Lehmann ( 1978) Easson and Stedman ( 1933) Koch and Gilliland ( 1977), Leitereg er a / . ( 1976) Eccles et al. (1988d.b) Barton (1986) Mei and Barton (1988) Sengupta et a / . (1988) Little ef a!. (1982) Fischer et a/. (1985) Nies et al. (1976) Arne et a / . ( 1 988) Dayer et a / . ( 1985) Eichelbaum el a/. (1988) Brunner and Muller (1987) Hendel and Brodthagen (1984)

USARs, Structure activity relationships

Mention has also been made of the poor absorption of D-methotrexate in man because it is not a substrate for active transport. This feature enabled Hendel and Brodthagen (1984) to use the D-enantiomer as a marker to estimate the enterohepatic recycling of the L-enantiomer. The D-enantiomer is unlikely to have either active secretion or reuptake in the renal tubules. This suggests that the Lenantiomer also is not actively secreted or reabsorbed since it has the same renal clearance. A number of studies have used the enantiomers of the P-blockers to distinguish between pharmacological actions which are mediated by P-blockade and those mediated by other effects such as membrane stabilization. Thus, it appeared, for example, that the protective effects of propranolol in ischemic tissue

110

Kenneth M. Williams

following experimental middle cerebral artery occlusion were not secondary to P-blockade (Little et al., 1982). Similarly, the anticonvulsant action of propranolol was not dependent on a p-antagonist action as the (+ )=enantiomer was more efficacious than the P-antagonist, (-)-propranolol (Fischer et al., 1985). Reference has already been made to the studies of Nies et al. (1976), who showed that the differences in the effects of the enantiomers of propranolol on liver blood explained why, although (+)-propranolol and the racemic drug were cleared equally by the liver in v i m , the clearance of the racemate was less than that of the (+)-enantiomer in vivo. It is clear from earlier discussion that there have been useful insights into polymorphic drug disposition from the study of the enantiomers of such chiral marker compounds as mephenytoin and debrisoquin. Thus, the enantioselectivity displayed by the 4-hydroxylation of debrisoquin may be a more sensitive criterion for phenotypic assignment than the use of the current metabolic ratio (Eichelbaum et al., 1988). A further recent suggestion has been that a study of the relative rates of clearance of the enantiomers of mephenytoin may be used as a marker of hepatic function (Arne et al., 1988). While double-stranded DNA normally adopts a right-handed helical conformation (A- and B-DNA), some sequences take on a left-handed helical form (ZDNA; Dickerson et al., 1982). Chiral metal complexes, such as those formed between phenanthroline or 4,7-diphenylphenanthroline and ruthenium, have been found to be useful for probing the local helical structure of DNA and to distinguish between the left- and right-handed helical conformations (Barton et al., 1984;Barton, 1986). By combining the selective intercalation of chiral metal complexes to the DNA with subsequent photoactivation, DNA can be cleaved at specific sites (Mei and Barton, 1988). Alternatively, the chiral complex can be used to introduce a redox active metal, such as ferrous or cuprous ions, to specific DNA sites. These artificial nucleases cleave the DNA by generating local high concentrations of hydroxyl free radicals (Barton, 1986). Even in the absence of a metal complex, chiral intercalating agents, such as derivatives of actinomycin D, can be DNA-acting antitumor agents (Segupta et a l . , 1988).

IX. Therapeutic and Regulatory Considerations In 1925, even Cushney, whose important contributions to understanding the actions of “optically isomeric substances” have been discussed, did not see the potential importance of his data to therapeutics. He wrote “I have often been asked how research in such fundamental questions as I have been discussing . . . [i.e., the pharmacological actions of enantiomerically related substances] is to be justified; of what profit is it to us, and how does it benefit medicine? And I must admit that it is of minor practical importance, it has little direct application to the use of these remedies in therapeutics to know that the

111

Molecular Asymmetry

natural adrenaline is twice as powerful as the synthetic, for this can be compensated by doubling the concentration of the latter” (Cushney, 1926). Cushney’s lack of insight on this matter is one which still lingers in the literature. However, the various data presented in preceding discussion should allow no other conclusion but that enantioselective differences in drug action and disposition are of the utmost relevance and importance to therapeutics. The much cited example of thalidomide is one which has been used to highlight this conclusion. The events surrounding the development and release of thalidomide, followed by the recognition of its teratogenic action and its subsequent withdrawal, were the impetus for significant ammendments to the Food and Drug Act by the FDA in 1962 (Kumkumian, 1988). Thalidomide is a racemic drug whose enantiomers appear to have equal hypnosedative activities. Subsequent data suggested that not only was it the metabolites of thalidomide which were teratogenic, but more precisely only the metabolites of the (S)-enantiomer were toxic (Faigle et al., 1962; Blaschke et al., 1979). There has been some argument about the validity of these conclusions for thalidomide because of factors such as species variability and the potential for racemization (Schmahl et al., 1988; for more detailed discussion see Williams, 1990b). Whatever, the final conclusion, the data do illustrate that there are important therapeutic and regulatory implications to be addressed when dealing with chiral drugs. For the specific case of thalidomide, these questions are still relevant with the reemergence of interest in the drug because of its effectiveness in indications such as graft-versus-host disease and rheumatoid arthritis (Kaitin, 1988; Gutierrez-Rodriguez, 1984). Enantiomeric drugs may thus have similar potencies, but enantioselective metabolism may lead to differential toxicities of the enantiomers via formation of toxic metabolites. It is logical to raise the question as to the therapeutic and regulatory implications of this knowledge, i.e., should we be using only enantiomerically pure drugs or are racemic drugs acceptable in some or even the majority of situations? This question has already received some attention in the literature (Rahn, 1983; Kumkumian, 1988; Baldwin and Abrams, 1988; Carter, 1988; Reidenberg, 1988; De Camp, 1989). The therapeutic options are really 4-fold (Table XIII). Besides enantiomers and Table X l l l Chiral Drugs-The

Therapeutic Options

Composition

Examples

Archiral Enantiomer Nonracemate Racemate

Fentanyl, Indomethacin Dopa. Methotrexate Indacrinone Dobutamine, propranolol

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Kenneth M. Williams

racemates there are the two further options, namely, nonracemic mixtures and achiral drugs.

A. Nonracemic Drugs There are data to suggest that a ratio of between 1:4and 1:8 of the enantiomers of the loop-acting diuretic indacrinone may be optimal because this gives a clinically improved balance between the uricosuric and natriuretic activities of the individual enantiomers as contrasted with the racemic drug (Tobert et al., 1981; Vlasses et al.. 1981, 1984; Blaine et al., 1982; Field et al., 1984). However, fixing the ratio in a single formulation still may not be optimal, because presumably one combination is not likely to be ideal for all patients.

B. Achiral Drugs It has been argued that, rather than complicating issues by using chiral drugs, use of an achiral drug should be the first option. For example, fentanyl is an achiral drug whose analgesic and anesthetic activities are much greater than those of the chiral morphine (Soudijn, 1983). Similarly, if anti-inflammatory activity is required, it could be argued that indomethacin is a potent achiral anti-inflammatory agent which could be used instead of the chiral 2-arylpropionates. However, although this may be the case, it is clear that indomethacin, while very potent, has more than its share of central side effects and fentanyl is not the perfect agent for induction of anesthesia. Similarly, ibuprofen is effective in the management of pain and inflammation associated with the rheumatic diseases but it is chiral and is administered as the racemate, although activity resides in the (S)-enantiomer. We can make an analogous achiral anti-inflammatory drug, ibufenac, by replacing the 2-methyl substituent with hydrogen (Fig. 28). However, ibufenac, which actually preceded

%COO.

\ /

CH3

%COO.

\ / lbufenac

(S)-i buprofen

2-methyllbuprofen

Fig. 28 (S)-Ibuprofen and two achiral derivatives. Ibufenac had anti-inflammatory activity but was hepatotoxic. The 2-methyl-substituted derivative was devoid of anti-inflammatory activity.

Molecular Asymmetry

113

ibuprofen in clinical development, has reduced anti-inflammatory activity, but more importantly, this drug was associated with hepatotoxicity and was withdrawn from clinical use (Adams er al., 1970). The similarly achiral 2-methylibuprofen (Fig. 28) has no anti-inflammatory activity. Chirality is integrally and inseparably an aspect of molecular geometry (a point clarified in Easson and Stedman, 19331, which means that we cannot, therefore, necessarily remove the asymmetry without a change in the desired activity. There are circumstances where an achiral drug is the drug of choice and chirality is not always essential for good activity, as appears to be the case for some of the platelet-activating factor antagonists (Grue-Sorensen el al., 1988). However, in general, it may be better to use a pure enantiomer which is specific for the desired action.

C. Racemic Drugs The therapeutic index for some drugs, it is argued, will not be improved by use of the distomers. Two examples which have been cited are dobutamine and propranolol. Because the a and p activities of dobutamine primarily reside in the opposite enantiomers, the racemic drug has the combination of pharmacological actions which makes it suitable for use in congestive heart failure, since it increases cardiac output with little effect on heart rate or diastolic pressure, i.e., in this instance, the pressor effect of (-)-dobutamhe is offset by the combined P,-agonist and a,-antagonistic activities of the (+)-enantiomer (Ruffolo et al., 1981; Ruffolo and Yaden, 1983; Ruffolo and Messick, 1985). (R)-Propranolol on the other hand, apparently contributes nothing significant to the pharmacological action of the racemate, but at the same time appears to have no discemable toxicity. Therefore, it has been suggested that, for this reason, there is little value in resolving the enantiomers (Rahn, 1983). Flecainide might also be added to this list, as there is no apparent difference in the antiarrhythmic activities (Smallw6od et al., 1989; Hill et al., 1988) of its enantiomers, although there is still the potential for enantioselective metabolism and its sequelae to consider. Clearly, if the asymmetric center is removed from the critical binding areas of the molecule, both in terms of the pharmacodynamic interactions and in terms of metabolic reactivity, there will be no difference in the therapeutic index for the enantiomers of the drug.

D. Enantiomeric Drugs and the Considerations In a purely intrinsic, scientific sense, with relatively few exceptions, the use of racemates is an untenable position. The requirement that drug formulations must be of the highest purity, in terms of absence of starting materials and by-products of synthesis, is one which is hard to sustain when we are ready to accept at least a 50% presence of an entirely different pharmacological species, the distomer. Such arguments have been vigorously pursued in the literature (Ariens, 1984,

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1988, 1989; Ariens et al., 1988) and need no further elaboration here. However, to conclude that enantiomers are, therefore, clinically superior and should always be used is an extrapolation which is not so easy to sustain given our present state of scientific ignorance. Those who advocate a somewhat more cautious and pragmatic approach suggest that we must more closely assess the clinical superiority of enantiomeric preparations and the feasibility and costs associated with their preparation. It cannot be reasonably argued that it cannot be cost effective to use enantiomers, after all there are a significant number of drugs which have been developed as enantiomers (Table XIV). Some of these, such as thyroxine and propoxyphene (Darvon, narcotic analgesic; Novrad, antitussive) are used as each of the individual enantiomers. Some are derived from natural products (pethidine) but others are resolved from the racemate (naproxen) or are prepared by enantiospecific synthesis (timolol). There are several questions which need to be addressed in the decision as to whether to use an enantiomer rather than the racemate. 1. Is toxicity associated with the distomer (a basis for using enantiomerically pure drug) or is toxicity an extension of the therapeutic effect of the eutomer?

Not uncommonly, the toxicity that is of concern is that mediated by mechanisms common to the action of the drug. For example, the major toxicity of the 2arylpropionates is the gastrointestinal disturbances and the adverse effects on the kidney in patients with compromised renal function. These effects are mediated by inhibition of prostaglandin synthesis, the action which also induces analgesia and is the basis for the anti-inflammatory action. That is, toxicity is simply an extension of the therapeutic efficacy of the drug. Preparation of the (S)-enantiomers of the 2-arylpropionates will not overcome this particular problem. 2. Is there interconversion of the enantiomers in vivo, either by inversion or racemization?

Table XIV Some Examples of Enantiomeric Drugs Amphetamine Arbaprostil Codeine Deprenyl Dopa Isoprenaline Methotrexate

Methyldopa Naproxen Penicillarnine Pethidine Propoxyphene Thyroxine Timolol

Molecular Asymmetry

115

Some drugs (Table VII) racemize in solution at a biologically significant rate, as discussed earlier (Section II,B,3,c). Clearly, there is no point in resolving a drug such as oxazepam. Similarly, the natural anticholinergic product is (S)hyoscyamine, but the drug rapidly racemizes in solution to give atropine which is racemic (Ariens, 1988). However, that is not to say that all drugs which racemize should be used as racemates. Arbaprostil, a stable prostaglandin analog, is administered as the inactive ( 1 5R)-enantiomer but undergoes pH-dependent epimerization in the stomach. The acidity of the stomach controls the rate of epimerization and in this respect it is self-titrating with regard to the amount of active (S)-enantiomer present in vivo. This approach limits to some extent the amount of active drug available for systemic absorption and therefore limits potential systemic toxicity. Chiral inversion of the 2-arylpropionates may also be a contraindication for use of the (S)-enantiomer, except that presumably this would remove one more factor likely to contribute to variability in response (Williams and Day, 1985, 1988; Day et af., 1988b) and the potential problems of hybrid lipid incorporation of the (R)-enantiomers have yet to be resolved. The efficacy of (S)-ibuprofen has recently been demonstrated not unexpectedly in patients with rheumatoid arthritis (Geisslinger er al., 1990), although it has yet to be shown to be superior to the racemate. 3. HOWmuch contamination by the distomer is acceptable? This is a question which needs to be addressed for each individual drug and requires assessment of the pharmacokinetic and pharmacodynamic characteristics of the distomer. The earlier discussion of thyroxine is an important illustration in point. 4. Defining the pharmacokinetics of the eutomer

For those drugs which are presently used as racemates or for which there is reasonable argument to use as racemates (such as lack of any demonstrable toxicity of the distomer), should we insist that the pharmacokinetics of the eutomer be defined'? This would appear to be a reasonable expectation, after all it is expected that assay methodology used for pharmacokinetic studies should be capable of separating a parent drug from metabolites, whether these are active or inactive. It should now be clear that this is no different to the situation with enantiomerically related drugs. 5. Which enantiomer?

Discussion will now focus specifically on this last and apparently most obvious of questions by addressing data for penicillamine, a representative drug already used as the pure D-enantiomer. The aim is to highlight a few of the

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implications, both therapeutic and regulatory, for the use of chiral drugs generally. Why D-penicillamine? Why is it that we use D-penicillamine and not the Lenantiomer? After all, there is an “intuitive feeling” that D-amino acids and their derivatives may cause toxicological problems. D-Glutamic acid has been found to be a potent immunosuppressant, D-serine to cause renal tubular necrosis, and the D-glutamate metabolites produced from thalidomide have been implicated in the teratogenicity of this drug. Penicillamine was first introduced for the treatment of Wilson’s disease, where the chelating properties of penicillamine effectively promoted excretion of the accumulating copper. Clinical data, while limited (Table XV), suggested, however, that L-penicillamine was responsible for the nephrotic syndrome occasionally observed in patients being treated with the racemate. Thus, in a review of eight such cases of patients treated with the racemate (Sternlieb, 1966), three patients rechallenged with ~,~-penicillamine again developed the syndrome while two recommenced on the D-enantiomer were able to continue with this

Table XV A Comparison of the Activities of

and L-Penicillamine Adverse effect

Rx

Clinical data Wilson’s disease

D-

Reference

Sternlieb ( I 966)

Wilson’s disease

DL

Nephrotic syndrome nil Optic neuritis

Wilson’s disease

DL

Thrombocytopenia/leukopenia

DL D

Relative activities

Tu et a/. ( I 963). Goldstein et al. (1966) Scheinberg (1968) Reference

Animal data LDSO

Toxicity

D:

>1200 mg/kg;

DL: =

365 mglkg

weight loss, fitting, death no symptoms (choline deficient) L is 8 times more mutagenic than D The same or L > D L:

D:

Mutagenicity Antipyridoxal

Collagen synthesis

Uptake Of DL > D Increased soluble collagen

DL

>D

Aposhian (1958). Aposhian and Aposhian (1959) Wilson and du Vigneaud (1948, 1950) Glatt and Oesch (1985) Kuchinskas and du Vigneaud (1957). Kuchinskas er al. ( 1957) Planas-Bohne (1981) Ruiz-Torres (1974). Nimni and Bavetta (1965), Nimni et al. (1969)

Molecular Asymmetry

117

therapy without the renal complications. Other more tenuous data have linked use of the racemate with optic neuritis and blood discrasias. Various animal data also generally indicated a greater toxicity for the Lenantiomer (Table XV). Thus, it was reasonably concluded that because the chelating properties of the enantiomers were equal, the therapeutic index was better for the D-enantiomer. However, the primary indication now for use of penicillamine is not Wilson’s disease but rheumatoid arthritis and for this indication the relative activities of the enantiomers are unknown. That is, while having chosen to use D-penicillamine for rheumatoid arthritis, presumably on the grounds of the apparent toxicities of the L-enantiomer, this was done without any data establishing the efficacy of the L-enantiomer for the primary indication. For future drug development, no assumption should be made that because an enantiomer is the eutomer for one application, it will be the eutomer for another. For a more detailed discussion of the enantiomeric disposition of penicillamine the reader should refer to other reviews (Howard-Lock et al., 1986; Williams, 1990b).

E. Racemate or Enantiomer? The Choice Which drugs presently used as the racemates might usefully be resolved? This is rather a difficult question because of the factors already discussed. Attention has been given to labetalol with preparation of (RR)-levalol (Baum et al., 1981; Sybertz et af., 1981; Baba et al., 1988). It has been suggested that there are sufficient data to suggest that many of the adverse postoperative effects of ketamine are mediated by the less potent anesthetic (- j-enantiomer (Marietta et al., 1977; Ryder et al., 1978; Meliska et a l . , 1980; White e f al., 1980) and, therefore, that consideration could be given to its resolution (Soudijn, 1983). Fenfluramine has also attracted some interest, with the side effects having a greater association with the (-1-enantiomer and the (+)-enantiomer being the anorexic agent (Pinder er af., 1975). On a pragmatic basis, racemic drugs which are already marketed will continue to be used. Additionally, we must accept that those drugs which have already reached a significant level of development should not now be rejected by the Regulatory Authorities simply because they are racemic. It must be recognized that these drugs have been thoroughly assessed and, in general, we know their limitations.

F. Bioavailability Studies It is generally true that regulatory agencies require the pharmacokinetics of a drug as the basis for safe and effective dosage regimes to be established. It follows that, if drugs are administrated as racemates, enantiospecific assays should be used to define the pharmacokinetics of the active enantiomer. In

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particular, bioavailability studies should compare the disposition of the active enantiomer. The first of such studies has recently been reported for ibuprofen (Fig. 29) where bioinequivalence of the active (S)-enantiomer in the two formulations was demonstrated (Cox et al., 1988), although it is not clear that the differences will be clinically significant.

TIME AFTER DRUG ADMINISTRATION (hours) Fig. 29 Average ibuprofen enantiomer concentration-time data from 1 1 subjects following ingestion of a single tablet (400 mg) of one of two (R,S)-ibuprofen preparations: 0, (S)ibuprofen; A , (R)-ibuprofen. (From Cox er al., 1988. Reprinted with permission of the authors and the copyrighf holder, John Wiley and Sons, Sussex.)

Molecular Asymmetry

1 I9

X. Therapeutic Drug Monitoring A significant contribution to improvement in the rational use of drugs has been the advent of blood concentration monitoring of drugs. This has been based on the thesis that there should be a better correlation between the plasma concentration of a drug and its effect than between the dose of a drug and effect. Studies have now established therapeutic ranges for a number of commonly prescribed drugs with narrow therapeutic indices, such as the anticonvulsants, antidepressants, and antiarrhythmics. It was recognized that, to establish therapeutic ranges, it was important that the assay methodology was specific for the parent drug and did not suffer from interference by metabolites. Thus, chromatographic assays replaced colorimetric and spectrophotometric assays and such “nonspecific” assays were rightly deemed to be unacceptable for such studies. Chromatographic assays also allowed the monitoring of active metabolites, with ranges thus being established for both parent and metabolite, e.g., procainamide and N-acetylprocainamide, amitryptiline and nortryptiline. However, it is equally important that, for those drugs administered as racemates, the concentration-response relationships be based on the measurement of the active enantiomer (Drayer, 1988a,b; Walle and Walle, 1986). While it is true that in some cases there may not be very significant enantioselective elimination, e.g., ketoprofen (Foster et al., 1988a,b), it is more likely to be the case that each enantiomer makes a variable contribution to the total concentration. Indeed, the active enantiomer may contribute little to the total racemic drug concentration, e.g., etodolac (Jamali et a / ., 1988). For this more usual scenario, monitoring the drug concentrations nonenantiospecifically is akin to using a colorimetric assay which does not distinguish between drug and inactive metabolite. The problems associated with pharmacokinetic analysis of racemic drugs using nonenantioselective assays have already been mentioned (Evans et al., 1988) (Section II,B,3,c). Racemic drugs commonly monitored by achiral techniques include disopyramide, flecainide, tocainide, verapamil, mianserin, propranolol, rnexiletine, warfarin, mephenytoin, mephobarbitone, and ethosuximide. Of these, it appears that the enantiomers of flecainide have equal potency and toxicity. Under such circumstances, achiral monitoring of plasma concentrations is reasonable. Additionally, while enantiospecific analyses have led to an understanding of the interactions which occur with warfarin, this drug is more frequently monitored by determining the pharmacodynamic response, viz, the prothrombin time. Generally, however, the active enantiomer should be monitored in any situation where a racemic drug is administered.

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XI. Conclusions The importance of molecular asymmetry impinges on all areas of pharmacology. This is a reflection of the asymmetric environment of the basic biochemical milieu, which means that the biological macromolecules, whether plasma proteins or proteins involved in the active transport of substrates, enzymes, or neuronal receptors, frequently can distinguish between enantiomerically related drugs. This article has illustrated how often both pharmacokinetic and pharmacodynamic differences are expressed by enantiomers. Because of these dissimilarities, enantiomers may interact selectively with other drugs. At the same time, the similarities between enantiomers may lead to interactions between the enantiomers themselves, such that the racemic drug is not the simple sum of the activities of the individual enantiomers. It is clear that interpretation of both pharmacokinetic and pharmacodynamic data based on achiral techniques is fraught with the potential for misinterpretation and erroneous conclusions. Pharmacological enantioselectivities leave some question as to the rationale for the continued use of racemic drug preparations, although the question as to which racemic drugs might reasonably be resolved is one associated with some controversy. Regardless of the significant therapeutic benefit likely to be attained by the use of enantiomerically pure drug preparations, the use of enantiomers as probes is an invaluable asset to further our understanding of the mechanisms of drug action. A chiral approach is required if we are to understand an intrinsically chiral discipline.

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Amos, H. E., Lake, B. G . , and Artis, J. (1978). Possible role of antibody specific for a practolol metabolite in the pathogenesis of oculomucocutaneous syndrome. Br. Med. J. 1, 402-404. Aposhian, H. V. (1958). Protection by D-penicillamine against the lethal effects of mercuric chloride. Science 128, 93. Aposhian, H. V., and Aposhian, M. M. (1959). N-Acetyl-DL-penicillamine,a new oral protective agent against the lethal effects of mercuric chloride. J. Pharmacol. Exp. Ther. 126, 131-135. Ariens, E. J. (1963). Steric structure and activity of catecholamines on (I- and P-receptors. In “Modem Concepts in the Relationship Between Structure and Pharmacological Activity” (K. L. Brunnings and P. Lindren, eds.), Vol. 7, pp. 247-264. Pergamon, Oxford. Ariens, E. J. (1984). Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur. J . Clin.Pharmacol. 26, 663-668. Ariens, E. J. (1986a). Stereochemistry: A source of problems in medicinal chemistry. Med. Res. Rev. 6, 45 1-466. Ariens, E. J. (1986b). Chirality in bioactive agents and its pitfalls. TIPS 7, 200-205. Ariens, E. J. (1988). Stereospecificity of bioactive agents. In “Stereoselectivity of Pesticides. Biological and Chemical Problems” (E. J. Anens, J. J. S. van Rensen, and W. Welling, eds.), pp. 39108. Elsevier, Amsterdam. Ariens, E. J. (1989). Stereoselectivity in the action of drugs. Pharmacol. Toxicol. 64, 319-320. Ariens. E. J., Soudin, W.. and Timmermans. P. B. M. W. M., eds. (1983). “Stereochemistry and Biological Activity of Drugs.” Blackwell, Oxford. Ariens, E. J., Wius, E. W., and Veringa, E. J. (1988). Stereoselectivity of bioactive xenobiotics. Biochem. Pharmacol. 37, 9- 18. Arne, P. A , , Wilkinson, G . R., and Branch, R. A. (1988). The stereoselective disposition of mephenytoin provides a probe of hepatic function and development of porta-systemic shunts in liver disease. Hepatology ( N . Y . ) 8, 1277. Amett, E. M., Gold, J. M., Harvey, N., Johnson, E. A , , and Whitesell, L. G. (1988). Stereoselective recognition in phospholipid monolayers. Adv. Biol. Med. 238, 21-36. Aula, P., Kaila, T., Huupponen, R.. and Salminen, L. (1988). Timolol binding to bovine ocular melanin in vitro. J. Ocular Pharmacol. 4, 29-36. Baba, T., Murabayashi, S., Aoyagi, K . , and Ishizaki, T. (1988). Effects of dilevalol, an R,R-isomer of labetalol, on blood pressure and renal function in patients with mild-to-moderate essential hypertension. Eur. J. Clin.Pharmacol. 35, 9- 15. Bagwell, E. E., Webb, J. G., Walle, T., and Gaffney, T. E. (1989). Stereoselective uptake of atenolol into storage granules isolated from PC12 cells. J. Pharmacol. Exp. Ther. 249, 476-482. Bai, S . A , , Walle, U. K., Wilson, M. J., and Walle. T. (1983). Stereoselective binding of the (-)-enantiomer of propranolol to plasma and extravascular binding sites in the dog. Drug Metab. Dis. 11, 394-395. Baillie, T. A,. Adams, W. J.. Kaiser, D. G., Olanoff, L. S . , Halstead, G. W., Harpootlian, H., and Van Giessen, G . J. (1989). Mechanistic studies of the metabolic chiral inversion of (R)-ibuprofen in humans. J. Pharmacol. Exp. Ther. 249, 517-523. Baldwin, J. J., and Abrams, W. B. (1988). Stereochemically pure drugs: An industrial perspective. In “Drug Stereochemistry. Analytical Methods and Pharmacology” (I. W. Wainer and D. E. Drayer, eds.), pp. 31 1-356. Dekker, New York. Banfield, C., O’Reilly, R., Chan, E., and Rowland, M. (1983). Phenylbutazone-warfxin interaction in man: further stereochemical and metabolic considerations. Br. 1. Clin. Pharmacol. 16, 669675. Barlow, R. B . , Franks. F. M . , and Pearson, J. D. M. (1972). The relation between biological activity and the degree of resolution of optical isomers. J. Pharm. Phurmacol. 24, 753-761. Barton, J. K. (1986). Metals and DNA: Molecular left-handed complements. Science 233, 727-734.

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Toon, S., and Trager, W. F. (1984). Pharmacokinetic implications of stereoselective changes in plasma-protein binding: warfarin/sulfinpyrazone. J . Pharm. Sci. 73, 1671- 1673. Toon, S . , Low, L. K., Gibaldi, M . , Trager, W. F., O’Reilly, R. A., Motley, C. H., and Goulart, M. A. (1986). The warfarin-sulfinpyrazone interaction: Stereochemical considerations. Clin.Pharmacol. Ther. 39, 15-24. Toon, S., Hopkins, K. J., Garstang, F. M., Aarons, L., Sedman, A., and Rowland, M. (1987). Enoxacin-warfarin interaction: Pharmacokinetic and stereochemical aspects. Clin. Pharmacol. Ther. 42, 33-41. Toon. S . . Davidson, E. M., Garstang, F. M., Batra, H., Bowes, R. J., and Rowland, M. (1988). The racemic metoprolol H2-antagonist interaction. Clin. Pharmacol. Ther. 43, 283-289. Topiol, S . (1989). A general criterion for molecular recognition: Implications for chiral interactions. Chirality 1, 69-79. Trager, W. F., and Testa, B. (1985). Stereoselective drug disposition. In “Drug Metabolism and Disposition: Considerations in Clinical Pharmacology” (G. R. Wilkinson and M. D. Rawlins, eds.), pp. 35-61. MTP Press, Boston, Massachusetts. ’h, J.-B., Blackwell, R. Q., and Lee, P.-F. (1963). DL-peniCihmine as a cause of optic axial neuritis. J . Am. Med. Assoc. 185, 119-122. Van Breemen, R., and Fenselau, C. (1985). Acylation of albumin by I-0-acyl glucuronides, Drug Metab. Dispos. 13, 318-320. Verbeeck, R. K., Wallace, S. M., and Loewen, G. R. (1984). Reduced elimination of ketoprofen in the elderly is not necessarily due to impaired glucuronidation. Br. J. Clin. Pharmacol. 17, 783784. Vermeulen, N. P. E. (1986). Stereoselective biotransformation: Its role in drug disposition and drug action. In “Innovative Approaches in Drug Research” (A. F. Harms, ed.), pp. 393-416. Elsevier, Amsterdam. Vlasses, P. H., Irvin, J. D., Huber, P.B., Lee, R. B., and Ferguson, R. K. (1981). Pharmacology of enantiomers and (-)-p-OH metabolite of indacrinone. Clin. Pharmacol. Ther. 29, 798-807. Vlasses, P. H . , Rotmensch, H. H., Swanson, B. N., Irvin, 3. D., Johnson, C. L., and Ferguson, R. K. (1984). Indacrinone: natriuretic and uricosuric effects of various ratios of its enantiomers in healthy men. Pharmacotherapy (Carlisle, Mass.) 4, 272-283. Vogelgesang, B., Echizen, H., Schmidt, E., and Eichelbaum M. (1984). Stereoselective first-pass metabolism of highly cleared drugs: studies of the bioavailability of L- and o-verapamil examined with a stable isotope technique. Br. J . Clin. Pharmacol. 18, 733-740. von Euler, U. S . (1949). Identification of the sympathomimetic ergone in adrenergic nerves of cattle (Sympathin N) with laevo noradrenaline. Acra B i d . Scand. 16, 63-74. Wade, D. N., Meanick, P. T., and Moms, J. L. (1973). Active transport of Ldopa in the intestine. Nature (London) 242, 463-465. Wainer, 1. W., and Drayer, D. E., eds. (1988). “Drug Stcrewhemistry. Analytical Methods and Pharmacology.” Dekker, New York. Walle, T., and Walle, U. K. (1986). Pharmacokinetic parameters obtained with racemates. TIPS 7, 155-157. Walle, U. K., Walle, T., Bai, S . A,, and Oranoff, L. S . (1983). Stereoselective binding of propranolol to human plasma, a,-acid glycoprotein. and albumin. Clin. Pharmacol. Ther. 34, 718123. Wedlund, P. J., Aslanian, W. S., Jacqz, E., McAllister, C. B., Branch, R. A., and Wilkinson, C. R. (1985). Phenotypic differences in mephenytoin pharmacokinetics in normal subjects. J . Pharmacol. Exp. Ther. 234, 662-669. Weir, D. C., Brown, J. P., Freedman, D. S., and Scott, J. M. (1973). The absorption of the diastereoisomers of 5-methyltetrahydropteroylglutamatein man: a carrier-mediated process. Clin. Sci. Mol. Med. 45, 625-631.

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White, P. F., Ham, J . , Way, W. L.,and Trevor, A. J . ( 1980). Pharmacology of ketamine isomers in surgical patients Anesthesiologv 52, 23 1-239. Wiley, R. H . , and Wiley, P. (1964). “The Chemistry of Heterocyclic Compounds. 12. Pyrazolones, Pyazolidones, and Derivatives.” Wiley (Interscience), New York. Williams, K. M. (1984). Kinetics of misonidazole enantiomers. Clin.Pharmacol. Ther. 36, 817823. Williams, K. M. ( 1987). Inversion of enantiomers: 2-arylpropionic acid anti-inflammatory drugs. Pharmacol. f n t . Congr. Ser. (M. I. Rand and C. Raper, eds.), pp. 795-797. Elsevier, Amsterdam. Williams, K. M. (1990a). Metabolic chiral inversion. I n “Problems and Wonders of Chiral Molecules” (M. Simonyi, ed.). pp. 181-204. Akademiai Kiad6, Budapest. Williams, K. M. (1990h). Enantiomers in arthritic disorders. Pharmacol. Ther. 46, 273-295. Williams, K. M., and Day, R. 0. (1985). Stereoselective disposition-basis for variability in response to NSAID’s. Agents Actions, Suppl. 17, 119-126. Williams, K . M., and Day, R. 0. (1988).The contribution of enantiomers to variability in response to anti-inflammatory agents. Agenrs Actions, Suppl. 24, 76-84. Williams, K. M . , and Lee, E. (1985). Importance of drug enantiomers in clinical pharmacology. Drugs 30, 333-354. Williams, K. M.. Day, R . , Knihinicki, R., and Duffield, A. (1986). The stereoselective uptake of ibuprofen enantiomers into adipose tissue. Biochem. Pharmucol. 35, 3403-3405. Wilson, B . K., and Thompson, J. A. (1984). Glucuronidation of propranolol by dog liver microsomes. Drug Mefab. Dispos. 12, 161-164. Wilson, J. E., and du Vigneaud, V. (1948). L-Penicillamine as a metabolic antagonist. Science 107, 653. Wilson, J . E., and du Vigneaud, V. (1950). Inhibition of the growth of the rat by L-penicillamine and its prevention by aminoethanol and related compounds. J . Biol. Chem. 184, 63-70. Wingard, L. B..Jr., and Levy, G . (1977). Comparative pharmacokinetics of coumarin anticoagulants XXXVI: Predicted steady-state patterns of prothrombin complex activity produced by equieffective doses of (R)-(+]- and (S)-(-)-warfarin in humans. J. Pharm. Sci. 66, 1790-1791. Wu, D.-F., Yu, Y.-W., Tang. Z.-M., and Wang, M.-Z. (1986). Pharmacokinetics of (?), ( + ), and (-)-gossypol in humans and dogs. Clin. Pharmacol. Ther. 39, 613-618. Yacobi, A , , and Levy. G . (1977). Protein binding of warfarin enantiomers in serum of humans and rat!,. J . Pharmacokinet. Biopharm. 5, 123- 13 I . Yacobi, A . , Lai, C.-M., and Levy, G . (1984). Pharmacakinetic and pharmacodynamic studies of acute interaction between warfarin enantioniers and metronidazole in rats. J . P harmacol. Exp. Ther. 231, 72-79. Young, W. F., Gornian, C. A , , Jiang, N.-S., Machacek, D., and Hay, I . D. (1984). L-Thyroxine contamination o f pharmaceutical D-thyroxine: Probable cause of therapeutic effect. Clin. Phurmacol. Ther. 36, 781-787.

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Blood- Brain Barrier: Transport Studies in Isolated Bruin Capillaries and in Cultured Bruin Endotheliul Cells Yoshinobu Takakura,* Kenneth 1. Audus, and Ronald T. Borchardt Department of Pharmaceutical Chemistry The University of Kansas Lawrence, Kansas 66045

I . Introduction 11. Establishment and Characterization of an in V i m Blood-Brain Barrier Model A. Isolated Brain Capillaries B. Cultured Brain Microvessel Endothelial Cells 111. In Virro Transport Studies A. Nutrients B. Ions C. Neurotransmitters D. Peptides and Proteins E. Drugs F. Fluid-Phase Endocytosis G. Adsorptive Endocytosis IV. In V i m Studies on Regulation of Blood-Brain Barrier Transport A. Peptide Regulation B. Astrocyte Interactions C. Developmental Changes D.Pathological Changes E. Alteration of Blood-Brain Barrier Permeability by Hyperosmotic Treatment V. Summary References

1. Introduction At the beginning of this century, Paul Ehrlich discovered that distribution of intravenously injected dyes to the brain was highly restricted, in contrast to the rapid distribution of the dyes to all other tissues. Since that time, many concepts *Present address: Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Advuncei m PhurmacoluXy. Volume 22

Copyright 0 1991 by Academic Press, Inc All righls of reproduction in any form reserved

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concerning the barrier between blood and brain have been proposed (Davson, 1989). It is now generally accepted that the cerebral microvessel endothelium is an integral part of the blood-brain barrier (BBB). Unlike peripheral endothelium, brain microvessel endothelial cells are characterized by the presence of highly resistant, tight intercellular junctions, minimal pinocytic activity, and the virtual absence of fenestrations (Betz and Goldstein, 1984). These characteristics endow the endothelial cells with the ability to restrict the passage of most large and small polar blood-borne molecules from the cerebrovascular circulation to the brain. In addition, the cerebral endothelium possesses a high activity of metabolic enzymes (Pardridge, 1983). Therefore, the BBB is not only a physical barrier but also a significant metabolic barrier. As the life expectancy of humans has increased with the development of medical sciences, there has been increased interest in developing drug therapies to treat central nervous system (CNS) disorders associated with old age. For a better understanding of the biochemical, physiological, and pathological characteristics of the BBB, scientists have investigated the way in which endogenous and exogenous substances traverse the interface from the circulation to the brain parenchyma. From a pharmacological perspective, scientists have also been concerned with developing strategies for improving the delivery of drugs to the CNS (Scheld, 1989). Many studies of the BBB have been done using in vivo animal models (Oldendorf, 1971). However, there are limitations to the use of whole animal models for the detailed investigation of the BBB at the cellular level. In vitro models (i.e., isolated brain capillary preparations and appropriate tissue culture systems) offer a promising alterative, which might enable scientists to define the characteristics of the brain capillary endothelium at the molecular and cellular level (Betz and Goldstein, 1984; Joo, 1985; Audus et al., 1990; Audus and Borchardt, 1991). The purpose of this review is to describe studies of the BBB that employ in vitro BBB models consisting of either isolated brain capillaries or cultured brain microvessel endothelial cells.

II. Establishment and Characterization of an in Vitro Blood-Brain Barrier Model

A. Isolated Brain Capillaries The development of methods for the isolation of brain capillaries from animal and human brains has provided a valuable resource for studying transport properties of the BBB. There are several different methods available for isolation and purification of brain capillaries (Brendel et al., 1974; Orlowski et af., 1974; Goldstein et al., 1975; Mrsulja et al., 1976; Williams et al., 1980). Generally,

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suspensions of brain capillaries are prepared from brains by a combination of techniques, including mechanical homogenization, enzyme treatment, filtration through nylon meshes, density gradient centrifugation, and glass bead column filtration. The disadvantages of isolated brain capillaries include contamination of the preparation by other brain cells (Williams et a l . , 1980; White et al., 1981) and the potential for metabolic deficiencies induced by the isolation procedures (Lasbennes and Gayet, 1984). The metabolic deficiency is a particularly significant problem for transport studies. For example, receptor-mediated exocytosis, which requires ATP, cannot be detected in isolated brain capillaries because of ATP depletion due to cellular damage caused by a mechanical or an enzymatic homogenization, while some receptor-mediated endocytosis can be demonstrated in isolated brain capillaries through an energy-independent process (Pardridge, 1988). The advantages of this system include availability and ease of use. Relatively pure and metabolically active populations of microvessel can be conveniently obtained. With isolated capillaries, numerous studies have been done on the physiology, biochemistry, and developmental biology of the BBB; transport studies have been done as well, as reviewed by Joo (1985). From the viewpoint of transport studies of the BBB, it should be noted that it has been possible to study only solute uptake into the capillary cells and not transendothelial transport across the wall of the isolated tubular segments (Fig. 1). When isolated brain capillaries are used, solutes are allowed easy access to the antiluminal membrane. The uptake of solutes across the luminal membrane would be negligible, although the solutes might gain access to the luminal surface via the partially collapsed lumens. Therefore, it seems likely that phenomena identified in the isolated capillary preparation should be considered to be

u Endothelial cells

Fig. 1 Diagram of isolated brain capillaries.

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those on the antiluminal side (i.e., the brain side of the BBB) rather than the luminal side (the blood side of the BBB). Thus, solute uptake by isolated capillaries is not equivalent to transport across the BBB; however, this preparation can provide an opportunity to look at aspects of BBB transport.

B. Cultured Brain Microvessel Endothelial Cells Since Panula el al. (1978) demonstrated that rat brain microvessel endothelial cells could be maintained in tissue culture, various kinds of both primary and passaged cultures of isolated brain microvessel endothelial cells have been established from mouse (DeBault et al., 1979, 1981; Robinson et al., 1986; Dropulic and Masters, 1987), rat (Phillips et al., 1979; Spatz et al., 1980; Bowman et al., 1981; Diglio et al., 1982), bovine (Phillips et al., 1979; Bowman et al., 1983; Goetz et al., 1985; Audus and Borchardt, 1986a, 1987; Meresse et al., 1989), human (Vinters et al., 1987; Gerhart et al., 1988), canine (Gerhart et al., 1988), and porcine (Mischeck et al., 1989) brain. In general, either enzymatic or mechanical dispersal, or a combination of both techniques, followed by either filtration or centrifugation steps are employed to isolate a homogeneous population of brain microvessel endothelial cells from the extremely heterogeneous population of cells found in brain tissues. For example, isolation of a viable, homogeneous population of brain capillary endothelial cells for establishment of a tissue culture system is accomplished by a two-step enzymatic digestion with dispase and a dispase/collagenase mixture of cerebral gray matter and successive centrifugation over dextran and Percoll gradients (Bowman et al., 1983; Audus and Borchardt, 1986a, 1987). In our laboratories, primary cultures of bovine brain microvessel endothelial cell monolayers have been shown to retain morphological properties typical of the BBB in vivo, such as tight junctions, attenuated pinocytosis, and lack of fenestra (Audus and Borchardt, 1986a, 1987; Guillot et al., 1990). In addition, specific BBB enzyme markers (y-glutamyl transpeptidase and alkaline phosphatase), endothelial cell markers (angiotensin-converting enzyme and factor VIII antigen), catecholamine-degrading enzymes (monoamine oxidase A and B, cytosolic catechol 0-methyltransferase, and thermostable phenol sulfotransferase) (Audus and Borchardt, 1986a; Baranczyk-Kuzma et al., 1986, 1989a; Scriba and Borchardt, 1989a,b), choline esterases (A. M. Trammel and R. T. Borchardt, unpublished data), aminopeptidases (Baranczyk-Kuzma and Audus, 1987), and acid hydrolases (Baranczyk-Kuzma et al., 1989b) are retained in the model. All biochemical properties of the in vitro model are consistent with present understanding of the BBB in vivo. Basically, two types of experimental systems have been employed for study of transport phenomena using cultured brain microvessel endothelial cells: the first is the uptake study, and the second involves a transcellular transport study. The

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Endothelial Cell Monolayer

Endothelial Cells Collagen Matrix

\

Culture Plate

A

I

I

B

Uptake

I

Microcarrier

Fig. 2 Experimental systems for uptake study using brain microvessel endothelial cells in v i m . (A) Culture dish system; (B) microcanier system.

former system uses microvessel endothelial cell monolayers grown in culture dishes (Fig. 2A). Uptake experiments can be also performed using cerebral microvessel endothelial cells cultured on microcarriers (e.g., dextran beads) (Bottaro et al., 1986; Kempski et af., 1987) (Fig. 2B). This system allows examination of the first step of the transport process, that is, the uptake of the solutes into the brain capillary cells from the lumen. The most sophisticated in vitro system for transport study is the transcellular transport study system, consisting of cultured brain microvessel endothelial cell monolayers grown on microporous membranes. The system includes the side-by-side diffusion system (Fig. 3A) and Transwell system (Fig. 3B). It affords an opportunity to look at bidirectional transendothelial movement (transfer from brain to blood and that from blood to brain) of solutes across the BBB in vitro since, at least for primary cultures of bovine microvessel endothelial cells, the cells are shown to be morphologically and functionally polarized in terms of rich recycling (Raub and

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Side-by-Side Diffusion Cell

Endothelial Cells Collagen Matrix

A

-

Microporous Membrane

Endothelial Cell Monolayer in Transwell

Endothelial Cell

6

Microporous Membrane

Fig. 3 Experimental systems for transport study using brain microvessel endothelial cells in vitro. (A) Side-by-side diffusion cell system; (B) Transwell system.

Audus, 1990), transfemn transport (Newton and Raub, 1988), and angiotensin I1 responsiveness (Guillot and Audus, 1989a, 1990). Although bovine brain microvessel endothelial monolayers retain tight junctions, the tight junctions are not identical to those observed in vivo with regard to extent and complexity. This leads to higher leakiness in vitro than in vivo, which is a disadvantage in the use of the tissue culture system to study transcellular transport. As discussed later, cocultures with astrocytes hold promise for restoring tight junction integrity in v i m . Nonetheless, normalizing corrections for leakiness in the monolayers can be made with impermeant marker molecules (e.g., sucrose, fluorescein, inulin, dextran) when transport of a specified solute is studied. Thus, cultured brain microvessel endothelial cells can offer a good system for the investigation of transport across the BBB in vitro.

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111. In Vitro Transport Studies A. Nutrients 1. Glucose Since glucose is an important source of energy for the brain, the mechanism of glucose transport across the BBB has been particularly well studied in vivo (Lund-Anderson, 1979; Pardridge, 1983). These studies support the concept that glucose is transported through the cerebral microvessel endothelium mainly by the mechanism of carrier-mediated facilitated diffusion, which is similar to that described for red blood cells. It was also shown that the equilibrating glucose transport process is not rate-limiting for its metabolism under normal conditions, and that the transport system may exist on both sides of the BBB. Goldstein et uL. (1975) and Mrsulja et aL. (1976) reported the uptake of 2deoxy-D-glucose (2DG), a glucose analog that is phosphorylated but not further metabolized, by isolated rat brain capillaries. A saturable (Knl = 9.3 M ) ,temperature-dependent, and stereospecific uptake of 2DG by isolated brain capillaries was demonstrated by Goldstein et al. (1977a); the uptake was sodium independent, more sensitive to phloretin than phlorizin, minimally affected by ouabain and 2,4-dinitrophenol (DNP), and insensitive to insulin. Similar results were reported for 3-O-methyl-~-glucose(3MG), a nonmetabolizable glucose analog, in isolated brain microvessels (Betz et al., 1979). Kolber et al. (1979) studied 2DG and 3MG uptake in isolated microvessel preparations and estimated affinity constants for these glucose analogs to be I8 mM, which was comparable to values of 6-9 mM for glucose uptake determined in vivo. Glucose transport characteristics were also studied in cultured brain microvessel endothelial cells. Vinters et ul. (1985) demonstrated that the properties of 3MG and 2DG uptake in established lines of cultured mouse cerebral microvessel endothelium are similar to those observed in vivo. They also reported an increased 3MG uptake after insulin treatment in the cultured cells. Drewes et ul. (1988) showed that phorbol ester stimulates the uptake of hexoses (3MG, 2DG, and glucose) by primary and passaged cultured microvessel endothelial cells isolated from human and canine brains, suggesting that BBB glucose transport may be regulated by phorbol ester-activated protein kinase C. Recently, characteristics of both uptake and transendothelial transport of 3MG were studied in primary cultures of bovine brain microvessel endothelial cells (Takakura and Borchardt, 1990). The uptake characteristics of 3MG (e.g., K,, = 16 mM) are shown to be identical to those observed in vivo and in vitro using isolated capillaries. Transport rates from the luminal to basolateral side and from the basolateral to luminal side, measured across the brain microvessel endothelial

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cells grown onto polycarbonate membranes, were almost the same, suggesting symmetrical glucose transport across the BBB.

2. Amino Acids The passage of amino acids across the BBB was found to be saturable and stereospecific in vivo (Oldendorf, 1971). Sershen and Lajtha (1976) first reported the existence of a sodium-independent uptake of neutral amino acids in cerebral capillaries isolated from rat brains. Hjelle et al. (1978) demonstrated the temperature-dependent, saturable, and ouabain-insensitive uptake of large neutral amino acids by isolated bovine brain microvessels. Betz and Goldstein (1978) found that both Na+ -independent L (leucine-preferring) and Na -dependent A(alanine-preferring) systems of neutral amino acid uptake exist in isolated brain capillaries. Based on the fact that in vivo studies had demonstrated that a transport system for small neutral amino acids is not present on the blood side of brain capillaries, they concluded that the A-system is located on the brain side. Characteristics of cysteine (Hwang et al., 1980) and proline (Hwang et al., 1983) uptake have also been studied using isolated rat brain capillaries. Cangiano et al. ( 1983) reported that increased intercellular glutamine affects the uptake of different categories of amino acids by isolated bovine brain microvessels and hypothesized that the A- and Lsystems can cooperate in the uptake of the large neutral amino acids. Uptake of phenylalanine and other neutral amino acids has been characterized by using isolated human brain capillaries (Choi and Pardridge, 1986; Hargreaves and Pardridge, 1988); the results show that K , values determined in the human capillary in vitro correlate significantly with K , values determined in the rat brain capillary. Cancilla and DeBault (1983) showed the presence of A- and Lsystems for uptake of neutral amino acids in cultured mouse cerebral endothelial cells. The A-system was slower than the Lsystem, and each was inhibited by other amino acids. Using primary cultures of bovine brain microvessel endothelial cell monolayers grown onto microporous membranes, transport of a large neutral amino acid, leucine, was shown to be saturable (K, = 0.18 mM; V,,, = 6.3 nmol/mg/min), bidirectional, competitive with other amino acids, and energyindependent (Audus and Borchardt, 1986b). The kinetic parameters appear to be in good agreement with true kinetic parameters of the in vivo BBB. The transport of several amino acid drugs, including baclofen (van Bree et al., 1988), amethyldopa (Chastain and Borchardt, 1989), and acivicin (Chastain and Borchardt, 1990), by the amino acid carrier has also been explored in this system. The y-glutaniyl transpeptidase (y-GTP), which catalyzes the transfer of the yglutamyl residue of glutathione to amino acids, has been detected in isolated cerebral microvessels (Orlowski et al., 1974; Goldstein et al., 1975; Mrsulja et +

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al., 1976) and in cultures of brain microvessel endothelial cells (Spatz et al., 1980; Audus and Borchardt, 1986a; Vinters et al., 1987; Mischeck et al., 1989). This enzyme was suggested to be located on both the luminal and antiluminal membranes of the brain capillary endothelial cells (Betz et al., 1980). It has been postulated that y G T P is involved in the transport of amino acids (Orlowski and Meister, 1970), but the precise mechanism by which amino acids are transported across the BBB is not understood.

3. Monocarboxylic Acids Generally, the degree of BBB passage of monocarboxylic acids depends on their lipophilicity; however, Oldendorf ( 1973) showed a specific carrier-mediated transport of short-chain monocarboxylic acids in vivo. Spatz et al. (1978) demonstrated a saturable ( K , = 2.5 mM) uptake of lactate, which was cross-inhibited by pyruvate in the isolated rat cerebral capillaries. They observed that lactate uptake in the capillaries isolated from young brain was greater than that in the capillaries isolated from adult brain.

4. Choline Because of a limited capability for de novo synthesis of choline, an important precursor to acetylcholine and phospholipid, the brain must depend on the blood for its supply of choline. Cornford et al. (1978) demonstrated the saturability of brain uptake of choline after intracarotid injection in rats. The specific uptake of choline into isolated rat brain microvessels was studied by Shimon et al. (1988), who suggested the existence of endocrine modulation by the observation of inter- and intrasex variations ( K , range: 10.6-54.9 pM) in choline uptake. Recently, Estrada et al. (1990) studied choline uptake by bovine cerebral capillary endothelial cells in culture, demonstrating that these cells were able to incorporate choline by a camer-mediated mechanism ( K , = 7.6 pM). The choline uptake was temperature-dependent and was inhibited by choline analogs, but was not affected by ouabain or DNP. Transendothelial transport of choline in cultured bovine brain microvessel endothelial cells has been characterized (Trammel and Borchardt, 1987); it has been shown that the transport has a K , of 0.2 mM and is insensitive to ouabain and sodium azide, suggesting a facilitated diffusion mechanism.

5 . Nucleotides and Bases Since the brain is one of the most active tissues for carrying out nucleotide and nucleic acid synthesis, the transport of purine and pyrimidine nucleosides and bases across the BBB has been of interest. By employing the intracarotid injec-

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tion technique in rats, Cornford and Oldendorf (1975) demonstrated the presence of two independent carrier systems for nucleic precursors, a nucleoside carrier with a measurable affinity for purine nucleosides [e.g., adenosine (Km = 18 pM), guanosine, inosine, and the pyrimidine nucleoside, uridine], and a purine base carrier with an affinity for purine bases [e.g., adenine ( K , = 27 pM), guanine, and hypoxanthine]. Wu and Phillis (1982) reported that adenosine uptake by isolated rat brain capillaries is a carrier-mediated ( K , = 4.74 pM), temperature-sensitive, and pH-sensitive process. The uptake was inhibited by several nucleosides (e.g., the adenosine analogs), and some drugs (e.g., papaverine, hexobendine, and dipyridamole). A similar carrier-mediated uptake of adenosine ( K , = 5 pM) into mouse cerebral capillary endothelial cells in tissue culture has been reported (Beck er al., 1983). Hypoxanthine transport and metabolism were characterized by Betz (1985) using microvessels isolated from rat brain. He concluded that hypoxanthine is carried into brain capillaries by a transport system shared with adenine. Characterization of the nucleoside uptake (e .g., adenosine, thymidine) into monolayers of cultured bovine brain endothelial cells was also studied, and the results suggest the presence of a carrier-mediated uptake of adenosine ( K , = 2.9 pM) and thymidine ( K , = 0.8 pM) (Shah and Borchardt, 1989). Adenosine uptake is primarily via the carrier-mediated pathway, whereas thymidine enters by both a carrier-mediated and a passive pathway. Both nucleosides are extensively metabolized (e.g., phosphorylated) in the cultured bovine endothelial cells.

B. Ions Studies on ion transport across the BBB are important in order to understand the mechanisms for regulating ion concentration in cerebrospinal fluid (CSF) better. Of particular interest are the transport mechanisms by which major cations, such as sodium and potassium ions, are transported across the BBB. A Na+ ,K+-ATPase, which is similar to that in choroid plexus and the ouabain-sensitive uptake of potassium analog 86Rb, was found in capillaries isolated from rat brain (Eisenberg and Suddith, 1979; Eisenberg et al., 1980). These results suggest that the brain microvessels may play an active role in brain potassium homeostasis and subsequent extracellular fluid formation. A possible role for the brain capillary in the regulation of extracellular fluid potassium was also suggested by Goldstein (1979), who observed that isolated brain capillaries show saturable ( K , = 3 mM), energy-dependent, and ouabain-inhibitable uptake of 86Rb. These results are similar to those observed in vivo (Bradbury et a l . , 1972). Goldstein proposed that the K + pump (Na+ ,K+-ATPase) is located on the antiluminal plasma membrane of brain capillary endothelial cells; the polar distribution of this pump was confirmed by cytochemical localization and by

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fractionation of membranes prepared from isolated brain capillaries (Betz er af., 1980; Betz and Goldstein, 1980). Chaplin et al. ( 1 98 1) reported that K transport by the isolated capillaries was inhibited by steroids in relation to their lipid solubility, suggesting a direct membrane action for high-dose steroid therapy, possibly on Na ,K -ATPase, rather than a nuclear-mediated change in cell function. They postulated that inhibition of active sodium and potassium transport in brain capillaries and choroid plexus produces a secondary decrease in CSF secretion and that this may be a mechanism for the beneficial action of high doses of steroids in brain edema. In addition to Na+ , K + -ATPase, Betz (1983a) demonstrated the presence of a transport system capable of mediating Na+/Na+ and Na+/H+ exchange in brain capillaries. Since a similar transport system does not appear to present on the luminal membrane of the brain capillary endothelial cells in vivo (Betz, 1983b), he proposed that Na-+/H exchange occurs primarily across the antiluminal membrane. +

+

+

+

C. Neurotransmitters Generally, the BBB passage of monoamine neuotransmitters has been shown to be insignificant under physiological conditions in vivo (Oldendorf, 1971). This can be attributed in part to their low lipophilicity and subsequent poor permeability to BBB, and also to the high level of metabolic enzymes in brain capillary endothelium. Hardebo et al. ( 1979) reported that isolated microvessels take up L-Dopa in a stereospecific and energy-dependent manner. L-Dopa was shown to be subsequently converted to dopamine within the capillary wall, and the dopamine was degraded to inactive compounds by monoamine oxidase, which is present in the capillaries. Abe et al. (1980) and Spatz et al. (1981) showed that monoamines such as L-norepinephrine (NE) and 5-hydroxytryptamine (5-HT) were taken up and metabolized in the isolated cerebral capillaries. The uptake into the capillanes was saturable (Kn, = 14.5 for NE; K , = 2.3 IJ.M for 5-HT), and Na+ ,K -, and temperature-dependent. It was suggested that this canier-mediated process was shared with other monoamines but not by amino acids that can be transported by either the A- or the L-system. +

D. Peptides and Proteins 1. Insulin and Insulin-Like Growth Factors Although the physiological role of insulin in the regulation of brain functions remains to be elucidated, van Houten and Posner (1979) revealed that blood

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vessels throughout the CNS of the rat bind plasma insulin rapidly and with considerable specificity in vivo. A BBB receptor for insulin was first characterized using isolated rat brain capillaries in vitro by Frank and Pardridge (1981), who demonstrated a rapid and specific binding of insulin to the microvessel directly. Specific insulin receptors have been also identified in microvessels isolated from porcine and bovine brains (Haskell et al., 1985). Pardridge et al. (198%) also showed the existence of an insulin receptor with a high affinity dissociation constant (KO = 1.2 nM) in isolated human brain capillaries and suggested that the human BBB insulin receptor may participate in a receptor-mediated, endocytosis-exocytosis system for the transport of the peptide through the BBB. In addition, specific receptors for insulin-like growth Factor I (IGF-I) ( K , = 2.9 nM) and IGF-I1 ( K , = 3.3 M),which are separate from that for insulin, have been identified using isolated bovine brain capillaries (Frank et al., 1986). Frank et al. (1986) observed the internalization of these peptide hormones but did not conclude that the BBB functions as a selective transporter because of ATP depletion of the capillaries. Similar results were obtained for IGFs using isolated human brain capillaries (Duffy et al., 1988). The binding and receptor-mediated endocytosis of insulin and IGF-I were also studied using cultured bovine brain microvessel endothelial cells (Keller and Borchardt, 1987; Keller et al., 1988). Rosenfeld et al. (1987) showed the similarity between the characteristics of the specific receptors for IGF-I and IGF-I1 in the cultured bovine brain microvessel endothelial cells and in isolated rat brain microvessels.

2. Transferrin Using monoclonal antibodies to the transferrin receptors, Jefferies et al. (1984) first reported that rat and human brain capillary endothelia have receptors for transfemn, an iron-transport protein in the circulation. The BBB transferrin receptor has been identified by Pardridge er al. (1987a) using the isolated human brain capillary preparation: the kinetics of binding (KO = 5.6 nM) and endocytosis of human transferrin have been examined. Newton and Raub (1988) characterized the transferrin receptor in primary cultures of brain capillary endothelial cells, indicating suturable binding ( K O = 5 nM) and internalization. They also demonstrated the transcytosis and the polarized efflux of transferrin using brain microvessel endothelial cells grown onto polycarbonate filters of a Transwell diffusion cell; these findings are in good agreement with the in vivo observations of Fishman et al. (1987) and Banks et al. (1988).

3. Atrial Natriuretic Factor Atrial natriuretic factor (ANF), which is a 28-amino acid peptide produced by cardiac myocytes and is released in response to increases in atrial pressure,

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expresses its natriuretic, diuretic, and hypotensive effects by acting on renal and vascular tissues. ANF receptors have been identified in isolated bovine brain microvessels (Chabrier et al., 1987), demonstrating the presence of a single class of ANF-binding sites with high affinity ( K , = 0.1 nM). A specific receptor for ANF was also identified using primary cultures of bovine brain capillary endothelial cells (Smith et al., 1988). The binding of ANF was rapid, reversible, and unaffected by the presence of insulin, vasopressin, and angiotensin 11, but was inhibited by atriopeptins. The dissociation constant for ANF was calculated to be 400 pM. Furthermore, cultured bovine microvessel endothelial cells rapidly internalized ANF by a temperature-dependent process. A specific receptor for brain natriuretic peptide (BNP), which is found in the brain and has similarity to ANF in its structure and biological activities (Sudoh et al., 1988), was identified in primary cultures of bovine brain microvessel endothelial cells (M. Fukuta, Y. Takakura, and R. T. Borchardt, unpublished data). These studies suggested that ANF and BNP shared the same receptor in bovine brain microvessel endothelium.

4. Angiotensin I1 Speth and Harik (1985) reported that angiotensin I1 (Ang 11) binds to microvessels isolated from dog brain in a specific, saturable, and reversible manner and with high affinity ( K , = 1 nM). It was suggested that specific Ang I1 receptor binding sites are present in brain microvessels and that these receptors may have an important role in regulating the microcirculation of the brain. Unpublished work from our laboratories indicates that bovine brain microvessel endothelial cell monolayers retain a high-affinity Ang I1 binding site ( K , = 3 nM) that can be competed for by Ang I1 peptides and internalized (F. L. Guillot and K. L. Audus, unpublished data).

5 . Enkephalins Pardridge and Mietus (198 1 ) studied leucine-enkephalin uptake by isolated brain microvessels and found a rapid degradation of the peptide. No high-affinity receptor or transport mechanism for leucine-enkephalin was suggested in this study. Similarly, somatostatin was shown to be rapidly degraded by bovine brain capillaries (Pardridge et al., 1985b). Recently, the transport of leucine-enkephalin across the BBB by a carriermediated mechanism has been demonstrated in vivo (Zlokovic el al., 1987). In addition, Thompson and Audus ( 1989) showed that leucine-enkephalin transfer across the brain endothelium occurs at a relatively high rate, which is consistent with a facilitated diffusion mechanism.

6. Vasopressin Vasopressin transport across the BBB has been examined with primary cultures. of brain microvessel endothelial monolayers (Reardon and Audus, 1989). Re-

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sults suggest the existence of facilitated transport of the peptide from the antiluminal to the luminal side of the monolayers. This finding is consistent with the in vivo characterization of a vasopressin BBB transport system (Banks et al., 1987). Van Bree et al. (1989) have also studied transport of vasopressin using an in vitro system and suggested that no carrier mediation is involved over a higher concentration range. Confirmatory studies are required to clarify the transport mechanism in detail.

7 . Delta Sleep-Inducing Peptide Raeissi and Audus (1989) characterized the BBB permeability to delta sleepinducing peptide (DSIP) in cultured microvessel endothelial cells. The results support in vivo observations indicating that intact DSIP crosses the BBB by simple transmembrane diffusion (Banks and Kastin, 1987). Recently, Zlokovic et al. (1989) presented evidence in support of a facilitative BBB carrier for DSIP in vivo. Further work is ongoing concerning the solution structure of DSIP, which may help to explain its ability to readily penetrate the BBB (Audus and Manning, 1990).

8. Modified Albumin Native albumin, which is an acidic protein in plasma, is considered to pass through the BBB very slowly (Pardridge etal., 1985a). However, increased BBB uptake or transport has recently been reported when it is chemically modified. Kumagai et al. (1987) demonstrated the enhanced binding and adsorptive-mediated endocytosis of cationized albumin by isolated bovine brain capillaries. The binding was saturable (K, = 0.8 nM) and inhibited by other polycations (e.g., protamine, protamine sulfate, and polylysine). Similar results have been reported for other types of polycationic proteins, including cationized immunoglobulin G (Triguero et al. 1989) and histone (Pardridge ef al., 1989). The use of cationized albumin in directed delivery of peptides through the BBB was examined by coupling P-endorphin to cationized albumin via a disulfide linkage (Kumagai et al., 1987; Pardridge et al., 1987b). The authors showed that the P-endorphincationized albumin chimeric peptide was rapidly bound and endocytosed by isolated bovine brain capillaries, and the binding was inhibited by unlabeled cationized albumin but not by unconjugated P-endorphin and native bovine albumin. Smith and Borchardt (1989) studied the binding, uptake, and transcellular transport of bovine serum albumin (BSA), cationized BSA (cBSA), and glycosylated BSA (gBSA) in cultured bovine brain microvessel endothelial cells. This study demonstrated that both cBSA and gBSA bind to the cells specifically (K, = 27 nM for cBSA; K D = 3.7 nM for gBSA) and are transported by an adsorptive-phase endocytotic mechanism.

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E. Drugs In contrast to water-soluble nutrients and peptides, which are transported by specific carrier- or receptor-mediated systems as mentioned above, most watersoluble solutes, including drugs, pass through the BBB by a passive diffusion mechanism. From in vivo studies, it has been well established that the permeability of these molecules across the BBB depends directly on their lipophilicity and inversely on their molecular size (Oldendorf, 1974; Levin, 1980; Cornford et al., 1982). Rim et al. (1986) studied the relationship of lipid solubility to the rate of passage of drugs across bovine brain microvessel endothelial monolayers grown onto regenerated cellulose membranes for nine compounds, including propranolol, antipyrine, and caffeine. They observed a significant positive correlation between transcellular diffusion and octanol/buffer or octanol/water partition coefficients and demonstrated that these compounds undergo passive diffusion across the monolayers in which the rate of transfer depends directly on their lipid solubility and indirectly on molecular weight. Using a similar in v i m BBB model consisting of cultured bovine brain microvessel endothelial cells grown on polycarbonate membranes, an excellent correlation was established between the permeability coefficients of the solutes and their lipid solubility for drugs with higher lipophilic nature, such as progesterone, haloperidol, testosterone, and esterone (Shah et al., 1989).

F. Fluid-Phase Endocytosis Guillot et al. (1990) studied the kinetics of fluid-phase endocytosis in the bovine brain microvessel endothelial cell monolayers with Lucifer Yellow (LY), a fluorescent, soluble marker for fluid-phase endocytosis. They observed the timedependent and biphasic uptake of LY by the cells; the total turnover rate was shown to be much slower than those reported for other types of cells, suggesting an attenuated pinocytosis analogous to the BBB in vivo.

G. Adsorptive Endocytosis Nonspecific adsorptive endocytosis and membrane recycling were examined in primary cultures of brain microvessel endothelial cells (Raub and Audus, 1990). Using a lectin, Ricinus cornrnunis agglutinin 1 (RCAI), as a tracer of adsorptive endocytosis, the half-life values for RCAI binding, internalization, and efflux were determined to be 5, 18, and 13- 14 min, respectively. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured brain microvessel endothelial cells (Guillot et al., 1990). Moreover, endocytosis of membrane by the brain microvessel endothelial cells in culture was proved to be similar to that reported for brain endothelium in vivo

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(Broadwell et al., 1988) in that a fraction of the cell surface membrane was routed to the trans-Golgi network.

IV. In Vifro Studies on Regulation of Blood-Brain Barrier Transport

A. Peptide Regulation 1 . Regulation of Blood-Brain Barrier Glucose Transport by Insulin It is well known that the peptide hormone insulin plays a central role in the regulation of glucose transport and metabolism in mammalian cells. Most notably, insulin enhances glucose transport in its target tissues, such as fat and muscle, within minutes (Simpson and Cushman, 1986). In spite of a number of in vivo studies of the effect of insulin on BBB glucose transport, conflicting results have been reported (Hertz and Paulson, 1983; Pardridge, 1983). Although in vitru systems generally offer more controlled conditions in which to clarify the matter than do in vivo experiments, clear answers have not been obtained with regard to effect of insulin on glucose uptake in brain endothelium. Negative insulin effects on the uptake of the glucose analogs 2DG and 3MG have been reported by several groups using isolated brain microvessels (Goldstein et al., 1977a; Betz et al., 1979) and using cultured brain endothelial cells (Drewes et al., 1988). On the other hand, Djuricic et al. (1983) showed that insulin greatly enhanced 2DG uptake ( 1%fold) in isolated cerebral capillary preparations, while Vinters et al. (1985) demonstrated that insulin treatment stimulated 3MG uptake by cultures of rnouse brain microvessel endothelial cells.

2. Regulation of Blood-Brain Barrier Permeability by Vasoactive Peptide Under certain pathophysiological conditions, vasoactive peptides may play an important role in mediating changes in BBB permeability. To elucidate angiotensin peptide regulation of BBB permeability, studies have been done with brain microvessel endothelial cell monolayers (Guillot and Audus, 1989a,b, 1990; Audus, 1990). Pinocytosis, measured as LY uptake, was stimulated on exposure to nanomolar concentrations of angiotensin agonists, Ang I1 and Saralasin, and this effect was blocked by pretreatment with indomethacin, suggesting that angiotensin peptide effects on BBB pinocytosis are probably mediated by prostaglandins. The authors also studied the effects of angiotensin peptides on transcellular permeability by measuring the flux of fluorescein-conjugated dextran across the monolayer, and suggested the polarity of the responsiveness of the

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bovine brain microvessel permeability to the peptides. Exposure of the luminal side of the monolayers to the peptides resulted in a significant decrease in permeability in the luminal-to-abluminal direction, yet the permeability in the abluminal-to-lurninal direction was not changed when the abluminal side was exposed to the peptides.

B. Astrocyte Interactions 1. Regulation of Blood-Brain Barrier Nutrient Transport It is widely accepted that the brain microvessel endothelial cells form the structural and functional bases of the BBB; however, some of the BBB functions are known to be regulated by a kind of glial cell, astrocytes, which encircle the microvessel endothelial cells with their foot processes in vivo. In vitro cell culture systems of brain capillary endothelial cells offer a good opportunity to examine how astrocytes regulate the functions of microvessel endothelial cells in vivo. For instance, induction of specific marker enzymes, including y-GTP (DeBault and Cancilla, 1980; DeBault, 1981; Maxwell et al., 1987; Bauer el a l . , 1990) and Na+ ,K +-ATPase (Beck et a l . , 1986; Bauer et al., 1990), has been demonstrated in passaged brain capillary endothelial cell cultures when they are cocultured with glial cells or treated with astrocyte-conditioned medium. Cancilla and DeBault (1983) demonstrated that contact with glial cells (a rat line of neoplastic astrocytes designated as C6 glioma cells) or exposure to glialconditioned media enhances neutral amino acid uptake by passaged mouse cerebral endothelial cells in culture. The influence of glial cells on the polarity of Asystem neutral amino acid transport has been studied by Beck et ul. (1984). A situation similar to the endothelium-astrocyte relationship existing at the BBB was produced by growing passaged cerebral endothelium on one side of a filter and C6 gliorna cells on the other side. Transport of a-methylaminoisobutyric acid, which is transported by the A-system, was demonstrated to be more rapid from the glial surface across the endothelium (from the brain side to the blood side) than in the opposite direction. Maxwell et al. (1989) demonstrated that conditioned media prepared from both astrocytes and C6 glioma cells stimulated glucose uptake by passaged mouse cerebral endothelial cells. The effect of astrocytes was exposure timedependent and blocked by the presence of cyclohexamide, a protein synthesis inhibitor. Treatment of the conditioned medium with trypsin destroyed its effectiveness. These results suggest that the enhancement of glucose uptake by the cells was induced by a protein released by the astrocytes. Similar effects of astrocyte- and glioma cell-conditioned media on glucose uptake by primary cultures of bovine brain microvessel endothelial cells have been reported (Tak-

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akura et al., 1990). However, the factor(s) from astrocytes and glioma cells was shown to be insensitive to trypsin treatment, suggesting that the glucose uptake stimulating effects are mediated by a factor which is not proteinaceous in nature.

2. Regulation of Blood-Brain Barrier Permeability Although primary cultures of brain endothelium alone can form tight intercellular junctions (Bowman et al., 1983), Tao-Cheng et al. (1987) showed that when brain endothelial cells are cocultured with astrocytes, the endothelial tight junctions were enhanced in length, width, and complexity. Gap junctions, common in brain endothelium in vitro but absent in mature brain capillaries in vivo, were markedly diminished in area among the enhanced tight junctions of the cultures. Arthur et al. (1 987) reported that passaged rat brain capillary endothelial cell cultures, which no longer possess the tight junctions, had numerous, elaborated complex, tight junctions which were identical to those displayed in vivo when these cells were exposed to rat brain astrocyte-conditioned media on an endothelial cell matrix-coated substrate. These data suggest that astrocytes in vitro play a role in the formation, extent, and configuration of the junctional complexes in brain endothelium and, therefore, may affect their permeability to solutes. Recently, Trammel and Borchardt ( 1989) reported that conditioned media from rat astrocyte cultures or C6 glioma cell cultures decreased the permeability (>50%) to radiolabeled inulin of bovine brain microvessel endothelial cell monolayers grown on polycarbonate membranes. Coculturing astrocytes with endothelial cells also caused a decrease in the permeability of the endothelial cell monolayers, whereas glioma coculture experiments increased the permeability by more than 50%. Using conditioned media from C6 glioma cells, Raub et al. (1989) showed similar results for the permeability of solutes through bovine brain endothelial cell monolayers grown in Transwells. However, the data reported by the authors concerning the effects of coculturing brain endothelial cells with C6 glioma cells differ from those observed by Trammel and Borchardt (1989). Raub et al. (1989) demonstrated that cocultures of bovine brain endothelial cells with C6 glioma cells result in a decrease in the permeability and an increase in the transendothelial electric resistance as well. The reason for the different experimental results remains to be elucidated, yet these experiments clearly show that astrocytes are producing factors that can alter the permeability of brain endothelium in vitro. Recently, Dehouck et af. (1990) studied the permeability of the BBB to some solutes using an in vitro model system of culturing passaged brain microvessel endothelial cells grown on one side of a filter and astrocytes on the other.

C. Developmental Changes There have been several reports of changes in the BBB transport of some substrates during development. Developmental changes of lactate and 2DG uptake

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have been studied using cerebral capillaries isolated from postnatal and adult rats (Spatz et al., 1978). The uptake of lactate was higher, while that of 2DG was lower in capillaries from the newborns when compared to the uptake by capillaries from adults. Betz and Goldstein ( I 98 1) studied developmental changes in the metabolism and transport properties of capillaries isolated from the brains of rats of various ages. Capillary glucose uptake was similar at 10 and 30 days of age and the activity of the K + pump, which was determined as ouabain-inhibitable 86Rb+ uptake, was relatively constant at all ages. In contrast, Na+-dependent neutral amino acid (a-methylamino acid) transport was not present until 21 days of age, suggesting that this transport system for neutral amino acids (A-system) does not occur in the immature BBB. Frank et al. (1985) demonstrated enhanced specific insulin binding to isolated cerebral microvessels in newborn rabbits (60.6%/mg protein) compared with 3week-old (23.8%/mg protein) and adult animals (13.6%/mg protein). Similar results were observed for in vivo brain uptake of insulin after a single-pass carotid injection in this study. Scatchard analysis revealed that the difference was due to an increase in receptor number with minimal changes in affinity. They speculated that insulin has increased access to the newborn brain, where it may function as a growth factor.

D. Pathological Changes 1. Ischemia and Hypoxia It is important to understand the BBB’s reactivity to reduced blood flow and oxygen tension in order to elucidate the pathophysiology of cerebrovascular disease (Spatz, 1984). The ischemic and postischemic effects on uptake of 2DG (Spatz et al., 1977, 1979) and neutral amino acids (Spatz et al., 1979) were studied in cerebral microvessels obtained from gerbils with bilateral occlusion of the common carotid artery. Spatz et al. demonstrated an ischemic decrease with postischemic recovery and a transient increase in 2DG uptake in the capillaries. In contrast, cerebral ischemia led to a transiently increased uptake of some neutral amino acids. These results suggested an oxygen-dependent uptake of 2DG and oxygen-independent entry of neutral amino acids under pathological conditions. Similar effects on the capillary uptake of these substances were observed when isolated cerebral microvessels were exposed to a nitrogen atmosphere in vitro (Spatz et al., 1979). Decreased uptake of 2DG was also reported by Nell and Welch (1979) in microvessels isolated from gerbils that were subjected to ischemically induced seizures.

2. Aluminum Effects Aluminum has been implicated as a potential neurotoxin or contributing factor in the development of several CNS disorders, including Alzheimer’s disease and

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other dementias. Aluminum effects on the growth (Audus et al., 1988a) and permeability (Audus et al., 1988b) of primary cultures of bovine brain microvessel endothelial cells have been studied. Aluminum salts have been shown to stimulate DNA synthesis. Aluminum increased the transendothelial permeability to fluorescein sodium in a rapid manner, and also attenuated the pinocytosis as quantitated by the uptake of LY. On the other hand, aluminum had no effect on the fluidity of the cell membrane. These results support in vivo findings of aluminum-induced increases in BBB permeability (Banks and Kastin, 1983), and suggest that the potential site of alterations of BBB permeability may originate at the luminal surface of the microvessel endothelial cells.

3. Lead Intoxication In an attempt to investigate changes in cation transport that may underlie the pathogenesis of brain edema in acute lead encephalopathy, Goldstein et al. (1 977b) have investigated cation uptake by isolated brain capillaries. The steadystate uptake of calcium by the capillaries was tripled after exposure to lead nitrate, whereas potassium uptake was not altered. The increased uptake of calcium was suggested to be due in part to inhibition of active calcium efflux. The effect of lead intoxication on monosaccharide transport in isolated rat brain microvessels has been investigated (Kolber et al., 1980). A block in facilitated transport of 3MG, as well as damage to the endothelial cells resulting in increased passive permeability, was observed in microvessel preparations isolated from the brains of rats exposed to lead acetate. Microvessels from younger rats were more sensitive to lead treatment than those from adults. Abolition of facilitated transport of 3MG was also found when microvessels isolated from untreated rats were preincubated with lead acetate in vitro.

4. Miscellaneous Uptake of amino acids has been characterized using brain microvessels isolated from rats after portacaval anastomosis (PCA), which can be a model of the spontaneous portal-systemic shunting of the circulation that occurs in patients with liver cirrhosis and portal hypertension (Cardelli-Cangiano et al., 1981). The results suggested that brain capillaries from rats with PCA take up neutral amino acids more rapidly than those from control rats, which supports the hypothesis that high concentrations of neutral amino acids in brain after PCA in vivo reflect changes in transport of the amino acids at the BBB.

E. Alteration of Blood-Brain Barrier Permeability by Hyperosmotic Treatment It has been reported that the permeability of the BBB in vivo is transiently enhanced by infusion of hypertonic solution through the carotid artery by the

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opening of tight junctions. This technique has been used to deliver drugs that normally are poorly permeable to the BBB and is considered to be one of the useful strategies for the drug delivery to the brain (Pardridge, 1986). Bowman er al. (1983) demonstrated that a hypertonic solution of 1.6 M arabinose increased transcellular permeability ( 14C-labeled sucrose flux) by 40% in a reversible manner across the monolayer of bovine brain microvessel endothelial cells cultured on nylon mesh. Similar results were observed by Audus and Borchardt ( 1986a) in primary cultures of bovine brain microvessel endothelial cells and by Dehouck et al. (1990) in passaged bovine brain microvessel endothelial cells cocultured with astrocytes. Dorovini-Zis et al. (1984) reported that hyperosmotic arabinose solutions open the tight junctions and permit the penetration of horseradish peroxidase, which is an impermeable marker, between the junctions in bovine brain capillary endothelial cells in primary cell culture.

V. Summary The development of in vitro BBB models consisting of isolated brain capillaries and cultured brain microvessel endothelial cells has made possible the study of BBB transport phenomena at the cellular level. Basic characteristics of BBB transport of endogenous and exogenous solutes and their biochemical, pharmacological, ontogenic, and pathological regulation mechanisms have been investigated. This information has led not only to a better understanding of BBB transport but also to the construction of strategies for improving drug delivery to the CNS for diagnosis and therapeutics. To elucidate the complexity of BBB transport, in vivo studies are always necessary at some point; however, in vitro systems can be useful complements to the in vivo systems. The tissue culture systems seem to be especially important in the clarification of cellular, biochemical and molecular features of BBB transport. Appropriate systems should be selected or combined, depending on the purpose of the investigation.

Acknowledgments The authors’ research on bovine brain microvessel endothelial cells as a model of the blood-brain barrier was supported by grants from The Upjohn Company, Merck Sharp & Dohme-INTERx C o p . , The American Heart Association, and The American Heart Association-Kansas Affiliate. The authors thank Nancy Harmony for editorial assistance.

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Protein Kinase Inhibitors: Probes for the Functions of Protein Phosphor ylation John

E. Casnellie

Department of Pharmacology and Cancer Center University of Rochester School of Medicine Rochester, New York 14642

1. Introduction 11. Inhibitors of Protein SerineiThreonine Kinases A. Inhibitors Competitive with ATP

B . Inhibitors Competitive with Activating Cofactors or Second Mehsenger, C. Inhibitors Competitive with ProteiniPeptidc Substrate 111. Inhibitors of Protein Tyrosine Kinasea A . Inhibitors Competitive with ATP B . Inhibitors competitive with ProteiniPeptide Substrate C. Inhibitors Designed to Be Transition State Analogs D. Protein Tyrosine Kinase Inhibitors that Have Other Modes of Action IV. Concluding Remarks References

1. Introduction The observations of Krebs and Fisher and of Sutherland and co-workers that the activity of glycogen phosphorylase was regulated through phosphorylation catalyzed by a glycogen phosphorylase kinase was the first demonstration that protein phosphorylation has an important role in cell physiology (see Krebs, 1972, 1983, for reviews of the early literature of this field). Since those discoveries, the study of protein kinases and their substrates has expanded into virtually every area of biology and medicine. Studies on the mechanisms of signal transmission by hormones and neurotransmitters, the mechanism of signal transduction by growth factor receptors, the mechanisms of actions of oncogenes and tumor promoters, the mechanisms of gene regulation, and the study of cell cycle mutants in yeast and of developmental mutants in mice and drosophila, have all coalesced into the study of protein kinases. The demonstration that the insulin receptor is a protein tyrosine kinase gave the first real insight into how the insulin I61

168

John E. Cosnellie

receptor transmits a signal-certainly one of the most intractable if not important questions in the field of endocrinology. The observations on the regulation of glycogen phosphorylase were made in the 1950s. However, a general role for protein kinases in cellular regulation was not appreciated until the discovery of the cyclic AMP-dependent protein kinase in 1968 (Walsh ef al., 1968). By the time of this discovery, Sutherland and other workers had established that cyclic AMP was a second messenger not only for the P-adrenergic receptor but also for several other hormones. These observations, together with the universal presence of the cyclic AMP-dependent protein kinase in eukaryotic cells, suggested that, unlike the specialized role of glycogen phosphorylase kinase, the cyclic AMP-dependent protein kinase was involved in a multitude of responses. The challenge then was to establish a role for this enzyme in physiological responses that appeared to involve cyclic AMP and to determine the physiological substrates for this kinase. Virtually all of the research in the field of protein kinases that was performed in the 10 to IS years following the discovery of the cyclic AMP-dependent protein kinase was either on the cyclic nucleotide-dependent enzymes or on other protein kinases that served as intracellular receptors for second messengers, such as protein kinase C and the calcium/calmodulin-dependentenzymes. These enzymes are characterized by having a broad distribution throughout different tissues and cell types as well as an involvement in diverse responses; their discovery and characterization were achieved through the use of techniques of protein purification and characterization. In contrast, much of the more recent work on protein kinases has depended on the techniques of molecular biology to identify first the genes and then their protein products. This has led to the discovery of protein kinases with much more specialized functions and more limited cellular distribution. It has also led to the realization that the genome of higher organisms contains well over a 100 and perhaps as may as 1000 distinct genes for protein kinases (Hanks et al., 1988; Hunter, 1987). While the molecular biology of protein kinases has rapidly progressed, it has been much more difficult to increase the understanding of their function in the cell. The need to study protein kinases in intact cells in order to understand their function in a given biological response has been a major challenge since the time the field was largely confined to the study of the cyclic AMP-dependent protein kinase. For this reason, workers studying protein kinases have long sought the development of specific protein kinase inhibitors that would permit the definitive determination as to whether a particular protein kinase is involved in mediating a specific response. Indeed, the discovery of the cyclic nucleotide-dependent enzymes stimulated some of the first systematic attempts to synthesize cyclic nucleotide antagonists. The availability of such compounds would have permitted a much more rapid understanding of the role of cyclic nucleotides and cyclic nucleotide-dependent protein kinases in cellular responses. In a similar manner, a

Protein Kinase lnhibiton

169

specific antagonist of protein kinase C would permit the determination as to whether protein kinase C is indeed solely responsible for all the complex biological effects seen with tumor promoters. As research on protein kinases has accelerated so too has the search for specific inhibitors. In the past decade a large number of structurally diverse compounds have been found which inhibit protein kinase activity. Most of these inhibitors act by binding to either the ATP-binding site, the peptide binding site, or an allosteric regulatory site. Of these three sites, the greatest specificity would be expected for inhibitors directed at the peptide or the regulatory sites since these sites reflect the biological specificity of protein kinases. The emphasis of this review is on low-molecular-weight inhibitors, especially those that can be used for'studies with intact cells. Since the specificity of these inhibitors is a major concern when using them with intact cells, their effects on isolated enzymes-rather than on intact cells-is the major focus of discussion. However, some comments are made on how the specificity of the inhibitors against isolated enzymes affects their utility for studies of protein kinases using intact cells. Site-specific inhibitory antibodies, although useful for some studies, are not considered. Although no general classification scheme has yet been devised for the protein kinase family, there is at least one functional attribute which is useful for classifying these enzymes and is germane to this review. Thus, protein kinases that phosphorylate proteins on tyrosine residues form a structurally homologous group and the serineithreonine protein kinases likewise form their own distinct group. Since the residue specificity is absolute, this implies that all protein kinases have one of two types of peptide-binding sites and that it should be possible to direct inhibitors specifically to one of these two sites.

11. Inhibitors of Protein Serine/Threonine Kinases Given the large number of protein serine/threonine kinases that are present in the cell, inhibitors of these enzymes are only useful if they are relatively specific for one or a few enzymes. More studies have been done on or with inhibitors of protein kinase C than of any other enzyme. Indeed, there have been more studies on inhibitors of protein kinase C than on inhibitors of all the other protein kinases combined. The interest in developing and using inhibitors for protein kinase C has been in part because of its ability to serve as a receptor for tumor-promoting phorbol esters (Castagna el a l . , 1982; Niedel et al., 1983). Thus, inhibitors of this enzyme might provide for new therapeutics for the treatment of cancer. Moreover, the widespread occurrence of this enzyme (Nishizuka, 1986), the diverse physiological effects of phorbol esters (Blumberg, 198I), and the large number of receptors linked to the production of diacylglycerol, the second messenger for protein kinase C (Nishizuka, 1986), together suggest that protein

170

John E. Casnellie

kinase C is involved in the regulation of a large number of different cellular responses. Because of the intense interest in protein kinase C it is almost always included in tests of the efficacy of any putative inhibitors. Since it is present in virtually all eukaryotic cells and because of its role in a multitude of physiological processes, the cyclic AMP-dependent protein kinase is also usually included in tests of an inhibitor’s specificity. Three other protein kinases that have been frequently included in the in vitro screening of protein kinase inhibitors are the cyclic GMP-dependent protein kinase and the two calcium/calmodulindependent protein kinases, myosin light-chain kinase (MLCK) and calcium/calmodulin-dependentprotein kinase I1 (CAMK 11). MLCK is involved in regulating myosin function in a variety of cells, including smooth and skeletal muscle and platelets; the function of CAMK I1 has not yet been well delineated but its high content in brain suggests important roles in mediating calciumdependent signaling in nervous tissue (Schulman, 1988).

A. Inhibitors Competitive with ATP 1. Isoquinoline and Naphthalene Sulfonamides Hidaka and colleagues have undertaken an ambitious program to use synthetic chemistry to develop systematically specific protein kinase inhibitors. These workers had introduced the chloronaphthalene derivative W-7 as a calmodulin antagonist (see Hidaka et al., 1990, for a review). They observed that, while this compound is an effective inhibitor of calmodulin-dependent enzymes, it also weakly inhibits protein kinases by competing with ATP. Further research showed that replacement of the naphthalene ring in W-7 with the isoquinoline ring resulted in compounds that no longer bound to calmodulin but instead effectively bound to the ATP site of several protein kinases (Hidaka et al., 1984). Over the past few years these workers have synthesized several isoquinoline as well as naphthalene sulfonamides with differing specificities toward protein kinases. These compounds and their kinetic parameters toward several protein kinases are listed in Table I and the structures of these inhibitors are displayed in Fig. 1 and 2. The isoquinoline sulfonamides appear to be relatively specific for protein kinases, as tests against other ATP-utilizing enzymes such as ATPases and adenylate cyclase generally show little or no inhibition (Hidaka et al., 1984; Hagiwara et al., 1987; Asano et al., 1989). Although only limited systematic structure-activity studies have been published, it is possible to draw a few general conclusions for this table with regard to the specificities of these compounds. It is clear that most of them are more effective against the cyclic nucleotide-dependent protein kinases than the other enzymes listed in the table. Indeed, H-7, which has been frequently used as a

Table I Inhibition Constants (K, or IC,o*) of Naphthalene and lsoquinoline Sulfonamides for Various Protein Kinases”

Inhibitor H-9

H-8 H-88 H-87 H-89 HA-1004 HA-100 (C-I) H-7 HA- 156 HA-1077 HA-I40 CKI-7 A- 3 ML-9

PKA 1.9 1.2 0.4 0.04* 0.05 2.3 8* 3 8* 1.6

> loo

550 4.3 32

PKG 0.9 0.5 0.8

0.5 1.3 4* 5.8 6* 1.6 > 100

3.8

CAMK I1

MLCK 70 68 50

60

70 5.9* 30

> loo* 30 I50

>

loo* 97 22’ 36 19* 7.0 4

PKC 18 15

80

> loo*

CKI 110 133 60 -

30 40 12*

40

6 1I *

100

-

-

-

9.5 80 -

CKII

> 300 950 100

I40 -

780

-

w

5.1

-

References Hidaka et al. (1984), Chijiwa et 01. (1989) Hidaka et a/. ( 1984) Chijiwa ef al. (1990) Miyamoto et a/. (1 990) Chijiwa et a / . (1990) Hidaka er a/. (1984) Hagiwara et a/. ( I 987). Gerard er al. (1986) Hidaka er al. (1984) Hagiwara et al. (1987) Asano et al. (1989) Hagiwara er al. (1987) Chijiwa et a/. (1989) Inagaki er al. (1986) Saitoh et a / . (1987)

UPKA, Cyclic AMP-dependent protein kinase; PKG, cyclic GMP-dependent protein kinase; CAMK 11, calciumicalmodulin-dependent protein kinase 11; MLCK, myosin light-chain kinase; PKC. protein kinase C; CKI, casein kinase I; CKII, casein kinase 11. *Values marked with an asterisk are IC50 values. Those without an asterisk are K, values.

172

John E. Casnellie

lnhlbl lor

Rl

RZ

H-9

SO,NH(CH,),NH,

H

H-6

H

H-86

H

H-67 H-69

S O 2~ H ( C H

HA-I 004

SO,NH(

,

,N H c H

CH,),NHC

,c H =c

H-@

r

/rH ‘NH2

HA-100

S0,N

n

NH

U

H3C

hN H

H-7

SOzN

HA-156

S02N

HA-1 077

SOzN

HA-140

CI

S0,N

CK 1-7

CI

S02NH(CH,),NH2

u

H

n

NH

u

CI

uN H

H

n

n

NH

u

Fig. 1 Structures of the isoquinoline sulfonamides whose kinetic constants for inhibition of protein kinases are listed in Table I .

173

Protein Kinose lnhibiton

R

Cl

lnhlbl tor

R

w-7

S02NH(CH2),NH,

A-3

S02NH(CH2)2NH2

ML-9

SO,N

n W

NH

Fig. 2 Structures of the chloronaphthalene derivatives A-3 and M L 9 that inhibit protein kinases by competing with ATP. The structure of the calmodulin antagonist W-7 is shown for comparison. The inhibitory constants of A-3 and ML-9 for protein kinases are given in Table 1.

“specific” inhibitor of protein kinase C, is also an effective inhibitor of the cyclic nucleotide-dependent enzymes. Most of the inhibitors show little discrimination between the cyclic AMP- and cyclic GMP-dependent kinases. Comparisons of the structures in Fig. 1 with the kinetic parameters in Table I also reveal that large changes in the structures of the inhibitors can have dramatic effects on their specificities. For example, attaching the relatively bulky p-bromocinnamyl group to the amine of H-9 had little effect on the ability of the resulting compound (H-89) to inhibit MLCK, protein kinase C , or the casein kinases; however, H-89 does have a Ki for the cyclic AMP-dependent protein kinase that is 40-fold lower than that of H-9. Moving the ethylamine sulfonamide group that is at position 5 on the isoquinoline ring in H-9 to position 8 and placing a chlorine at position 5 results in a compound (CKI-7) that is only weakly inhibitory to all the enzymes tested except casein kinase I. Another compound that is more specific is ML-9. This compound has a chlorinated naphthalene ring instead of the isoquinoline ring (Fig. 2). Saitoh et al. (1987) studied a series of naphthalene derivatives and showed that the presence of a halogen on the ring lowered the K , of the compound for MLCK. These workers found that the K , for MLCK correlated with the hydrophobicity of these naphthalene derivatives, the greater the hydrophobicity,

174

John E. Casnellie

the lower the Ki. The increased specificities of H-89, ML9, and CKI-7 over H-9 and the other initial derivatives are impressive and provide a clear demonstration that it is possible to synthesize compounds that are able to discriminate effectively among the ATP binding sites of protein kinases. Another important property of the isoquinoline and chloronaphthalene sulfonamides is that they are permeable to cells and thus can be used for experiments with intact cells. Indeed, the isoquinoline sulfonamides were the first relatively specific protein kinase inhibitors that could be used in this manner. The successful development of these inhibitors greatly stimulated the interest in the development of specific protein kinase inhibitors that are permeable to cells. Since H-7 is relatively potent at inhibiting protein kinase C it has been widely used to evaluate the role of protein kinase C in a variety of cell types, and a large body of literature has developed on experiments with this compound. An extensive evaluation of this literature is beyond the scope of this review but a few general conclusions can be drawn. Obviously, a negative result with H-7 is more meaningful than a positive result. Thus, if H-7 does not inhibit a receptormediated event under conditions where it inhibits an effect of an active phorbol ester, then it seems reasonable to conclude that the receptor-mediated event does not involve protein kinase C. However, if H-7 does inhibit the receptor-mediated event then the conclusion is ambiguous. As can be seen in Table I, published work on the kinetics of inhibition of protein kinases by H-7 is limited to just a few enzymes. Thus, a positive result with H-7 has two interpretations. One is that protein kinase C is involved in regulating the process and the other conclusion is that there is some other protein kinase involved that is inhibited by H-7 but whose susceptibility to H-7 has not yet been documented. How probable is the second possibility? This is difficult to evaluate but several examples have been found where physiological stimuli activate multiple novel protein kinases (Ferrell and Martin, 1989; Ahn and Krebs, 1990; Ahn et al., 1990). One of the best examples was provided by the work of Ferrell and Martin (1989), who used sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by blotting and renaturation to demonstrate that thrombin treatment of platelets activated at least 10 novel protein serine/threonine kinases. Thus, the effects of H-7 on platelets could be due to inhibition of any of these enzymes. In a recent review, Hidaka and colleagues (1990) have cautioned that results with H-7 cannot be interpreted in isolation because it is not completely specific for protein kinase C. The incomplete specificity of H-7 and the involvement of multiple protein kinases may explain the lack of agreement among the large number of studies on the role of protein kinase C in the responses of neutrophils to the chemotatic factor F-Met-Leu-Phe. Indeed, there is a report that treatment of neutrophils with this factor does activate at least one protein kinase that is distinct from protein kinase C (Huang and Laramee, 1988). Another problematical aspect of the studies with H-7 is the wide variations in the concentrations of H-7 that are

Protein Kinose Inhibiton

175

needed in order to inhibit the responses to various stimuli. Nixon et al. (1991) have compared the effect of H-7 on a number of responses in T cells and platelets. They found that while H-7 was highly effective at inhibiting 1L2dependent proliferation, it was ineffective at inhibiting the down regulation of T cell surface antigens caused by treatment with phorbol dibutyrate. They argued that the effect of H-7 on T cell proliferation cannot be due to inhibition of protein kinase C since it did not effectively inhibit the responses to the phorbol ester which are known to be mediated by protein kinase C. This argument gains additional force from the fact that other data has shown that protein kinase C is not involved in IL-2-dependent T cell proliferation (Mills et al., 1988; Valge et al., 1988). Other workers have also observed that H-7 does not consistently inhibit the responses to phorbol esters (Nakadate ef al., 1988) or that it has effects on cells that are independent of its ability to inhibit protein kinase C or cyclic nucleotide-dependent protein kinases (Bedoy and Mobley, 1989; Birrell el al., 1989; Love et al., 1989).

2. Antibiotic Inhibitors Containing the Indole Carbazole Group Another fruitful approach for identifying inhibitors of protein kinases has been to screen natural products. Soil microorganisms have been found to elaborate several compounds that can inhibit protein kinases. A particularly fascinating group are those compounds that contain the indole carbazole chromophore (Fig. 3 and Table 11). Staurosporine was the first compound in this group shown to inhibit protein kinases. Staurosporine is a remarkably effective inhibitor of protein kinase C. Kinetic and binding data have shown that staurosporine inhibits protein kinase C with a Ki on the order of 1 to 3 nM (Tamaoki et al., 1986; Gross et al., 1990; Herbert et al., 1990); this is the lowest Ki for any protein kinase inhibitor thus far discovered. Initially described as a highly specific inhibitor of protein kinase C, further work has demonstrated that staurosporine inhibits several other protein serinekhreonine and protein tyrosine kinases as well, including the cyclic AMP-dependent protein kinase (Nakano et al., 1987), phosphorylase kinase (Meyer et al., 1989; Elliott et al., 1990), ribosomal protein S6 kinase (Meyer et al., 1989), pp60v-src(Nakano et al., 1987), and the receptor tyrosine protein kinases that are regulated by epidermal growth factor (EGF), insulin, and insulinlike growth factor (Meyer el al., 1989; Fujita-Yamaguchi and Kathuria, 1988). While it is difficult to make comparisons of IC,,s and Kis from different papers, the order of potency of staurosporine for these enzymes appears to be roughly protein kinase C > [cyclic AMP-dependent kinase, phosphorylase kinase, S6 kinase, CAMK 11, pp60v-src]> insulin receptor > epidermal growth factor receptor > insulin-like growth factor receptor. From binding data and enzyme inhibition studies it would appear that the affinity of staurosporine for protein kinase C is only slightly greater than its affinity for the cyclic AMP-dependent

& H

ye::, R,

R,

=

H,

=

OH,

=

H ,

R,

= OH:

= H ,

R,

=

R,

Staurotpor I ne

NH-CH,:

=

UNC- 01

NH-CH,:

=

RK-286C

N-CH3:

CGP

41 .25 1

I

Benzoy I H

R,

=

H ,

R,

= H ,

R,

=

R,

=

H ,

R,

= H,

R,

= H:

R,

=

H ,

R,

= H,

R,

=

R,

=

H ,

R 2 = CH,,

R,

= O(CH,),CH,,

R, R,

=

CH,:

K252a K252b

(CH,),CH,: =

H,

CH,: R,

KT5720 KT5822

=

CH,:

KT5926

177

Protein Kinose Inhibitors

Table II Inhibition of Protein SerineiThreonine Kinasesa by Indole Carbazole Alkaloids and Their Analogs

~

Inhibitor

PKC

PKA

Staurosporine

0.003*

0.008*

UCN-0 I RK-286C CGP 41.251 K252a

0.004* 3 0.05* 0.025

0.04*

K252b KT5720 KT5822 KT5926

0.02 >2 0.08 0.7

PIG

MLCK

~

CAMK I1

Tamaoiki et al. (1986), Nakano et al. (1989b) Takahaski et al. (1989b) Osada et al. (1990) Meyer er al. (1989) Kase et af. (1987), Nakanishi et al. (1990) Kase ef al. (1987) Kase er al. (1987) Kase er a/. (1987) Nakanishi et al. (1990),

2.4* 0.02 0.09 0.06 0.04 1.2

Nonplanar analogs, compound no. I 0.3* 16* 2 0.08* 5* 3 0.01* 2* 4 0.03* 3* 5 0.09* >20*

References

Davis er nl. (1989)

> loo* 15'

17* 14*

>loo*

'JPKC, Protein kinase C; PKA, cyclic AMP-dependent protein kinase; PKG, cyclic GMPdependent protein kinase; MLCK, myosin light-chain kinase; CAMK 11, calciumicalmodulindependent protein kinase 11. *Values marked with an asterisk are ICso values. Those without an asterisk are K, values.

protein kinase and the other enzymes in this group. However, its IC,,s for the insulin receptor, epidermal growth factor receptor, and insulin-line growth factor receptor are approximately 60, 600, and 6000 nM, respectively (Meyer et al., 1989; Fujita-Yamaguchi and Kathuria, 1988). Other than being a less effective inhibitor for receptor protein tyrosine kinases, it is difficult to discern a correlation between the affinity of staurosporine for protein kinases and the degree of sequence homology among the catalytic domains of these enzymes. Indeed, the insulin-like growth factor receptor and the insulin receptor have a very high degree of sequence homology-much greater than that between protein kinase C and pp60v-src(Hanks er a l . , 1988)-yet staurosporine binds to the catalytic domain of the insulin receptor 100 times more effectively than to the insulin-like growth factor receptor. Clearly, the degree of sequence homology is not a very Fig. 3 Structures of the indole carbazoles whose inhibition constants for protein kinases are listed in Table 11.

178

fohn E. Casnellie

reliable predictor of the relative susceptibility of protein kinases to the action of this inhibitor. The ability of staurosporine to bind to a variety of protein kinases with an affinity that is two orders of magnitude greater than that of ATP suggests that it is recognizing some three-dimensional aspect of the ATP binding site that is common-and therefore important to the catalytic process-in all of these enzymes. The high affinity of staurosporine in comparison to the substrate is reminiscent of the ability of transition state analogs to bind to active sites much more tightly than substrates. It is possible that some part of staurosporine’s structure resembles that of ATP in the transition state in the active sites of protein kinases and this is the source of its high binding affinity. If this is the case, then the tight binding of staurosporine suggests not only similar structures at the active sites of these protein kinases but similar mechanisms of phosphate transfer as well. Another indole carbazole that has been isolated from microbial organisms is K252a (Kase et al., 1986, 1987) (Fig. 3 and Table 11). Its structure is similar to that of staurosporine except that the sugar attached to the chromophore has a fivemembered instead of a six-membered ring. Like staurosporine, K252a has a broad specificity for inhibiting protein kinases, although in general it seems to be less effective than staurosporine, Several other indole carbazoles have either been isolated or prepared semisynthetically and some of these have reduced activity toward certain protein kinases and thus greater specificity toward others (Fig. 3 and Table 11). Staurosporine and the other indole carbazoles are obviously very interesting compounds. However, a cursory examination of the kinetic data on the effect of staurosporine and K252a on isolated protein kinases clearly shows that these agents are not specific inhibitors of protein kinase C. Indole carbazoles may inhibit the effects of phorbol esters because of their ability to inhibit protein kinase C; however, one can confidently draw this conclusion only because of the known specificity of phorbol esters for activating protein kinase C. Inhibition of the effects of receptor-mediated events by staurosporine is inconclusive because of the broad and as yet incompletely determined activity of this agent as an inhibitor of protein kinases or an effector of other enzymes. There have been several reports where staurosporine paradoxically appeared to augment or mimic the effects of tumor promoters, suggesting that it may have actions other than inhibition of protein kinases (Kiyoto et al., 1987; Sako et af., 1988; DierksVentling et al., 1989; D. J. Taylor et al., 1990; Yoshizawa et al., 1990). Although staurosporine has not been shown to have effects on enzymes other than kinases, K252a has been shown also to inhibit calmodulin-dependent enzymes (Kase et al., 1986; Matsuda et al., 1988) and cyclic nucleotide phosphodiesterase. No systematic attempts have been made to study how the structure of the indole carbazoles influences their ability to inhibit protein kinases. However,

Protein Kinose Inhibiton

179

there has been a preliminary report of structure-activity studies with compounds that are analogs of the indole carbazoles (Davis et al., 1989). These analogs are bis(indoly1)maleimides;they differ from the indole carbazoles in that they do not have the bond that directly connects the two indole rings; in addition, these analogs have a second carbonyl placed symmetrically on the five-membered lactam ring to give the maleimide group. Since the two indole rings in these analogs are no longer directly tethered, the two carbonyls of the maleimide group can sterically force the indole rings out of the plane of the maleimide ring. Thus, the bis(indoly1)maleimideshave a nonplanar structure in contrast to the flat, rigid indole carbazoles. An important consideration in the design of these analogs was the presence of an amine on the six-membered tetrahydrophyran ring in staurosporine. This amine is absent in K252a and its absence could explain the lower potency of this compound at inhibiting protein kinase C. Consistent with this idea are the results with compound RK-286C, whose structure is identical to that of staurosporine except that it has a hydroxyl group in place of the amine. RK-286C has a Ki for protein kinase C that is 1000 times higher than that of staurosporine (Table 11). In the analogs the tetrahydropyran ring of staurosporine is removed and is replaced by a series of linear functional groups. In spite of these changes in structure from the staurosporine molecule, some of the bis(indo1y)maleimide inhibitors-generally those that have an amine attached to the indole ring-retain low IC,,s for protein kinase C and they are 2 to 3 orders of magnitude less potent at inhibiting either cyclic AMP-dependent protein kinase, CAMK 111, or phosphorylase kinase (Davis et a / . , 1989; Elliott et al., 1990). Thus, these analogs are potentially much more specific inhibitors of protein kinase C . This approach may also open the way to a more rational approach for designing protein kinase inhibitors (Davis et a / ., 1991).

3. Miscellaneous Agents that Compete with ATP Screening for inhibitory activity has revealed the ability of sundry agents to antagonize the activity of protein serine/threonine kinases by competing with ATP. The inhibitor of the Na+/H+ exchanger amiloride has been found to inhibit protein kinase C as well as protein tyrosine kinases (Besteman et al., 1985; Davis and Czech, 1985). The narcotic antagonist apomorphine inhibits protein kinase C and cyclic AMP-dependent protein kinase with micromolar inhibition constants (Bruni et al., 1986; Wennogle et al., 1988). Zandomeni et a/. (1986) and Meggio et al. (1990) have shown that the nucleotide analog 5,6dichloro- 1 -(P-D-ribofuranosyl)benzimidazole is a relatively selective inhibitor of the casein kinases with micromolar Kis. Inhibition of casein kinase I1 by this compound is apparently responsible for its ability to inhibit mRNA transcription. Neither protein kinase C nor the cyclic AMP-dependent protein kinase is affected by this analog. Screening of several antitumor nucleoside analogs showed that

180

John

E. Cosnellie

among them only sangivamycin was an inhibitor of protein kinase C (Osada et al., 1989; Loomis and Bell, 1988). The K i of sangivamycin for protein kinase C was approximately 10 pM. Although sangivamycin is a more effective inhibitor of protein kinase C than the cyclic AMP-dependent proteiri kinase, it is not specific for protein kinase C since it also inhibits the EGF receptor protein tyrosine kinase (Osada et al., 1989).

B. Inhibitors competitive with Activating Cofactors or Second Messengers 1. Inhibitors of Protein Kinase C Although the importance of protein kinase C in cell physiology has provided one stimulus for studies of compounds that inhibit its activity, another reasons for the larger number of studies of inhibitors of protein kinase C is simply that the regulation of its activity is complex and, as a result, protein kinase C is subject to inhibition by a wide variety of structurally diverse compounds. For maximal activity protein kinase C requires calcium ion, phosphatidylserine, and diacylglycerol. Of these three cofactors only the diacylglycerol appears to have a significant role as a second messenger and it is increases in diacylglycerol that instigate protein kinase C activation (Nishizuka, 1986). The presence of binding sites for three cofactors in addition to the binding sites for ATP and peptide substrate means that, in principle, there are at least five sites where inhibitors could act on protein kinase C. An additional complicating factor is that there are several isozymes of protein kinase C and these isozymes have subtle differences in their regulation by the three cofactors (see Kikkawa et al., 1989; Parker et al., 1989, for reviews). There have been few attempts to examine inhibitors separately on each species and only one report of an inhibitor acting more effectively on one species than on another (Mahoney et al., 1990). a. Inhibitors that Are Competitive with Phosphatidylserine Protein kinase C has a strong preference for phosphatidylserine as a cofactor, most other lipids being inhibitory. Due to the nonspecific nature of the hydrophobic interactions, a large number of structurally diverse hydrophobic molecules are able to compete with protein kinase C for binding phosphatidylserine and thus inhibit its activity (Kuo eF al.. 1986; Nakadate et al., 1988; Rando, 1988). Many of these inhibitors are effective only at high concentrations. In order to provide an indication of the heterogeneity of the inhibitors that can compete for phosphatidylserine, some of these agents are listed in Table 111. In addition to the agents listed in this table a large number of lipid analogs have been found to inhibit protein kinase C by interfering with the binding of phosphatidylserine (Helfman et al., 1983; Charp et al., 1988; Daniel et al., 1988; Marx et al., 1988;

181

Protein Kinase lnhibiton

Table 111 Agents that Inhibit Protein Kinase C by Conipeting with Phosphatidylsenne Anthracyclines Vinca alkaloids Tamoxifen, other triphenylethylene antiestrogens Triphenylacrylonitriles Calrnidazoliurn Local anesthetics Trifluoperazine, other antipsychotic drugs Palmitylcarnitine Cytolytic polypeptide nielitten Calrnodulin antagonist W-7 Polyrnyxin B

Katoh et a/. (1981, Palayoor et al. (1987) Palayoor et a/. (1987) O’Brian ef a/.(1985, 1986), Su et a/.(1985) Bignon et a/. ( I 989) Mazzei et a/. (1984) Mori er a/.(1980) Mori er a/. (1980), Schatzman et al. (1981) Wise et a/. (1982, Wise and Kuo (1983) Katoh et a/.(1982) Wise ef a / . (1982) Mazzei et a/.(1982). Wise er al. (1982)

Wennogle et al., 1988; Marasco et a / . , 1990). Included in Table Ill are several drugs with important pharmacological properties. While it has often been suggested that inhibition of protein kinase C could be significant in regard to the therapeutic actions of these drugs, there have been few tests of this idea for any of these drugs. I t seems unlikely that drugs as diverse as antipsychotics and antiestrogens would have inhibition of protein kinase C as a common mechanism of action. Indeed, the therapeutic uses of tamoxifen are completely inconsistent with such a mechanism since this anticancer drug is effective only against estrogen-receptor-positive breast tumors. The competitive inhibition versus phosphatidylserine that is observed with compounds such as those listed in Table I11 is also compatible with their competing with phosphatidylserine for binding to protein kinase C, and evidence for directing binding to the enzyme has been presented for some of these compounds (O’Brian et al., 1987, 1988). Since protein kinase C binds phospholipid it must have a hydrophobic pocket for binding the lipid group, and it is possible that this pocket can be occupied by myriad drugs and hydrophobic compounds. In this manner, protein kinase C may resemble serum albumin, which can also bind a large number of drugs in a hydrophobic site that normally binds fatty acid. Many of the agents listed in Table I11 are also inhibitors of calmodulin, and this inhibition is due to their ability to bind to hydrophobic areas on the calmodulin molecule. b. Inhibitors that Compete with Diacylglycerol In principle, the greatest selectivity for inhibitors of protein kinases can be obtained by targeting the inhibitors toward the site of regulation. Synthetic approaches toward synthesis of antagonists of diacylglycerol have thus far been without success. While a large and diverse group of natural products have been found to bind to the diacylglycerol site, virtually all have agonist activity. However, inhibitors that act

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on the diacylglyceroi binding site have been isolated from a soil fungus (Iida et al., 1989; Kobayashi et al., 1989a,b,c). These compounds have a complex multiring quinone structure and are termed calphostins. The most potent of these compounds is calphostin C and its structure is shown in Fig. 4. Calphostin C at a concentration of 1 pM caused complete inhibition of the binding of 50 nM [ 3H]phorbol dibutyrate to protein kinase C and consequent inhibition of its activity, while at 50 p M calphostin C had no effect on the activity of either cyclic AMP-dependent protein kinase or pp60v-src (Kobayashi et al., 1989~).Calphostin C inhibited the a, p, and y isozymes of protein kinase C with equal potency. Calphostin C is highly toxic to tumor cell lines, although it is not clear whether this is related to its ability to inhibit protein kinase C. Two other compounds with structures similar to that of calphostin C have also been found to inhibit the activity of protein kinase C, apparently by binding to the diacylglycerol site. These are the plant products hypericin and pseudohypericin (Takahasi et af., 1989a). Their potency is considerably less than that of calphostin C. Hannun et al. (1989) have reported that the adriamycin-(Fe3+) complex inhibits protein kinase C apparently by competing at the diacylglycerol site. It inhibited phorbol ester binding in vitro to protein kinase C with an IC,, of 50 pM. At higher concentrations (350 pV), adriamycin-(Fe3 ) also inhibited protein kinase C by binding to a site on the catalytic domain. c. Inhibitors that Act on More Than One Site in Protein Kinase C The most important inhibitors in this group are sphingosine and its derivatives. Sphingosine is an 18-carbon chain lipid base. In addition to an amine group it has two hydroxyl groups at its hydrophilic end. These hydrophilic groups provide points of attachment for alkyl groups, fatty acids, and sugar molecules to give +

Fig. 4 Structure of the protein kinase C inhibitor calphostin C

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various sphingoid derivatives (Hannun and Bell, 1989). Sphingosine is a building block for the synthesis of sphingomyelin and other complex sphingolipids found in high concentrations in nervous tissue. It inhibits protein kinase C competitively with respect to diacylglycerol, calcium, and phosphatidylserine (Hannun el al., 1986). These results led to the proposal that sphingosine was a natural negative regulator of protein kinase C (Hannun et al., 1986, Hannun and Bell, 1989) and to the use of sphingosine as an inhibitor of protein kinase C to study the physiological functions of this enzyme. However, it has become apparent that sphingosine has other effects besides inhibition of protein kinase C. Sphingosine is also an antagonist of calmodulin (Jefferson and Schulman, 1988), a property consistent with its amphipathic character as a lipid base. It has been shown to activate the EGF-receptor protein tyrosine kinase (Faucher et al., 1988; Davis et a l . , 1988; Northwood and Davis, 1988; lgarashi et al., 1990), to inhibit the activity of pp60v-src(Igarashi et al., 1989), and to affect other biological processes in a manner that is independent of its ability to inhibit protein kinase C (Winicov and Gershengom, 1988; Sohal and Cornell, 1990; Winicov et al., 1990; Zhang e t a / ., 1990). Thus, the use of sphingosine appears to be inappropriate for studies of protein kinase C in intact cells. Other inhibitors of protein kinase C whose kinetics are complex and have been studied in detail are the aminoacridines (Hannun and Bell, 1988) and suramin (Hensey et al., 1989; Mahoney et al., 1990). Aminoacridines inhibit protein kinase C predominantly by interacting with its regulatory domain and competing with both phosphatidylserine and diacylglycerol. Aminoacridines have a number of biological effects, including DNA intercalation and antitumor activity, in addition to any that would result from inhibition of protein kinase C. As in the case of sphingosine, the aminoacridines have an amphipathic character, being large hydrophobic molecules with a charged amine and the amine is essential to their ability to inhibit protein kinase C. Suramin is also a complex amphipathic molecule. It consists of a string of six aromatic rings, two of the rings each modified with three negatively charged sulfite groups. The effect of suramin on protein kinase C is complex and concentration dependent. At high concentrations it was competitive with ATP, while at lower concentrations suramin was actually able to substitute for phospholipid and activate protein kinase C in the presence of calcium ion (Mahoney et al., 1990). Suramin does not inhibit the binding of phorbol ester to protein kinase C. It does show some discrimination in its effects on the three main protein kinase C isozymes. The inhibitory effects of suramin on protein kinases are not confined to protein kinase C.

2. Antagonists of Cyclic Nucleotide-Dependent Protein Kinases During the 1970s, a group at ICN synthesized a large number of different derivatives and studied their effects on cyclic nucleotide phosphodiesterase and cyclic

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nucleotide-dependent protein kinases (reviewed in Simon el al., 1973). While some of the denvatives could act as antagonists of phosphodiesterase, none of the derivatives were antagonists of the kinase, except at high concentrations where they were sometimes competitive with ATP. As mentioned in the introduction, an antagonist of the cyclic AMP-dependent protein kinase would have been extremely useful in the early studies on the role of this enzyme in signal transduction by cyclic AMP. One of the compounds made by this group was adenosine 3‘,5’-monophosphorothioate, in which one of the oxygens attached to the phosphorous is replaced by a sulfur atom. Eckstein et al. (1974) also synthesized this compound. The resultant analog is composed of two diastereomers with potentially different properties. However, neither group had the methodology to obtain the two diastereomers in purified forms and thus the characterization of these derivatives was incomplete. Baraniak et al. (1979) reported a stereospecific synthesis of both diastereomers. Examination of the effects of these diastereomers on the cyclic AMP-dependent protein kinase showed that the S, analog, in which the sulfur is in the axial position with respect to the sugar ring, activated the cyclic AMP-dependent protein kinase (de Wit et al., 1982, 1984; O’Brian et al., 1982). However, the R, analog, which has the sulfur in the equatorial position, is an antagonist of the enzyme with a K i of 12 pM, in comparison with the K , of cyclic AMP of 0.03 pM. Rothermel et al. (1983) showed that the R, could antagonize the effects of the S, derivative on glycogenolysis in isolated rat hepatocytes, thus demonstrating its potential for in vivo studies. They also noted the fact that the R, analog is the only one out of approximately 600 derivatives of cyclic nucleotides that has antagonist activity. In spite of its relatively low affinity for the cyclic AMP-dependent protein kinase, the R, antagonist has been used to suppress the effects on intact cells of glucagon, choriogonadotropin hormone, and parathyroid hormone (Pereira et al., 1987; Connelly et al., 1987; Lau and Bourdeau, 1989) and to suppress cyclic AMP-mediated increases in the mRNA levels of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase in rat hepatocytes (Buchler et al., 1988). Similarly, it was recently reported that (RJ-guanosine 3’ ,5’-monophosphorothioate is an antagonist of the cyclic GMP-dependent protein kinase, with a Ki of 20 pkf (Butt et al., 1990). The 8-chloro derivative of this compound was a more potent inhibitor, with a Kiof 1.5 pM, in comparison to the K, of cGMP of 0.2 pM for the cyclic GMP-dependent protein kinase. The low K iof 8-chloro-(I$)-guanosine 3’ ,5’-monophosphorothioateshould make it highly useful for studies on the physiological role of the cyclic GMP-dependent protein kinase.

3. Inhibitors of Calmodulin-Dependent Protein Kinases There are large numbers of different drugs and other relatively small molecules that can bind to calmodulin and thus inhibit the activity of calmodulin-dependent

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protein kinases as well as other calmodulin-dependent processes. Since these agents are not specific protein kinase inhibitors, they will not be discussed in this article and the reader is referred to a recent review (Ovadi, 1989). Another mode by which these enzymes could be inhibited is by the specific binding of an agent to the calmodulin binding site on the enzyme rather than binding to calmodulin. Hagiwara et al. (1989) and Mamiya et al. (1989) found that the thyroid hormones thyroxine and triiodothyronine inhibit MLCK by competing with calmodulin for binding to the kinase. Thyroxine has a Ki of 2.6 pJ4 and a column with covalently bound thyroxine could be used to affinity purify the kinase. Tokumitsu et al. (1 990) have reported that the isoquinoline derivative KN-62 (1- [ N ,0-bis(5isoquinolinesulfonyl)-N-methyl-~-tyrosyl]-4-phenylpiperazine)inhibits CAMK I1 by competing with calmodulin for the calmodulin binding site on the kinase. The interaction was specific in that KN-62 did not inhibit another calmodulindependent enzyme, MLCK. It also had few inhibitory effects on protein kinase C or cyclic AMP-dependent protein kinase.

C. Inhibitors Competitive with ProteWPeptide Substrate Numerous attempts have been made to make inhibitors of protein kinases by synthesizing peptides with the sequences of sites of phosphorylation in substrate proteins. By replacing the phosphorylatable residue by one that cannot form a phosphate ester, a substrate peptide can be converted into an inhibitor. However, this approach has not provided very potent inhibitors. Inhibitors with Kis that are submicromolar all have sequences either from inhibitor proteins or from the enzymes themselves. The first truly specific protein kinase inhibitor to be discovered was the heat-stable Walsh inhibitor of the cyclic AMP-dependent protein kinase. This small protein (8000 Da from the amino acid sequence; Scott et al., 1985b) was found in boiled extracts from rabbit skeletal muscle shortly after the discovery of the cyclic AMP-dependent protein kinase (Walsh et al., 1971). It specifically inhibits the catalytic subunit of the cyclic AMP-dependent protein kinases in a manner that is competitive with peptide (Demaille et al., 1977) with a K , on the order of 1 nM. The biological role of this protein is as yet unclear and its low levels made isolation and sequencing difficult. However, two groups eventually isolated and sequenced peptides derived from the amino terminus of the inhibitor that retained the inhibitory activity of the intact molecule (Cheng et al., 1985; Scott er al., 1985a). Eventually, the entire sequence was determined and the location of the inhibitory domain confirmed by the synthesis of peptides with sequences of the domain. (Cheng et al., 1986; Scott et al., 1986). The most potent of these peptides displayed Kis in the nanomolar range and thus were nearly as potent as the intact inhibitor molecule. The sequence of the inhibitory domain of the protein inhibitor of the cyclic AMP-dependent protein kinase has some features that are also found in the

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substrates for this enzyme. Moreover, structural studies of several other second messenger-regulated protein kinases have revealed the presence of apparent pseudosubstrate autoinhibitory domains (House and Kemp, 1987; Kennelly et al., 1987; Kelly et al., 1988; Payne et al., 1988; Colbran et al., 1989; Malinow et al., 1989; Soderling, 1990; S . S . Taylor et al., 1990). These domains are hypothesized to bind to the active site blocking access by substrates. Upon binding of second messenger (or autophosphorylation of the domain), a conformational change occurs which apparently moves this domain out of the active site so that the substrates can bind and undergo catalysis. These models have found support from the observations that it is possible to synthesize peptides with sequences based on these putative domains that are inhibitory to these enzymes. This has been accomplished for protein kinase C, MLCK, and CAMK 11. [The peptide with the sequence of the autoinhibitory domain of CAMK I1 is competitive with ATP and noncompetitive with peptide (Colbran er al., 1989). It is considered in this section because it is more similar to the inhibitors discussed in this section than inhibitors that compete with ATP.] Soderling (1990) and Smith et al., (1990) have recently reviewed these data and have performed comparisons between the relative abilities of peptide inhibitors for cyclic AMP-dependent protein kinase, protein kinase C, MLCK, and CAMK I1 to inhibit these four enzymes. They found that the peptide derived from the Walsh inhibitor was several orders of magnitude more effective against the cyclic AMP-dependent protein kinase than the other three enzymes. However, the peptide from MLCK showed little discrimination between MLCK and the two other calcium-dependent enzymes. The peptides from protein kinase C and CAMK I1 were most effective at inhibiting the enzyme from which they were derived but still showed significant inhibitory activity against the other calcium-dependent enzymes. These results suggest that the peptide inhibitor for the cyclic AMP-dependent protein kinase can be used with confidence with regard to its specificity while those derived from protein kinase C and CAMK I1 must be used cautiously. The peptide from MLCK inhibits all three calcium-dependent enzymes and therefore has limited value in studying the physiological roles of MLCK. Since the peptide inhibitors are relative large and are highly charged they are not generally permeable to cells. However, there have been several studies where they have either been injected or used with permeabilized cells to demonstrate the involvement of protein kinases in various physiological responses (Alexander er al., 1989, 1990; Heasley and Johnson, 1989; Malinow et al., 1989; Eichholtz et al., 1990; Shen and Buck, 1990).

111. Inhibitors of Protein Tyrosine Kinases Proteirl tyrosine phosphorylations are rare events in comparison with the large number of protein serine and threonine phosphorylations. For every thousand

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proteins in the cell that contain phosphoserine or phosphothreonine there are only one or two that contain phosphotyrosine. Because of its rarity, protein tyrosine phosphorylation is often studied without regard to the kinase responsible for the reaction and this is often the case with the development and use of inhibitors of these reactions. Although tyrosine phosphorylations are relative uncommon, the tyrosine protein kinase family is large and complex, having well over 24 members that can be categorized into two or three different subgroups (Hanks et a/., 1988).

A. Inhibitors Competitive with ATP As is the case with the protein serine/threonine kinases, natural products have been an important source of inhibitors for the protein tyrosine kinases. In the early 1980s it was observed that the flavonoid quercetin could inhibit protein tyrosine kinases as well as protein serinelthreonine kinases (Cochet et a/., 1982; Graziani et a / . , 1983; Gschwendt et al., 1983; Srivastava, 1985). These results encouraged several structure-activity studies with other flavonoid compounds (End et al., 1987; Hagiwara et al., 1988; Ferriola er al., 1989; Geahlen et a l . , 1989). As part of a screening program, Ogawara et a/. (1986) isolated the isoflavonoid genistein from a strain of Pseudomonas on the basis of its ability to inhibit the EGF-receptor protein tyrosine kinase. Genistein had been previously isolated from other sources; its structure is shown in Fig. 5 together with the flavonoid quercetin. Studies comparing the ability of quercetin and genistein to inhibit several protein kinases revealed that both compounds inhibit the EGF receptor and pp60v-srcwith micromolar K,s and they were competitive with ATP (Akiyama et a / ., 1987). However, quercetin can also inhibit protein kinase C and phosphorylase kinase while genistein had little or no effect on these enzymes. Apparently, the different location of the six-membered ring in genistein reduced its ability to inhibit protein serine/threonine kinases without affecting its ability to inhibit protein tyrosine kinases. In a study of a series of isoflavones it was found that genistein was the most effective at inhibiting the EGF-receptor and pp60v-srcprotein tyrosine kinases, all three hydroxyls on the rings in genistein being necessary for maximal inhibition (Ogawara e f al., 1989). Genistein has not been extensively characterized with regard to its inhibition of protein kinases. Geahlen and McLaughlin (1989) have found that it does not inhibit a protein tyrosine kinase purified from the thymus, and thus genistein is not effective against all protein tyrosine kinases. The only published work on protein serine/threonine kinases in vitro is that of Akiyama et a / . (1987). Most of the work with genistein has been with intact cells. Genistein has been reported to inhibit the protein tyrosine kinase that is stimulated on T cell activation (Mustelin et al., 1990). It has been found to inhibit protein tyrosine phosphorylation induced by stimulating cells with platelet-derived growth factor (Hill er a/.. 1990) and the tyrosine phosphorylation that occurs when keratinocytes are stimulated to

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A

B

OH I

Fig. 5 Structures of the isoflavonoid genistein (A) and the flavonoid quercetin (B).

differentiate with high calcium or a phorbol ester (Filvaroff et al., 1990). Genistein inhibited the tyrosine phosphorylation that occurs when platelets are stimulated with platelet-activating factor (Dhar et al., 1990). In each of the aforementioned cases, inhibition of tyrosine phosphorylation caused a concomitant inhibition of the physiological response, thus suggesting that the tyrosine phosphorylation was a necessary step toward these responses. Genistein inhibited increases in inositol phosphate and diacylglycerol formation and increases in cell calcium when cells were stimulated by mitogens whose receptors are tyrosine protein kinases; however, it did not inhibit these responses when cells were stimulated with either angiotensin or ATP, which do not work through a protein tyrosine kinase, suggesting that genistein's effect is confined to inhibition of tyrosine phosphorylation (Dean et al., 1989). However, this conclusion assumes a convergent rather than parallel pathway for second messenger generation by these two sets of activators. Another group did experiments with a similar rationale and came to the conclusion that genistein inhibits cell growth by means other than direct inhibition of protein tyrosine kinase activity (Linassier et al., 1990). Although there are no reports that genistein can inhibit any protein kinase other than protein tyrosine kinases, three groups have reported that genistein can

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inhibit topoisomerases (Okura el al., 1988; Markovits et al., 1989; Yamashita et al., 1990) and induce DNA strand breakage in vivo (Constantinou et al., 1990; Kiguchi et al., 1990), suggesting alternative mechanisms whereby genistein can be cytotoxic. One of these groups reported that genistein is much more toxic to ras-transformed NIH3T3 cells than to normal NIH3T3 cells (Okura et a . , 1988), a curious observation that is difficult to reconcile with a mechanism of toxicity involving inhibition of protein tyrosine kinase activity. Although genistein has already been shown to have some interesting properties, it simply has not been sufficiently well characterized to judge its specificity as a protein tyrosine kinase inhibitor. Its purported lack of activity for protein serine/threonine kinases is based largely on published work with only two protein serinekhreonine kinases. The observation that genistein can inhibit topoisomerases raises the possibility that it could have multiple biological effects. Taken together, these considerations would suggest that genistein must be used cautiously in studies on the role of tyrosine protein kinases in physiological responses. Another inhibitor of protein tyrosine kinases that competes with ATP was isolated from the culture filtrate of a strain of Streptomyces (Onoda et al., 1989). This compound is a tertiary amine substituted with three phenyl groups and is called lavendustin A. It inhibited the EGF-receptor protein tyrosine kinase with a Ki of approximately 12 nM and it was competitive versus ATP. Lavendustin A had no effects on protein kinase C or the cyclic AMP-dependent protein kinase, though it did inhibit phosphatidylinositol kinase with a Ki of 17

w.

B. Inhibitors Competitive with ProteidPeptide Substrates Inhibitors in this category have come from the screening of natural products and synthetic chemistry. In early 1986, Umezawa and co-workers reported the isolation of an agent from a strain of Streptomyces that inhibited the activity of the EGF-receptor protein tyrosine kinase (Umezawa et al., 1986). Since this enzyme is probably the normal homolog of the erbB oncogene the agent was called erbstatin. The structure of erbstatin is shown in Fig. 6. Erbstatin inhibited the EGF-receptor kinase at micromolar concentrations but inhibited the cyclic AMPdependent protein kinase only at 100-fold higher concentrations. This group published the chemical synthesis of erbstatin and several derivatives (Isshiki et al., 1987a,b). As tested on the EGF receptor, the results showed that the two hydroxyl groups on the ring of erbstatin were necessary for the compound to be an effective inhibitor. Erbstatin was further shown to be a competitive inhibitor of the EGF-receptor protein tyrosine kinase with respect to peptide substrate with a Ki of approximately 6 pM (Imoto e l al., 1987a). Since erbstatin is uncharged it can penetrate intact cells and it has been shown that erbstatin is toxic to some tumor cell lines (Imoto et al., 1987b; Toi et al., 1990), although such toxicity

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B

A

0

H

qrNCHO H O/

Fig. 6 Structures of the protein tyrosine kinase inhibitors erbstatin (A) and the 4hydroxycinnamamide ST 638 (B).

was not shown to have resulted from erbstatin's ability to inhibit tyrosine phosphorylation. Umezawa et al. (1990) have demonstrated that a derivative of erbstatin can specifically inhibit EGF-dependent DNA synthesis. Levitzki and co-workers have made a large number of analogs of erbstatin (Yaish et al., 1988; Gazit et al., 1989) and have studied the relative abilities of these analogs to inhibit the EGF and insulin receptor protein tyrosine kinases. Levitzki (1990) has reviewed this work. Levitzki et al. refer to these analogs as tyrphostins. In an initial paper, they showed that it was possible to develop inhibitors with Kis for the EGF-receptor kinase that are three orders of magnitude lower than their Kis for the insulin receptor (Yaish et al., 1988), and therefore that it was possible to synthesize inhibitors of protein tyrosine kinases that are potentially specific for one enzyme. They have further shown that these analogs can specifically inhibit EGF-dependent breakdown of phosphoinositides and EGF-dependent cellular proliferation (Posner et al., 1989; Lyall et al., 1989). Another group independently reported that certain synthetic 4-hydroxycinnamamides have the ability to inhibit the activity of the EGF-receptor protein tyrosine kinase without affecting the activities of protein serinehhreonine kinases (Shiraishi et al., 1987). The structure of one of the most potent of these compounds (ST 638) is also shown in Fig. 6, where it can be compared to that of erbstatin. It is clear from the figure that they hydroxycinnamamides are structurally similar to erbstatin, in particular the position of the vinyl group relative to the aromatic ring is identical in the two compounds. Characterization of these hydroxycinnamamide derivatives showed that several of them were effective against the EGF-receptor, pp6OV-"", and viral f g r protein tyrosine kinases with IC,,s on the order of 1 to 10 @ (Shiraishi I et al., 1987, 1989) and that they were

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competitive versus peptide. Shiraishi et al. (1990) have shown that ST 638 can also inhibit the EGF-receptor, pp60v-src,and p70gag-actin-v-fgr protein tyrosine kinases in intact cells. Thus, erbstatin, its analogs, and the 4-hydroxycinnamamides have many of the properties that are desired in a specific protein tyrosine kinase inhibitor. They bind to the peptide binding site and therefore are likely to be more specific than inhibitors that bind to the ATP site; they are relatively small and uncharged and thus can penetrate intact cells. Thus, it is likely that these compounds will be of extensive use for studies of the role of tyrosine phosphorylation in cell function. In addition to the biological studies cited above, these compounds have been used in the studies of the tyrosine phosphorylations that precede the activations of T lymphocytes (Stanley et a / . , 1990), platelets (Salari et a / . , 1990), and neutrophils (Gomez-Cambronero et al., 1989). In each case the compounds inhibited both the tyrosine phosphorylation and the physiological response, thus demonstrating a potential causal link between these two events. In addition to the inhibitors based on the structures of erbstatin and 4hydroxycinnamamide, Shechter et al. (1989) examined a variety of analogs of the tyrosine residue for their ability to inhibit the insulin-receptor protein tyrosine kinases. All the analogs had Kis in the millimolar range. Geahlen and McLaughlin ( 1989) have shown that tetrahydroxy-trans-stillbeneinhibits protein tyrosine kinases by acting competitively with regard to peptide. It had a micromolar K , and it had no effect on the cyclic AMP-dependent protein kinase.

C. Inhibitors Designed to Be Transition State Analogs Several attempts have been made to synthesize inhibitors that could potentially mimic the transition state of phosphate transfer from ATP to the tyrosine residue. The resultant compounds display different types of inhibition but are considered as a group because they share a common design rationale. Two groups (Kruse el a/., 1988a,b; Baginski et a / . , 1989) have made inhibitors that incorporate elements of both ATP and the tyrosine residue. However, these compounds seem to bind largely to the ATP binding site and did not display the high affinity characteristic of niultisubstrate inhibitors. Saperstein et a / . (1989) designed a phosphonic acid derivative of naphthalene that inhibited the insulin-receptor protein tyrosine kinase with an IC,,, of 200 phf. The inhibitor was converted to a “prodrug” by esterifying the acid and hydroxyl groups. The prodrug could apparently penetrate intact cells where esterases converted it to the active form which inhibited the insulin receptor and the physiological responses of intact cells to insulin. Yuan et al. (1990) synthesized a peptide inhibitor whose sequence was based on that of a peptide substrate with a relatively low K,n; however, in the inhibitor the tyrosine is replaced by tetrafluorotyrosine. The rationale for this was that the fluoro atoms are small and would not increase the

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size of the phenyl group but they would lower the pK, of the hydroxyl. By making the phenol more acidic, they hoped to increase its affinity for the base that they hypothesized is involved in phosphate transfer at the active site. The resultant peptide did have some of the properties of a transition state analog. In studies with the insulin receptor, the inhibitor shows competitive kinetics versus both ATP and peptide and has a Ki of 4 p M , a value that is the lowest for any peptide inhibitor of protein tyrosine kinases.

D. Protein Tyrosine Kinase Inhibitors that Have Other Modes of Action Perhaps one of the most curious inhibitors of protein tyrosine phosphorylation is the antibiotic herbimycin A. This compound is a benzoquinone ansamycin (Fig. 7) that was isolated from the culture broth of a strain of Streptomyces. It was isolated on the basis of its ability to cause fibroblasts transformed by the Rous sarcoma virus to revert from the rounded, transformed morphology to the normal, more flattened morphology (Uehara et al., 1985; see Uehara and Fukazawa, 1991, for a review of the use of this compound). Further examination of this phenomenon showed that incubation of cells for several hours with herbimycin A caused a loss of enzymatic activity of the transforming protein of the Rous sarcoma virus pp60v-src,with subsequent loss of in vivo levels of tyrosine phosphorylation (Uehara et al., 1985, 1986, 1989a). The loss of enzymatic activity by pp60v-srccaused the reversal of the transformed phenotype (Murakami et al., 1988). The decrease in enzyme activity involved both the loss of enzymatic activity and lower levels of the protein. Studies of the effect of herbimycin A on cells transformed by other oncogenes showed that it was relatively selective for causing reversion of cells transformed by oncogenes whose protein products are protein tyrosine kinases such as src, yes, fps, ros, abl, and erbB (Honma et al.,

CH3

C H

8

CH3

CH3

8

0

Fig. 7 Structure of the protein tyrosine kinase inhibitor herbimycin A

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1989; Uehara el ul., 1988). Herbimycin did not reverse the morphologies caused by transformation with raf, myc, or rus. In the case of cells transformed by src, ros, and erbB, it was shown that loss of the transformed phenotype correlated with reduction in the levels of proteins phosphorylated on tyrosine residues. Studies on the mechanism of how herbimycin A inhibits the activity of pp60v-src revealed that herbimycin A caused irreversible inactivation by reacting with a sulfhydryl group in a reaction that involves the quinone moiety of herbimycin A (Uehara et al.,1989a). This sulfhydryl is not essential for activity since it can be modified by iodoacetamide without inhibiting the activity of pp60v-src.Indeed, pretreatment with iodoacetamide can protect the enzyme from in vitro inactivation with herbimycin A (Fukazawa et al., 1990). These results suggest that inactivation by herbimycin A involves steric hindrance of the active site rather than destruction of catalytic activity per se. Obviously, herbimycin A has the potential for multiple interactions when applied to intact cells. It is unclear how specific it is for inactivating tyrosine protein kinases over other enzymes, although its effects on protein tyrosine kinases were observed at concentrations of herbimycin A that were not generally toxic. It has recently been shown that herbimycin A inactivates two protein tyrosine kinases in T cells while being relatively sparing of protein kinase C and another protein serinelthreonine kinase c-ruf (June et al., 1990). Herbimycin A apparently does not inactivate the cyclic AMP-dependent protein kinase (Uehara and Fukazawa, 1991). It would be useful to know if the mechanism of inactivation of the other tyrosine protein kinases is the same as that found for pp60v-src and whether there are structural features of herbimycin A that favor its inactivation of protein tyrosine kinases over other enzymes. It would also be interesting to know if herbimycin A can synergize with another inhibitor such as erbstatin which works by a different mechanism. Herbimycin A’s unique mechanism may make it useful for studies of protein tyrosine phosphorylation in intact cells. However, unambiguous results require the careful design of controls which eliminate the possibility that any observed effects are due to nonspecific actions of this agent.

IV. Concluding Remarks Several different approaches have been taken to find specific inhibitors of protein kinases. Many of the most interesting inhibitors have come from systematic screening of natural products, and these compounds have provided the basis for the synthesis of other, potentially more specific inhibitors. While a great deal of progress has been made and some very useful inhibitors have been discovered, the lack of specificity remains a problem. The results thus far can be said to provide a demonstration that the synthesis of specific inhibitors is a difficult but

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potentially attainable goal. In the future, greater cognizance must be taken of the fact that cells contain a large number of protein kinases and that in v i m results with only a few enzymes cannot be extrapolated to the complex situation in an intact cell. There are several examples where the term “specific protein kinase inhibitor” has proved to be a misnomer. The results with studies on the effects of protein kinase inhibitors on intact cells often have rather malleable interpretations. The desire for a specific inhibitor should not outweigh the necessary skepticism that is demanded by the fact that few of the current inhibitors have been demonstrated to be truly specific.

Acknowledgments The work in the author’s laboratory is supported by NIH grant CA38821. I thank Dr. Neal Bramson for his helpful comments on this manuscript.

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Renin Inhibitors Hollis D. Kleinert, William R. Baker, and Herman H. Stein Abbott Laboratories Cardiovascular Research Division Abbott Park, Illinois 60064

I . Introduction 11. Physical and Functional Properties of Renin A. The Role of Renin in the Renin-Angiotensin Aldosterone System B. Enzyme Classification, Characteristics, and Pharmacokinetics of Renin Ill. Biochemical Evaluation and Specificity of Renin Inhibitors A. Proteinase Specificity B. In V i m Test Systems for Evaluation of Renin Inhibitors C. Plasma Renin Activity Assays in the Presence of Renin Inhibitors D. Species Specificity of Renin Inhibitors IV. Design and Structure of Renin Inhibitors A. Immunological Renin Inhibition B . Rational Approach for Designing Renin Inhibitors V. Pharmacology of Renin Inhibitors A. Experimental Efticacy Models B . Acute Effects of Parenterally Administered Renin Inhibitors C. Chronic Adniinistrdtion of Renin Inhibitors D. Dissociation between Plasma Renin Activity and Hypotensive Effect E. Oral Efficacy V1. Conclusion References

1. Introduction Interference with the renin-angiotensin-aldosterone system (RAAS) has resulted in the development of an extremely important, beneficial, and successful class of therapeutic agents and research tools, the angiotensin-coverting enzyme inhibitors (ACEIs). One might have predicted that abnormal activity of this major hormonal regulatory axis would be involved in the etiology of some forms of 207

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cardiovascular-renal disease and that interference with the RAAS would be a good choice of therapeutic target, but the degree of applicability of this class of agents was unanticipated. ACEIs are effective antihypertensive agents in a wide variety of subpopulations of hypertensives, displaying a high degree of efficacy in high and normal renin forms of hypertension, but also demonstrating activity in low-renin hypertensive patients (Case et al., 1978; Brunner et al., 1979; Gavras et al.. 1978). Patients on ACEI therapy report a feeling of well being, an additional advantage of this type of therapy, which supports patient compliance (Croog et al., 1986). Also, ACEIs are used in the treatment of congestive heart failure (Cody, 1985; Ferguson et al., 1984) and have recently been proposed as therapy for the medical management of diabetic nephropathy, memory disorders, postmyocardial infarction, and posttransluminal angioplasty (O’Connor, 1990). Although this class of agents represents an impressive advance in therapy, ACEIs lack selectivity for the RAAS. Angiotensin-converting enzyme (ACE) is a versatile enzyme that is capable of hydrolyzing a variety of substrates, e.g., bradykinin, enkephalins, substance P, neurotensin, and luteinizing hormonereleasing hormone (LH-RH), in addition to angiotensin I (Erdos, 1990). The antihypertensive activity may be largely related to the inhibition of the RAAS (Lo et a/., 1990), but the side-effect profile, which includes reports of urticaria (S. M. Wood et al., 1987; Wilkin et al., 1980), angioneurotic edema (S. M. Wood et al., 1987), and, more frequently, the occurrence of coughing (Coulter and Edwards, 1987), may be linked to the nonspecific actions of ACE (Slater et al., 1988; Gavras and Gavras, 1988). Theoretically, interruption of the generation of angiotensin I1 (ANG 11) by inhibitors of renin should offer a more specific alternative to ACEI therapy, since renin has only one known substrate, angiotensinogen. Furthermore, the inhibition of renin allows for intervention of RAAS activity at the initial, rate-determining step of the cascade. Renin inhibitors may have therapeutic advantages over ACEIs and other antihypertensives with less precise mechanisms of action by eliciting fewer side effects. This article reviews the advances and challenges encountered in the process of discovering and developing renin inhibitors.

II. Physical and Functional Properties of Renin A. The Role of Renin in the Renin- Angiotensin-Aldosterone System The RAAS regulates blood pressure and fluid balance. The effector hormone of the system is ANG 11, which was known as the most potent vasoconstrictor hormone in the human body until the recent discovery of endothelin (Masaki, 1989). ANG 11 is generated as a result of two hydrolytic reactions (Fig. 1). The

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n+

ANGIOTENSIN I1 ANOPOTIENSHN Un ANTAQONHSTS

Fig. 1

An outline of the cascade of events leading to the formation and receptor binding of angiotensin I1 (ANG 11). Points of possible interference with this system are listed to the right of the vertical arrows.

liberation of the first 10 amino acids from the amino terminal of angiotensinogen, an a,-globulin originating from the liver and primarily found in the plasma, is catalyzed by renin and results in the formation of the decapeptide angiotensin I (ANG I). This step is the first and rate-limiting reaction of the cascade. In the second step, ACE catalyzes the cleavage of two amino acids from the C-terminal of ANG I, generating the octapeptide ANG 11. This reaction most often takes place at the site of the endothelial cells lining the pulmonary and peripheral vascular lumenal surfaces, but may also occur in epithelial cells, neuroepithelial cells, the male genital tract, and in various body fluids (Erdos, 1990). ANG I1 then binds to its specific receptors (Peach and Dostal, 1990).

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210

B. Enzyme Classification, Characteristics, and Pharmacokinetics of Renin Renin is a member of the aspartic proteinase family (See Section III,A), with two catalytic aspartic acid residues occupying positions 32 and 215 of the active site of this molecule (Bolis et al., 1987). Human active renin circulates in plasma as a glycoprotein (Yokosawa et al., 1980). The oligosaccharide moieties have been suggested to be attached by an N-glycosidic linkage at Asn-5 and Asn-75 (Morris, 1986). In its active form, renin is composed of 340 amino acids (Moms, 1986). The molecular weight of purified renal renin has been estimated to be about 40,000 (Slater and Strout, 1981). However, renin predominantly circulates in the form of prorenin (Hsueh, 1982), a zymogen believed to be physiologically inactive, which differs from active renin by a 43-amino acid prosegment (Moms, 1986). The elimination of renin from the plasma is handled primarily by the liver and, to a lesser extent, by the kidney and transpires in two phases, a rapid component, t,12 = 6.7 minutes, and a slow component, c , / ~= 65.7 minutes (Kim et al., 1987; Keiser et al., 1987). It is unclear as to whether the elimination pattern of renin influences the route and speed at which renin inhibitors are cleared from the circulation. Whether renin inhibitors are removed from the circulation independently or bound in a complex to renin is yet to be elucidated.

111. Biochemical Evaluation and Specificity of Renin Inhibitors

A. Proteinase Specificity A potential clinical advantage of a renin inhibitor compared to an ACEI is the unique substrate specificity of the former. It would be predicted that the lack of activity of renin toward other substrates, unlike ACEI, should result in a lower incidence of side effects. In order to maintain the advantageous specificity profile, it is essential that the renin inhibitor does not interact with other mammalian aspartic proteinases, namely, cathepsin D, gastricsin, and pepsin. A variety of potent inhibitors of human renin have achieved this milestone (see Table I).

B. In Vitro Test Systems for Evaluation of Renin Inhibitors 1. General Considerations Renin is an aspartic proteinase which demonstrates a remarkable specificity for its substrate. Not only is its proteolytic activity limited to the single peptide bond

21 1

Renin Inhibitors

Table I Aspartic Proteinase Specificity of Inhibitors of Human Renin

Compound" H-l I3 CGP 29 287 ES-305 SR 43845 Compound 22 Compound 13 ES 8891 Compound 53 CGP 38 560 Enalkiren ~~~~

Renin

Cathepsin D

Gastricsin

Pepsin

0 19

>6.000 40.000 > 10,000 5,000 I .700 > 10,000 > 10,000 > 10,000 600' > 10.000

NRh NR NR NR NR NR NR

NR 40,000 > 10,000 >20,000 42,000 >10,000 >10,000

I 9.2 <0 01 14 2.0 I 1 I .o

0 7' 0.80'

> 10,000 3,000' > IO.000

>10,000 5 ,OOW

> 10,000

Reference Szelke et a / . ( I 982a) Wood er a / . (1985) Kokubu et u/.(1986) Nisato et a/. (1987) Thaisrivongs et a/. (1986a) Dellaria ei a / . ( 1987) Kokubu et a/. (1990) Luly et a/. (1988) Wood et a/. (1989b) Kleinert er u/. (1990)

~~~

USee cited reference for structure hNR, Not reported. ' K , (d).

between the tenth and eleventh N-terminal amino acids of angiotensinogen, but the enzyme also demonstrates species specificity. Primate renin will cleave all mammalian angiotensinogens to form ANG I, but nonprimate renin will not cleave primate angiotensinogen (Ondetti and Cushman, 1981). This latter observation may be due in part to the fact that the scissile bond in the primate substrate is between Leu and Val, whereas it is between Leu and Leu in nonprimate angiotensinogen (Tewksbury et af., 1981). The species specificity imposes a constraint on an in vitro test system for the evaluation of the potency of renin inhibitors since a primate renin must be utilized for compounds destined for human therapy. Renin also differs from other aspartic proteinases in that its pH optimum for activity is 5.5-6.0, considerably higher than, for example, cathepsin D and pepsin. Renin retains activity at physiological pH, but it is of the order of one-half that observed at the pH optimum (Siater and Strout, 1981). Thus, there is a diversity of potential conditions for the establishment of an assay for the evaluation of the potency of renin inhibitors. These include the source of renin activity, choice and source of substrate, pH of the test system, as well as the measurement of the product of the enzymatic reaction, ANG 1.

2. Development of Assays Early workers in the field were limited by a lack of both pure human renin and angiotensinogen. Renin could be isolated from human kidney tissue (Haas et a l . , 1966), but the specific activity was less than I % of that accepted today for pure

212

Hollis D. Kleinerf et

01.

renin (Bangham et al., 1975). Angiotensinogen was present in plasma at the level of approximately 60 mg/liter, but procedures for the isolation of the pure protein were not described until 1977-1978 (Tewksbury et al., 1977; Dorer et al., 1978). A sensitive radioimmunoassay for ANG I had been developed in 1969 (Haber, 1969a), suitable for the determination of plasma renin activity (PRA) (Haber et al., 1969b). Assays in a more purified system initially utilized a tetradecapeptide substrate, isolated from tryptic digests of equine angiotensinogen (Skeggs et al., 1957). The peptide represented the first 14 N-terminal amino acids and it was cleaved by renin to form ANG I. The radioimmunoassay for ANG I was not applicable easily to this system because of the cross-reaction of the substrate with the ANG 1 antibody, and fluorescence procedures were devised for quantitation (Corval et al., 1977; Galen et al., 1978; Poe et al., 1984). Subsequent studies revealed that the fluorescence assay with lowmolecular-weight substrates could yield misleading and artifactual results (Bock et al., 1987). Moreover, there was some question concerning the potency and nature of the inhibition determined with the smaller substrates compared to data obtained with the physiological substrate angiotensinogen (Stammers et al., 1987; Stein et al., 1988). Pure human renin was ultimately isolated from kidney tissue in milligram quantities utilizing ammonium sulfate precipitation and laborious multiple chromatographic procedures (Yokosawa et al., 1980; Slater and Strout, 1981). With the availability of potent, specific inhibitors of human renin, affinity column material capable of producing pure renin from a kidney homogenate in a single affinity chromatographic step could be prepared (McIntyre et al., 1983; Stein et al., 1985). Thus, human renin inhibitors could be evaluated in purified test systems and the intrinsic inhibitor behavior could be evaluated at the pH of maximum enzymatic activity. Such data are most meaningful for the development of structure-activity relationships directed toward the synthesis of potent inhibitors. Renin inhibitors targeted for human therapy must be capable of inhibiting renin in the plasma compartment at physiological conditions. Although cogent evidence has been presented for the existence of renin-angiotensin systems in tissue (Dzau, 1989; Frohlich et al., 1989), the overall goal must include the reduction of circulating levels of ANG I1 resulting from the action of plasma renin on plasma angiotensinogen. For this purpose, it is most appropriate to assess inhibitor potency at pH 7.4 in a plasma milieu.

3. Potency Determinations of Renin Inhibitors Data for the potency of renin inhibitors have been obtained under a variety of experimental conditions. Tables I through X list IC,, values for compounds synthesized in different laboratories and the corresponding literary source. The individual papers should be consulted for the specifics of the respective assays.

Renin l n h i b i t o ~

213

C. Plasma Renin Activity Assays in the Presence of Renin Inhibitors The evaluation of renin inhibitors in animals has revealed that the inhibition of PRA is longer lasting than the pharmacological parameters measured (Blaine et al., 1984; Kleinert et al., 1988a,b). Studies in man have shown that recovery of ANG I1 to control levels after administration of a renin inhibitor is achieved before the PRAs return to the predrug values (de Gasparo et al., 1989; van den Meiracker el al., 1990). Since active plasma renin is the rate-determining step for the generation of ANG 11 and the peptide is degraded rapidly in the blood (Bumpus et al., 1964), it would be expected that the time course of the two variables should be relatively similar. To explain the apparent discrepancy, the hypothesis has been made that angiotensinase inhibitors in the PRA assay, which serve to prevent the degradation of ANG 1 product, artifactually result in lower PRA values when renin inhibitors are present (Nussberger et al., 1989). This concept is supported by clinical data with the renin inhibitor CGP 38 560A (compound 13, Table IV); the ANG I1 levels correlated better with PRA values determined by antibody trapping methodology, in which ANG I is protected by a large excess of ANG I antibody and ethylenediaminetetraacetic acid (EDTA) only (Poulsen and Jorgensen, 1974; Nussberger et ul., 1987), than with PRA determined in the presence of 2,3-dimercaptoethanoI and EDTA (de Gasparo et al., 1989; Nussberger et al., 1989). A proposed mechanism for the effect is the displacement by the angiotensinase inhibitor of renin inhibitor, previously bound to plasma protein. Thus, it is suggested that the effective concentration of renin inhibitor is artifactually increased by the assay methodology in vitro, resulting in apparently lower PRA in vivo (Derkx et al., 1990). Since a variety of different agents, such as 8-hydroxyquinoline and phenylmethylsulfonyl fluoride, have been utilized as angiotensinase inhibitors, and the possibility exists that ANG I1 could be generated from a source other than plasma renin (Boucher el al., 1974; Okunishi et al., 1984), additional data are required to resolve this complex issue.

D. Species Specificity of Renin Inhibitors The synthesis of the first substrate analog (see Section IV,B,2), nanomolar inhibitors of human renin based on the correct sequence of the primate substrate, resulted in compounds which were 2-3 orders of magnitude less potent against canine renin (Szelke et al., 1982a). Primate species specificity was retained in aldehyde derivatives of small peptides representing the C-terminal portion of ANG I (Kokubu et al., 1984), dipeptide and tripeptide inhibitors containing the unnatural amino acid statine (Kokubu et al., 1986), a difluorostatone (Thaisrivongs er al., 1986a), dipeptide glycols (Hanson et al., 1985), inhibitors containing the unnatural amino acid norstatine (Iizuka el al., 1988), and dipeptide analogs of angiotensinogen containing nonpeptidic replacemehts at the scissile bond (Bolis et al., 1987). On the other hand, varying degrees

214

Hollis D. Kleinert et ol.

of specificity were observed with other inhibitors containing statine (Boger el al., 1985b; Bock et al., 1987), pepstatin analogs (Gutgan et al., 1986), a dipeptide hydroxyethylene isostere (Biihlmayer et a/., 1988), and tetrapeptides with hydrophilic groups (Bock et al., 1988). An extensive species selectivity profile for enalkiren has been reported (Kleinert et al., 1990). There does not appear to be a specific determinant which confers primate specificity on a compound, but those substrate analog inhibitors based on the amino acid sequence of primate angiotensinogen at the scissile bond seem to possess greater inhibitory specificity for primate renin.

IV. Design and Structure of Renin Inhibitors A. Immunological Renin Inhibition The first inhibitors of renin were antibodies directed against the enzyme (Johnson and Wakerlin, 1940; Helmer, 1958; Deodhar et al., 1964). Crude renal extracts were utilized to generate the antisera and, because their specificity was questionable, no conclusive results were obtained. More recently, pure renin has become available and antibodies with a high degree of specificity have been generated. Administration of these antisera revealed that both blood pressure and PRA were lowered (Dzau et al., 1980). Similar data were obtained with Fab fragments and monoclonal antibodies (Michel et al., 1984; Dzau, 1985; J. M. Wood et al., 1987a). Studies with antibodies serve to generate basic information about the RAAS, but an immunological approach could not be viewed as a viable means of therapy. Antibody protein would not be absorbed intact from the gastrointestinal tract, and repeated parenteral administrations could potentially induce serious antigenic reactions. Thus, the search for orally active renin inhibitors has been directed along more conventional medicinal chemical lines.

B. Rational Approach for Designing Renin Inhibitors In the early 1950s, pioneering research by Skeggs et al. (1956) and Lentz et al. (1956, and references cited therein) led to the purification and structural determination of the decapeptide hypertensin I and the powerful vasoconstrictor octapeptide hypertensin 11. These two peptides are referred to today as ANG I and 11, respectively. Since ANG I1 was found in the blood of patients with hypertensive cardiovascular diseases, Skeggs postulated that preventing angiotensin’s action in vivo might lead to useful cardiovascular therapy. Skeggs also defined three approaches for inhibiting the action of ANG I1 in vivo: (1) the development of ANG 11 antagonists, (2) inhibitors of ACE, and (3) inhibitors of renin (Skeggs et al., 1957). Skeggs stated, “Since renin is the initial and rate-limiting substance in the renin-angiotensin system it would seem that the last approach would

215

Renin Inhibiton

be the most likely to succeed.” The purpose of Section IV,B is to review the development of renin inhibitors by examining specific compounds which were critical for the advancement of the field.

1. Substrates of Renin In order to utilize a rational approach for identification of compounds that were able to inhibit renin, it was necessary first to elucidate the structure of renin’s substrate. Partial trypsin degradation of equine angiotensinogen yielded a polypeptide renin substrate, tetradecapeptide (1) (Skeggs et al., 1957). The structure of the tetradecapeptide was later confirmed by chemical synthesis (Skeggs et al., 1958). Incubation of 1 with hog renin produced a polypeptide with an amino acid composition identical to ANG I. Further enzymatic degradation of the polypeptide with ACE yielded A N G 11. It was also determined by carboxypeptidase degradation that the four C-terminal amino acids of the tetradecapeptide were leucine, valine, tyrosine, and serine (Skeggs et al., 1957). For the first time, the tetradecapeptide established the required amino acid sequence needed for the renin reaction and became the standard from which other substrates and inhibitors were developed. Skeggs et al. (1968) identified the essential structural requirements for a renin substrate (Table 11). The in vitra kinetics of nine synthetic peptide substrates, which represented portions of the tetradecapeptide renin substrate molecule and contained the hydrolyzable Leu-Leu bond, were evaluated. The octapeptide, His-Pro-Phe-His-Leu-Leu-Val-Tyr (2), was prepared and possessed a K, of 55 phl. When the C-terminal tyrosine was removed, the resulting heptapeptide was no longer a renin substrate. The aromatic amino acid was essential for the action of renin on this substrate. Most renin inhibitors were

Table I / Human Substrate, Substrate Analogs, and Peptide Inhibitor of Renin Reference ~

PI0

p9

PX p7 p6 p5 p4 p3 p2 PI

PI’ p2’p3’

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Protein Human angiotensinogen substrate 1

Asp-Arg-Sr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser Skeggs

ct

al. (1958)

Tetradecapeptide substrate 2

His-Pro-Phe-His-Leu-Leu-Val-Tyr

Skeggs et al. (1968)

Minimum peptide substrate 3

Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys Substrate analog inhibitor (RIP) l C ~ 0= 9 . 6 p M

Burton et al. ( 1989) Zusman et al. (1983)

216

Hollis D. Kleinert et a/.

based on variants of this octapeptide. Stabilization of the Leu-Leu scissile bond and modification of the amino acids adjacent to the scissile bond have been utilized effectively in developing substrate analog inhibitors.

2. Substrate Analog Inhibitors In 1980, Burton and co-workers identified a substrate analog inhibitor of renin. The compound was the decapeptide Pro-His-Pro-Phe-His-Phe Phe-Val-T’yr-Lys (3), which was modeled after the minimum octapeptide substrate and given the name renin-inhibitory peptide (RIP) (Table 11). A Phe-Phe residue replaced the Leu-Leu dipeptide at the scissile bond in 2 and stabilized the molecule toward cleavage by renin. Lysine was added to the C-terminal end of the molecule to increase water solubility and Boc-Pro was added at the N-terminus. RIP possessed in vitro activity in the micromolar range. Although smaller peptide substrates had been shown to inhibit the formation of ANG I in v i m (Kokubu et al., 1968), RIP was the first compound to establish that stabilization of the scissile bond of a renin substrate would produce in vivo pharmacological effects (Burton et al., 1980; Zusman et al., 1983). Efforts to identify other stabilized Leu-Leu or Leu-Val peptide bonds were then instituted.

3 . Peptide Transition-State Inhibitors The next development in the evolution of renin inhibitors was the introduction of a transition-state structure into the molecules. The concept of transition-state analogs in studying enzymatic reactions has been reviewed (Wolfenden, 1972). Inhibitors which mimic the transition structure of the hydrolysis of the Leu-Val amide bond should form strong interactions with the enzyme. Increased binding interactions were predicted for transition-state analogs in comparison with substrate analogs. Application of this concept, as a means for increasing inhibitor binding to renin, was advanced by the development of nonhydrolyzable inhibitors (Fig. 2). Umezawa and co-workers (1970) isolated and Morishima et al. (1970) identified a low-molecular-weight fermentation product, pepstatin, from the culture filtrates of various species of actinomycetes. Pepstatin, isovaleryl-Val-Val-StaAla-Sta (4), was a general aspartic proteinase inhibitor containing the modified amino acid statine. Pepstatin was an extremely potent inhibitor of pepsin, with an IC,, of 4.6 nM, and also a weak competitive inhibitor of renin (Gross et a / . , 1972; Miller et al., 1972). It was proposed that the 3s-hydroxyl group of the internal statine residue was a transition-state mimic of the enzyme reaction and allowed for efficient binding to the active site of the enzyme. Peptide transition-state inhibitors of renin are listed in Table 111. Szelke and coworkers ( 1982a,b) developed three renin inhibitors which contained nonhydrolyzable replacements for the scissile bond. The first two inhibitors were the

217

Renin lnhibiton

amidc bond

bond

unstable

transition state for amide bond hydrolysis

R

~

R

bond stable

a stable transition-state mimic

Fig. 2 Transition-state structure for amide bond hydrolysis and a stable transition-state mimic.

reduced isosteres (CH,-NH) of the equine (H-77, 5 ) and human (H-142, 6) substrate. The in vitro inhibitory potencies of H-77 and H-142 against canine renin were 0.02 and 10 phi’, respectively. However, against human renin, H-77 and H-142 exhibited an IC,, of 1 and 0.01 phi’, respectively. The reversal of activity from canine to human was explained by the closer sequence homology of H- 142 to human angiotensinogen. Thus, the simple modification of the peptide backbone at the scissile site increased the binding affinity to renin. H-261 (7), the third inhibitor developed by Szelke, incorporated a hydroxyethylene isostere (CHOHCH,) at the cleavage site of the human substrate (Leckie et al., 1984). The in vitro potency of H-261 was 0.6 nM, 1700 times more potent than its reduced amide counterpart and 17 times more potent than H-142 toward human renin. The secondary hydroxy group mimicked more closely the transition-state structure for amide hydrolysis and consequently bound tighter at the active site. In 1983, researchers at Merck incorporated statine at the scissile site of a peptide sequence more closely resembling that of human angiotensinogen. These compounds were referred to as statine-containing renin-inhibitory peptides (SCRIP). They were able to prepare potent inhibitors of human renin based on the pepstatin structure. The Merck group improved the in vitro potency of inhibitor 8 (Boger et al., 1983) by incorporating a cyclohexylmethyl group for the isobutyl group into the statine amino acid (Boger et al., 1985b). The new “cyclohexyl” statine inhibitor, 9, was 100 times more potent than 8 and was also

Hollis D. Kleinert et 01.

218 Table 111 Peptide Transition-StateInhibitors of Human Renin lC50 (nhf)

Inhibitor

Reference Umezawa er al. (1970)

4 pepstatin

B

D-His-Pro-Phe-His- N

5

NH-Val-Tyr

1,000

H-77 NH-Ile-His-Lys

6

10

Szelke ef al. (1982a,b)

H-142

Pro-His-Pro-Phe-His- N 'SNH-Ile-His

47

Szelke er al. (1982a)

H-261

0.6

Leckie ef al. (1984)

0

I

7

Iva-His-Pro-Phe-His- N&

OH 0

NH-Leu-P,,e-NH2

8

R = isopropyl

13

Boger ef al. (1983, 1985a)

9

R = cyclohexyl

0.17

Boger er a/. (1985b)

effective in reducing blood pressure in a dose-dependent manner when infused into beagle dogs. The discovery of the cyclohexylmethyl substituent at the P, position and its effect on in vitro potency had a great influence on subsequent renin inhibitor developments. The design of renin inhibitors based on transition-state structures was very successful, and the opportunity to reduce blood pressure with renin inhibitors

219

Renin lnhibibn

was very clear (Plattner and Kleinert, 1987). Thus, the quest for smaller, stable, potent, and orally active dipeptide transition-state inhibitors began. Transitionstate inhibitors improved the in vitro potencies over substrate analogs and greatly improved the in vivo efficacy in experimental animals and man (see Section V,B). The hydroxy-containing isostere “cyclohexyl” statine and the hydroxyethylene isostere found in H-26 I were the most promising dipeptide isosteres devised (Leckie et al., 1984). While these inhibitors possessed the inherent potency and pharmacological characteristics of medicinally useful compounds, they were not practical therapeutic agents from a drug development point of view. The compounds were minimally bioavailable after oral administration, and after intravenous (iv) administration only short in vivo half-lives were noted. These peptide inhibitors were considered investigational tools only.

4. Dipeptide Transition-State Inhibitors Four criteria were considered in developing potentially useful clinical dipeptide renin inhibitors: (1) high in vitro plasma potency at pH 7.4, (2) oral bioavailability and activity, (3) stabilization of the P,-P, amide bond, and (4) low molecular weight. The chemical structure of dipeptide inhibitors was divided into four substructure segments and an example of each is depicted in Fig. 3. The first segment was that portion of the molecule binding to the aspartic acid residues in the active site and adjacent lipophilic binding pockets of renin. This N-Terminus

P3Amino

P, Amino

Acid

Acid

Dipeptide Isostere

:

transition-state stmcture

~

P,’side chain

P, side chain

Fig. 3 Structure of dipeptide renin inhibitors. A: Carbonyl [Biihlmayer er al. (1988)], SO2 [Buhlmayer et al. (1988)l. B: 0 [Plattner er ul. (1988)], CH2 [Plattner et al. (1988)], NH [Plattner et ul. (1988)l. C: H [Luly er al. (1988)). CH3 [Thaisrivongs er al. (1986b)J. D: CHOH [Bolis et al. (1987)l. Carbonyl [Thaisrivongs et al. (1986a)], CH2 [Szelke er al. (1982a)l. E: CH2 [Rosenberg et al. (198811, CF2 [Sham et al. (1987a)], NH [Szelke et al. (1982b)l. F: H [Kleinert er al. (1988c)], CONHR [Kempf et al. (1987)], Isoleucine [Thaisrivongs e l al. (3986b)l. R: 4-Morpholino [Plattner er al. (1988)], r-Butyl [Biihlmayer et al. (1988)l. R,: CH2(Inaphthyl) [Iizuka el al. (1990)l. R2: Propyl [Karlsson et al. (1987)], CH2(4-thiazole) [Hiwada er al. (198811. Rl: CHzcyclohexyl [Sham et al. (1987a)], Isobutyl [Sham er al. (1987a)I. R4: Isopropyl [Buhlmayer er al. (1988)l.

220

Hollis D. Kleinerfet a/.

segment was referred to as the dipeptide isostere. Elements that comprised the dipeptide isostere were the transition-state structure and lipophilic side chains at the P, and P’, positions. Some inhibitors contained additional functionality at the C-terminus. The transition-state structure was a noncleavable group which mimicked the transition state of amide bond hydrolysis. Examples included ketomethylene, reduced amide, and hydroxyethylene isosteres. The second segment, the P, position, contained an amino acid which lends specificity to the inhibitor. Histidine, thiazoylalanine, leucine, and norleucine are amino acids that have been successfully employed at the P, position of renin inhibitors. The third and fourth segments, the P, and N-terminus, also influence in vitro potency. A lipophilic aryl side chain, normally I-naphthylalanine, phenylalanine, or modified phenylalanine, resided at the P, position of the inhibitor. Aryl amino acids were recognized by degradative enzymes (e.g., pancreatic chymotrypsin) and the amide bond adjacent to the aryl amino acid was susceptible to enzymatic cleavage. Therefore, stabilization of the P,-P, amide bond was necessary. The Nterminus was usually reserved for a lipophilic amide or an amino acid. The development of dipeptide transition-state inhibitors focused primarily on the discovery of small dipeptide isosteres. a. Hydroxyethylene Dipeptide Isosteres and Carboxy-Terminal Modifications The dipeptide transition-state inhibitors shown and referenced in Table IV incorporate a secondary hydroxyl group and lipophilic side chains in the dipeptide isostere structure. As observed in the statine-based peptide inhibitor series, an increase in potency was achieved when the isopropyl group was replaced with cyclohexyl at P, . Potent inhibitors were prepared by replacing the dipeptide Ile-His and C-terminal butyl amides (compare 10-11 with 12-13). The potency of renin inhibitors containing the hydroxyethylene dipeptide isostere were influenced by the chirality of the Pi position. Inversion of the stereochemistry of inhibitor 14 produced a 10-fold decrease in activity. However, removal of the chiral center by dehydration produced an inhibitor with an IC,, of 1.5 nM. These data suggested that a Pi substituent and C-terminal carboxamide were important for enzyme binding. When the structure of the hydroxyethylene isostere was simplified by inclusion of smaller molecular fragments (17-22 and 24), potent inhibitors were also produced. However, these compounds were not as potent as inhibitors which maintained the chiral Pi side chain and C-terminal carboxamide (13) or sulfone (23) groups. The simplification of the hydroxyethylene dipeptide isostere structure was an important contribution toward the development of dipeptide transition-state renin inhibitors. However, additional smaller isosteres were also developed. b. Dihydroxy Dipeptide Isosteres Dipeptide transition-state inhibitors employing a dihydroxy peptide isostere were designed to bind more tightly to the active site aspartic acid residues in renin (Table V). The data showed that the second hydroxyl group increased in virro potency (17 versus 26) and the ster-

22 1

Renin Inhibitors Table lV

Small Dipeptide Transition-State Inhibitors Containing Hydroxyethylene lsoteres and Carboxy Terminal Modifications

Y

X

2

Rl

G o (M) Reference

R2

0

0

10 11 12 13

r-BuO t-Bu t-Bu t-Bu

C=O C=O C=O SO2

Isopropyl lsopropyl Cyclohexyl Cyclohexyl

NH NH CH2 CH2

Ile-His-NH2 Ile-His-NH2 NHC,H,-n NHC,H,-n

15 20

6 2

Biihlrnayer e t a / . Biihlrnayer ei ul. Biihlrnayer et a/. Biihlmayer et a/.

(1988) (1988) (1988) (1988)

IC50

Rl

R2

R3

(nM)

Reference

5.5 50

Kernpf et al. (1987) Kernpf er d.(1987) Kernpf et al. (1987)

Boc-Phe-His- N 0

14 15 16

CH3 OH R , and R*=CH2

OH CH,

Isopentyl Isopentyl Isopentyl

1.5

H OH Boc-Phe-His-N&R1

17 18

Isobutyl CH2S02isopropyl

10 7.6

Kleinert et al. ( 1 9 8 8 ~ ) Bolis et a/. (1987) (continued)

Hollis D. Kleinert et 01.

222 Table IV (Continued)

RI 19 20 21 22

ICSO (M)

Reference

7.5 20 17 50

N3

CH2N3 NHCH2Ph OH

Rosenberg et al. (1988) Rosenberg et al. (1988) Natarajan et al. (1988) Kleinert et at. ( 1 9 8 8 ~ )

0.5

23

H

Karlsson et al. (1987)“

OH

Boc-Phe-Leu- N&&F2,

CF3

d 24

52

Sham er al. (1987b)

O F o r the synthesis of the dipeptide isostere see Karlsson et al. (1989).

eochemistry of the second hydroxy group was important for enzyme inhibition (compared 26 and 28). When the dihydroxy isostere was incorporated into the hydroxyethylene isostere series (29), inhibitors were produced which had IC,, values in the nanomolar range. Only the syn diols were reported. However, to achieve subnanomolar potencies, additional amino acid residues were needed at Pi (30). The discovery of the dihydroxy isostere was an important achievement. For the first time, a small and structurally simple renin inhibitory dipeptide isostere was developed. c. Statine Dipeptide Isosteres Dipeptide transition-state inhibitors incorporating the statine isostere also were reported (Table VI). The statine inhibitor design was based on the original SCRIP and “cyclohexyl” SCRIP, with the goal to identify small C-terminal peptide replacements. C-Terminal modifications in the statine series were successful. The potent inhibitor 36 possessed a C-terminal

223

Renin Inhibitors

Table V Small Dipeptide Transition-State Inhibitors Containing Dihydroxy lsosteres Rl

ICso (nM) Reference

R2

H OH R 2 - P h e - H i s - N. \ l L.R 1

.

22 25 26 27

H Et Isobutyl CH2N3

Boc Boc Boc Isobutyl

-

50 0.6 1.5 0.6

Kleinert et a/. ( 1 9 8 8 ~ ) Luly et af. (1988) Luly er al. (1988) Rosenberg er al. (1988)

Boc-Phe-His- N H a R

=-

OH

U 28 29 30

H CONH-Ile-NHCH2(2-pyridinyl) CONH-2(S)-rnethylbutyI

35 0.35 3.4

Kleinert et al. ( 1 9 8 8 ~ ) Thaisrivongs er al. (1987b) Thaisrivongs er a / . ( 1987b)

isopentyl amide and a 1,3-dithiolane P, side chain. The cyclohexyl analog 35 was also a potent renin inhibitor. More potent statine analogs were synthesized (31), but they required an additional amino acid at the C-terminus. Both the amino statine and difluoro statine analogs 32 and 33 were less potent than 31. Again, an improvement of in vitro potency was achieved using the cyclohexyl group at P, . A modified statine renin inhibitor analog was designed by Iizuka et af. (1990). Inhibitor 37 contained an isopropyl ester at the C-terminus. The isopropyl group was postulated to mimic the P; side chain and bind to the S; binding site of the enzyme. A modified phenylalanine amino acid was also incorporated into the inhibitor. d. Ketone Dipeptide Isosteres Table VII shows a series of dipeptide transi-

Hollis D. Kleinert et al.

224

Table VI Small Dipeptide Transition-State Inhibitors Containing Modified Statine Isosteres RI

R2

I;'

Rl

Boc-Phe-His- *N

Reference

IC50 (nM)

R3

0

0

Ile-NHCH2-

Y 31

OH

H

32 33

NH2

H F

OH R

34 35 36

37

lsopropyl Cyclohexyl I ,3-Dithiolane

H H F

I .7 15 12

ICSO(nM)

70 4 I

4.7

Thaisrivongs et al. (1987a) Thaisrivongs et al. (1987a) Thaisrivongs et al. ( I986a) Reference

Sham et a / . (1987a) Sham et a/. (1987a) Sham et al. (1987a)

Iizuka et al. (1990)

225

Renin lnhibitors

Table VII Small Dipeptide Transition-State Inhibitors Containing Modified Ketone Isosteres

38 39 40

41 42 43

Rl

R2

X

H H F

H H F

c=o

Rl

R2

His Leu Leu

CF2CO-Ile-NHCH2(2-pyridinyl) CHZCH2CH3 CFZCF~CF~

IC50 ( M ) Reference

Val

C

4

3.1

34 0.52 ICs0 (nM)

2.5 870 3

Thaisrivsongs et a/. (1987b) Thaisrivsongs ef a/. (1986a) Thaisrivsongs e r a / . (1986a) Reference

Thaisrivsongs ef a/. (1986a) Sham er a / . (1987b) Sham er a/. (1987b)

tion-state inhibitors employing ketone transition-state mimics. The ketone isosteres were designed to exist in the hydrated form in the active site of the enzyme. The a-difluoroketone isostere further stabilized the hydrated form of the ketone. Thus, inhibitor 40 was 68 times more potent than its hydrogen analog 39. Increasing potency using a-difluoroketones was illustrated in another ketone isostere series (42 versus 43). e. Heterocyclic Dipeptide Isosteres A series of small, potent dipeptide transition-state inhibitors employing heterocycles at the P; position have been reported (Table VIII). Molecular modeling suggested that the heterocyclic oxygen atom hydrogen bonds as an acceptor to the flap region of the enzyme. Hydroxyethylene, dihydroxy, and statine dipeptide isosteres have emerged

Hollis D. Kleinert et a/.

226 Table Vtlt

Small Dipeptide Transition-State Inhibitors Containing Heterocyclic Isosteres

X

Reference

Ic50 (nM)

H OH Boc-Phe-His- N&

d 44 45

46

C=CH2 NEt (S)-CH(CH3)

H OH Boc-Phe-His- N . A

47

Rosenberg et al. (1990) Rosenberg et al. (1990) Rosenberg et al. (1990)

0.71 0.63 0.32

1.2

Rosenberg et al. (1990)

from many novel dipeptide isostere designs as stable mimics of the transition state of amide bond hydrolysis. When these isosteres were incorporated into renin inhibitors, potent and efficacious antihypertensive agents were produced (see Section V). The structure of dipeptide isosteres was now optimized for in vitro potency. Research in renin inhibitor design was then directed toward stabilizing the dipeptide amide bond against enzymatic degradation. f. P,-P, Stabilization Several approaches were developed to stabilize the Phe-His amide bond of dipeptide transition-state inhibitors (Table IX). The P,-P, amide bond in inhibitor 48 (enalkiren) was stabilized by the introduction of the para-methoxy substituent on the phenyl ring. The half-life of inhibitor 48 toward chymotrypsin degradation was greater than 4 hours (Kleinert et al., 1988~).N-Methylation of the P2-P3 amide bond, inhibitor 49, or removal of the nitrogen atom and replacement with a methylene, inhibitors 50,37, and 13, were other strategies utilized to stabilize renin inhibitors toward chymotrypsin cleavage (Plattner et al., 1988). The five dipeptide renin inhibitors 48, 49, 50 (Table IX), 37 (Table VI), and 13 (Table IV) were effective in reducing mean arterial

227

Renin Inhibiton

Table JX Small Dipeptide Transition-State Inhibitors Containing Modified P2 and P3 Amino Acids G o (pH 7.4) Reference N4

NH

14

Kleinert er al. (1988a)

0.26

Thaisrivongs et a / . (1986b)

6.9

Hiwada e t a / . (1988)

CH30 48

B o c - P r o - Phe-H i s-

I

49

H

50

pressure when administered to experimental animals, but they all possess questionably low oral bioavailability. However, the four criteria considered for the development of dipeptide transition-state renin inhibitors had been met.

5 . Nonpeptide Renin Inhibitors Poor oral bioavailability is the major problem that must be overcome before a renin inhibitor can be developed as an orally effective therapeutic agent. A possible solution is the discovery of new inhibitors structurally divergent from

Table X Nonpeptide Transition-State Inhibitors Containing Novel P,-P, Dipeptide Replacements

51 52 53 54

55 56 57

X

Rl

NH NH NH

CH2Ph CH2Ph CHzPh CHz-l-naphthyl

R

i-Butyl i-Butyl n-Propyl

0

R2

ICS0(nM)

Reference

CH3

Fung er al. ( 1990)

n-Bu

85 15 13 3.5

RI

R2

ICso(nM)

Reference

Ph Ph 3-Pyridinyl

Cyclohexyl Isopropyl Cyclohexyl

n-BU

n-Bu

I .7 6.8 1.6

Fung et a/.( 1990) Fung et a/.(1990) Fung et a!. (1990)

Roberts et al. (1990) Roberts et al. (1990) Roberts et al. (1990)

'Y H

58 59

0.2 0.3

Bradbury et al. (1990) Bradbury er al. (1990)

Renin Inhibitors

229

their dipeptide predecessors. Table X shows two examples of new inhibitor designs in which the P,-P, dipeptide moiety has been replaced with simple structures that do not contain amino acids. Inhibitors 51-54 employed a novel design strategy. By replacing the P,-P, amide bond with a heteroatom and modifying the P, and P, side chain, potent nonpeptide renin inhibitors were obtained. When inhibitor 51 was administered intraduodenally to anesthetized monkeys at 10 mg/kg, an oral bioavailability of 15% was achieved (Fung et al., 1990). However, no hypotensive response was produced due to weak plasma renin inhibition at pH 7.4 (IC50 > 10 phi’). The nonpeptide transition-state inhibitors 55-59 possessed a novel heterocyclic ring system for the Phe-His dipeptide moiety. Inhibitors 55-57 were reported to cause a marked reduction in mean arterial pressure in sodium-deplete marmosets following iv administration; however, oral studies in bile duct-cannulated rats indicated that these compounds were not absorbed. Consequently, compounds 55-57 showed no oral activity. In contrast, oral activity of the nonpeptide renin inhibitor 58 was observed after oral dosing of 30 mg/kg in the same animal model. Oral bioavailability was not reported. Nonpeptide renin inhibitors have provided new leads for structure-activity relationship studies. In addition, they have offered new direction for developing bioavailable renin inhibitors. Possible strategies for increasing oral bioavailability of nonpeptide renin inhibitors include reducing the molecular weight, modifying water solubility, and adjusting the lipophilicity of the compounds. The opportunity exists to modify further the nonpeptide inhibitor structure and develop potent, efficacious, and bioavailable compounds.

V. Pharmacology of Renin Inhibitors A. Experimental Efficacy Models Most inhibitors of human renin selectively inhibit primate renin. This selectivity imposes restrictions on the animal species employed in the evaluation of in vivo efficacy. The nonhuman primate has become the model of choice, and saltdepletion is employed to enhance the sensitivity of the animals to the effects of renin inhibition. Testing in nonhuman primates has curtailed the quantity and type of information derived from the screening process. An obvious fault of saltdepleted primate models stems from the fact that, although baseline activity of the RAAS is enhanced in this state, baseline blood pressures range from below normal to normal. Yet, this model is employed often to determine hypotensive activity in as much as alternatives, such as the 2 kidney-1 clip model which mimics clinical renovascular disease, are not feasible to set up and maintain routinely. Renin originating from nonprimate species of common laboratory animals demonstrates varying degrees of sensitivity to human renin inhibitors (see Sec-

230

Hollis

D. Kleinert et ol.

tion III,D), and manipulation of endogenous renin dependency may allow for some nonprimate species, such as the dog, to serve as models for studying pharmacological activity. Rat renin appears to be especially insensitive to the majority of human renin inhibitors (Kleinert et af.,1988a,b). In order to use a rat model, exogenous hog or human renin must be infused into the rat for the duration of the test (Pals et af., 1990; Blaine et af., 1984). This model successfully detects renin inhibitory activity in vivo, but the activity of the drug may be limited to interacting with plasma renin or plasma-derived renin pools, excluding any possible tissue-derived renin involvement. This may be a possible explanation for the shorter duration of action of the renin inhibitor ES 6864 in hog renininfused rats than in marmosets (Hiwada et al., 1988).

B. Acute Effects of Parenterally Administered Renin Inhibitors 1. Hemodynamic and Endocrine Responses in Experimental Studies Parenteral administration of renin inhibitors to salt-depleted monkeys (Kleinert et af., 1988a,b; Schaffer et al., 1988; Nisato et af., 1987), baboons (Tree et al., 1983; declaviere et af., 1985), marmosets (Iizuka et al., 1988; Wood et al., 1985), dogs (Szelke et af., 1982b; Blaine et af.,1984), and renin-infused rats (Pals et al., 1990) causes reductions in blood pressure. Several generalizations regarding the hypotensive profile can be drawn. First, the onset of action is rapid (5 minutes post-dosing) and the peak hypotensive effect usually occurs within approximately 30 minutes following a bolus iv delivery. Second, the magnitude of the reductions in blood pressure and the recoveries to baseline are dose and compound dependent. The differences among compounds in their ability to reduce blood pressure cannot be explained simply by differences in potency. For example, SR 43845, a subnanomolar (<0.01 nM) inhibitor of primate renin reduced blood pressure in the cynomolgus monkey for less than 2 hours at a dose of 0.1 mg/kg, iv (Nisato et al., 1987), while CGP 29 287 (ICs0 = 5 nM) at the same dose induced hypotension for a comparable length of time in the marmoset (Wood et al., 1985). The brevity of the pharmacological effect of renin inhibitors may reflect the volume of distribution of the compound, as well as the limitation imposed by rapid hepatic extraction and/or metabolism (Luly et al., 1988; Greenfield et af., 1989; Kleinert et af., 1990). Third, both the systolic and diastolic blood pressures are affected (Wood et al., 1985; Kleinert et af., 1990; Pals et al., 1986). A typical iv dose-response curve is shown in Fig. 4. This illustration summarizes the effects of renin inhibitor A-64662 (enalkiren) when administered intra-

MABP Normal

0

PRA Normal 120

80 40

0 120

80

h

40

3 0 f

0

-

5

120

a -E

80

a F

40

c .->

-

v

0 120

80

w.

2

.-C

c

2

2

% 40

0 0

120

-20

80

-40

-60

Dose

=

40

10 mglkg

-80

0

5

15

30

60

120

1a0

Time Relative to Dose Administration (Min) Fig. 4 Effect of intravenous administration of vehicle or enalkiren (0.01-10 mglkg) on mean arterial blood pressure (MABP) and plasma renin activity (PRA) in salt-replete ( n = 25, five per group) and salt-deplete ( n = 25, five per group), sodium pentobarbital-anesthetized cynomolgus monkeys. Values are shown as mean -t SE. Statistical differences are depicted as *, P < 0.05 versus baseline values. (Reproduced with permission from Kleinerf ei al., 1988a.)

232

Hollis D. Kleinert et a/.

venously to normal and sodium-depleted, anesthetized cynomolgus monkeys (Kleinert et al., 1988a). One could conclude from this study that (1) the baseline PRA predicted the minimum effective dose, (2) dose-related falls in mean arterial pressure occurred in both pretreatment groups and were virtually identical at high doses, and (3) the hypotensive response occurred at a time when PRA was concomitantly inhibited, but the relationship between the two responses was not well correlated.

2. Hemodynamic and Endocrine Responses in Clinical Studies Early clinical experiences with renin inhibitors which were structurally pure peptides produced varied results (Zusman et al., 1983; Webb et al., 1983). However, the clinical evaluation of the more recently discovered transition-state substrate analogs has demonstrated that renin inhibition can induce predictable endocrine and hemodynamic changes in man. Normotensive subjects received iv infusions of either enalkiren (Delabays et al., 1989), RO 42-5892 (structure undisclosed; Camenzind et al., 1989), or CGP 38 560A (Nussberger et al., 1989) while in the supine position. As one might expect, considering the questionable state of renin dependency under the conditions of these experiments, e.g., normal baseline blood pressures and postural effects on renin secretion (Cohen et al., 1967; Sassard et al. , 1976), blood pressure was unaffected by renin inhibition, but PRA and plasma ANG I1 were suppressed in a dose-related fashion. Phase I1 clinical studies in hypertensive patients have been conducted with the same compounds. A direct comparison of the iv efficacy of these compounds is not feasible, since the experimental protocols differed among the studies. Statistically significant reductions were observed in all three studies. The infusion of CGP 38 560A induced a slight, yet significant fall in mean arterial blood pressure of the order of 5% in 12 patients (Jeunemaitre et al., 1989). When RO 42-5892 was given iv in two doses to uncomplicated essential hypertensives, average decreases in blood pressure of up to 13 mmHg were observed, but the responses were not clearly dose dependent (van den Meiracker et al., 1990). The first evidence of dose-related hypotension in response to a parenterally administered renin inhibitor was provided by a study employing enalkiren (Weber et al., 1990; Glassman et al., 1990). In the first part of the study, 18 patients with baseline diastolic pressures ranging between 95 and 115 mmHg, who were on an unrestricted sodium diet, were administered a series of four infusions of enalkiren in 0.03, 0.1, 0.3, and 1.0 mgfkg doses. The second part of the same study was identical in protocol except that the patients were pretreated with hydrochlorothiazide in order to enhance renin dependency of blood pressure. Just as the preclinical animal studies predicted, both systolic and diastolic blood pressures were affected. Dose-related falls in systolic blood pressure which were amplified by pretreatment with a diuretic (maximum mean decrease in pressure

Renin lnhibiton

233

of 10 mmHg in part 1 of the study versus 19 mmHg following diuretic therapy in part 2) were observed. Parallel responses in diastolic blood pressure were noted (maximum mean decrease in pressure of 6 mmHg in part 1 of the study versus 12 mmHg following diuretic therapy in part 2). PRA was inhibited in all three of these clinical trials. Plasma ANG I1 was measured in two of the studies and shown to be depressed after treatment with CGP 38 560A (Jeunemaitre et al., 1989) and RO 42-5892 (van den Meiracker et al., 1990).

3. Cardiac Effects a. Effect on Heart Rate When hypotension is elicited by an ACEI, a reflex increase in heart rate does not accompany the blood pressure response. This phenomenon may represent a general feature of inhibition of the RAAS. Recently, support for this hypothesis was provided by monitoring the heart rate response to renin inhibitors and ANG 11 antagonists (Wong et al., 1990). Acute and chronic renin inhibition reduces blood pressure without inducing reflex tachycardia in animal models (Blaine er a l . , 1985; Wood et al., 1985; Kleinert et al., 1988a,b; Verburg et al., 1989) and in patients (Glassman et al., 1990; van den Meiracker et al., 1990; Boger et al., 1990). The lack of a chronotropic compensatory response to falls in blood pressure of up to 60% below baseline values in normotensive, anesthetized, salt-depleted monkeys (Kleinert et al., I988a) is not fully understood, but it may be due to the inhibition of ANG I1 presynaptic facilitation of sympathetic neurotransmission (Starke, 1977) or enhanced cardiac vagal activity (Ajayi el al., 1985). b. Effect on Cardiac Function i. Experimental Studies Renin inhibitors exert their depressor activity by means of vasodilation. A reduction in blood pressure accompanied by a reduction in the calculated systemic vascular resistance with no alteration in cardiac output was observed when SR 42128 was given to baboons (declaviere et al., 1985), H-77 was given to dogs (Tree et al., 1989), or CP 80,794 was given to guinea pigs (Mangiapane et al., 1990). In experimental canine left ventricular failure, renin inhibition provided hernodynamic benefits as suggested by the reductions in left ventricular end-diastolic pressure and afterload (Sweet et ul., 1984). As a class, renin inhibitors do not exert direct inotropic or chronotropic effects (Kleinert, 1989). ii. Clinical Studies Enalkiren is the only renin inhibitor to have been studied to date in patients with chronic heart failure (CHF). This acute study suggests that renin inhibition has the potential to play a positive role in the treatment of this pathology. Neuberg et al. (1990) treated nine CHF patients on diuretics and digoxin with a 1 .O nig/kg iv dose of enalkiren. Results showed significant increases in cardiac index (17%) and stroke volume index (32%), and decreases

234

Hollis D. Kleinert et ol.

in mean arterial pressure (13%), left ventricular-filling pressure (20%), right atrial pressure ( l6%), systemic vascular resistance (39%), pulmonary vascular resistance (31%), and heart rate (6 beats per minute). PRA was inhibited at doses of enalkiren that were too low to cause hemodynamic alterations and PRA was not further inhibited at higher doses that were hemodynamically active (Neuberg et al., 1989).

4. Renal Effects of Renin Inhibitors Circulating titers of ANG I1 influence renal hemodynamics and electrolyte excretion (Navar and Rosivail, 1984). The antihypertensive efficacy of ACE1 is due in part to the renal vasodilation in hypertensive patients, a population often characterized by compromised renal blood flow (Hollenberg, 1986). The intrarenal infusion of low doses of renin inhibitors to sodium-depleted monkeys caused an increase in renal blood flow and glomerular filtration rate, urine flow rate, and fractional excretion of sodium (Verburg et al., 1990a). In this investigation, the effects of renin inhibition were restricted to the kidney proper, as no systemic alterations in hemodynamics and PRA were noted following intrarenal renin inhibition. In contrast, when renin inhibitors were administered intravenously at systemic depressor doses, the rise in renal blood flow was blunted and sodium excretion was reduced without significantly changing glomerular filtration rate (Verburg et al., 1990a,b). However, Pals et al. (1988) showed a reduction in glomerular filtration rate in salt-depleted nonhuman primates during renin inhibition. In general, enhancement of renal blood flow has been observed more consistently and, in addition to the cynomolgus monkey, has been demonstrated also in salt-depleted marmosets (Neisius et af.,1986) and conscious dogs (Siragy et al., 1988) during renin inhibition. Recently, the renal hemodynamic effects observed in animals were corroborated in 14 healthy men given doses of enalkiren that did not lower systemic blood pressure despite suppression of PRA, plasma ANG 11, and aldosterone (Fisher et al., 1990). Dose-related increases in renal plasma flow were observed in these subjects on a low-salt diet, the magnitude of which was diminished by a high-salt diet in the same subjects. These results suggest that renin inhibitors may have a clinically favorable effect on renal performance under some conditions.

C. Chronic Administration of Renin Inhibitors When the systemic blood pressure falls, renin is reflexly released from the kidney. Acute experiments have shown that the fall in blood pressure induced by renin inhibitors was accompanied by a reactive rise in immunoreactive renin (Hofbauer et al., 1985). Thus, it was conceivable that the reactive rise in active renin might compensate for the fall in blood pressure and normalize or elevate blood pressure with chronic treatment, invalidating renin inhibition as a viable new antihypertensive therapy. However, this has been shown not to be the case.

Renin lnhibiton

235

1. Constant Infusions in Animals Chronic iv or intraperitoneal infusion of renin inhibitors to nonhuman primates over a period of 1 or 2 weeks resulted in a sustained hypotension with no evidence of tachyphylaxis (J. M. Wood et al., 1987b; Verburg et al., 1989). PRA was inhibited throughout the treatment periods. Mean blood pressure did not return to control values until after the cessation of drug infusion, at which time a normal recovery was observed in both of these studies. Wood et al. (1989a) measured a reactive rise in active renin during 7 days of renin inhibition that was unable to overcome the pharmacological activity of the renin inhibitor.

2. Multiple-Dose Clinical Studies Only one study evaluating the tolerance and efficacy of chronic renin inhibition in hypertensive patients has been reported (Boger et al., 1990). Thirty-two hypertensive patients (seated diastolic blood pressures between 100 and 114 mmHg) were maintained on a 60 mEq/day sodium intake. Patients received a 10-minute infusion of placebo, 0.1, or 0.3 mg/kg four times daily or 1.2 mglkg of enalkiren given once a day for seven consecutive days. On the eighth study day, each dose was given only once. The first or only daily dose was administered at 6 AM routinely. Figure 5 shows the mean systolic blood pressures and Fig. 6 the mean diastolic blood pressure at 6-hour intervals. Statistically significant reductions in systolic and diastolic blood pressure compared to placebo were observed in the 0.3 q.i.d. and 1.2 4.d. dosing groups following the first study day, and these responses were actually magnified during one week of treatment. The percentage fall from baseline pressure was comparable for both the systolic and diastolic pressures. On day 8 , a single dose of either 0.3 or 1.2 mg/kg reduced blood pressure values for more than 12 hours by approximately 10 and 16% below baseline for each dose, respectively. Systolic pressures remained significantly lower than baseline 24 hours after 1.2 mg/kg dosing on study day 8. PRA was suppressed and remained so throughout the eight treatment days starting with the lowest dose of 0.1 mg/kg, q.i.d. Clearly, the blood pressure and PRA responses were dissociated.

D. Dissociation between Plasma Renin Activity and Hypotensive Effect The dissociation between the PRA and the hemodynamic response to renin inhibition has been described above in a number of studies under a variety of conditions. The explanation for this observation may be related to an artifact resultant of the conditions of the standard radioimmunoassay for determining PRA (see Section 111,C). On the other hand, the dissociation between these two parameters may be physiological. It is conceivable that the plasma RAAS may have a tissue-based counterpart (Campbell, 1987; Dzau, 1988a). The inhibition

236

n

-

I

I' -30

Time (24 hour clock)

Fig. 5 Average changes from baseline systolic blood pressure are shown for placebo or enalkiren treatment regimens in 6-hour intervals. *, P < 0.05, statistically significant change from baseline day. #, P < 0.05, statistically significant change from baseline in the enalkirentreated group compared to the corresponding change from baseline in placebo group. (Reproduced with permission from Glassman et al., 1990.)

0 n.

2400 - 0600

1800 .2400

1200.1800

0600 - 1200

2400 - 0600

1800 - 2400

1200- I800

0600. 1200

2400 - 0600

1800 - 2400

1200 - 1800

0600 . 1200

2400 - 0600

1800 - 2400

1200- 1800

0600. 1200

0

I 0 0

N

I -

0

I 0

Enalkiren 1.2 rnglkg qd W 0

I N 0

I A 0

I

Enalkiren 0.3 rnglkg qid 0

Enalkiren I

I

0.1 rnglkg qid I A 0 0

Placebo

238

Hollis D. Kleinert et ol.

of tissue renin pools may persist following the clearance of the renin inhibitor from the plasma. Accessibility of renin inhibitors to these putative pools of tissue renin may required high doses and/or specific physical properties. Some evidence for the existence of a tissue RAAS has been reported based on molecular biology and immunohistochemical techniques. It has been hypothesized that a tissue RAAS may function locally in an autocrine-paracrine capacity (Dzau, 1988b) and that an intracellular RAAS may function as a regulatory system separate from the circulating RAAS (Moms, 1986). The structure of the human renin gene has been determined and reviewed in detail (Morris, 1986). Gene expression is relatively high in all species in the renal juxtaglomerular cells (Catanzaro et al., 1983). These cells are the primary source of plasma renin (Catt et al., 1967; Semple et al., 1976; Waite, 1973). Renin is expressed to a lesser degree in other tissues, such as the submaxillary gland, adrenals, testicular Leydig cells, pituitary LH gonadotrophs and somatotrophs, thyroid, aorta, brain, neuroblastoma X glioma cell lines, and human chorion (Morris, 1986; Campbell, 1987; Dzau et al., 1987). Evidence of transcription and physiological relevance of gene expression in these various tissues derived from different species remains to be presented. Possible local cardiovascular (Lilly et al., 1985) or noncardiovascular roles for tissue renin have been proposed. The RAAS may be involved in the physiology of other organ systems, such as the reproductive system (Pellicer et al., 1988) and the nervous system (Ganten et af., 1983), and in the growth process (Ganten et al., 1975).

E. Oral Efficacy 1. Obstacles to Overcome The development of orally active renin inhibitors was far more challenging than was originally imagined. The problem of poor oral bioavailability has plagued the rapid development of these agents and can be attributed to limited absorption of parent drug and rapid hepatic extraction. The development of orally active agents with peptidic structures is not amenable to a general, systematic approach and solution. Little is understood concerning the absorption of peptides from the gastrointestinal tract and subsequent hepatic metabolism, and the literature offers no logical guidelines to solve the problem. Certainly the stability of the peptide bond(s) in the digestive tract is a major concern. Stabilization of susceptible amide bonds by chemical strategies, however, does not guarantee oral bioavailability (de Gasparo et al., 1989; Kleinert er al., 1990). Intravenous and/or oral administration of renin inhibitors described thus far has revealed a large degree of biliary extraction of the compounds, no doubt contributing to the observed short half-life (Boger et a l . , 1985a; Plattner er a l . , 1988; Luly et al.,

Renin lnhibiton

239

1988; Greenfield et a/., 1989). High concentrations of intact compound in the bile after oral administration would support the concept that gastrointestinal absorption per se is not the limiting factor. Rather, it is probably the high degree of biliary extraction which limits the bioavailability. Biliary extraction increases with the molecular weight (Klaassen and Watkins, 1984), and most potent renin inhibitors reported to date are in the range of 600-800 Da. Early compounds did not offer any sign of oral activity as assessed by hemodynamic or endocrine responses. However, stepwise progress with respect to bioavailability has been achieved with selected subsequent compounds.

2. Progress to Date Renin inhibitors, structurally diverse in nature, have been shown to demonstrate some degree of oral activity in that they lower blood pressure and PRA in animal models (Wood et a/., 1989b; Pals et nl., 1986; Luly et al., 1988; Morishima et al., 1989; Miyazaki et al., 1989; Kleinert et u / . , 1988~Painchard ; et al., 1990). Varying degrees of progress in improving systemic bioavailability have been achieved. BW- 175 (MW 688) exhibited dose-related increases in bioavailability, based on bioassay determinations of plasma drug levels, of 2.8% at a dose of 10 mg/kg and 9.7% at a dose of 30 mgfkg in rats. Oral activity has been defined loosely by the renin inhibitor literature as a detectable drug-related change in any pharmacological parameter, from partial suppression of PRA and plasma ANG 11, to falls in blood pressure of varying duration. However, the characteristics of an orally active therapeutic agent will have to include clear dose-related decreases in blood pressure of a long duration and a clearly measurable and predictable pharmacokinetic profile. No renin inhibitor with these characteristics has been reported in animal models to date. Enalkiren was administered as a single 10, 20, or 40 mg oral dose to 13 normal subjects (Cavanaugh et al., 1989). PRA showed dose-related suppression of more than 12 hours in duration with the highest dose. However, following the 40 mg dose, the mean peak plasma concentration was only 37 ng/ml. Likewise, after oral administration of 50, 100, or 200 mg of CGP 38 560 to normal subjects, PRA was inhibited but the plasma drug levels were low and bioavailability was estimated to be less than 1 % in man (de Gasparo et al., 1989). No changes in blood pressure or heart rate were noted in either of these studies, which may be a consequence of the low systemic drug blood levels or the fact that the subjects were normotensive. RO 42-5892 suppressed plasma ANG I1 in normal subjects when given orally (Camenzind et al., 1989) and was the first compound to be efficacious by this route of administration in phase 11 clinical studies. The most promising clinical results with regard to orally active renin inhibitors were reported recently by van den Meiracker et ul. (1990) in essential hyperten-

240

Hollis D. Kleinert et al. RO 42-5892 a i m uallv

RO 42-5892aim inhawnouslv 165

145

125

105

85

65

Time 01 day

Fig. 7 Average 24-hour systolic and diastolic blood pressures and heart rates in six essential hypertensive patients following administration of placebo (0); RO 42-5892 (0). 100 pg/kg 600 mg orally. intravenously; RO 42-5892 (A),1000 pg/kg intravenously; or RO 42-5892 (0). Asterisks indicate where patients either had lunch or were awakened. (Reproduced with permission from van den Meiracker et ul., 1990.)

sive patients on a normal salt intake. Figure 7 depicts the 24-hour blood pressures and heart rates following placebo, 100 kg and 1000 pg iv and a 600 mg oral dose of RO 42-5892. The higher iv dose, as well as the oral dose, induced a prolonged reduction in systolic and diastolic pressures without changing heart rate. This blood pressure reduction outlived the suppression of plasma ANC 11, implying that an extra plasma system may be involved in eliciting the hypotensive activity. No phannacokinetic or bioavailability data were reported for this compound. Further clinical evaluation will be needed to establish whether a predictable dose-response curve can be generated.

Renin Inhibiton

24 1

VI. Conclusion Renin inhibitors have emerged as agents capable of reducing blood pressure in man and experimental animals when administered intravenously. These compounds are more specific than ACE inhibitors, but like the latter, they exert their actions through vasodilation, e.g., peripheral, renal. Oral activity has been limited. The earlier renin inhibitors were high-molecular-weightpeptides. However, structural modification and physicochemical optimization have led to smaller dipeptide inhibitors which represent the most promising leads for drug candidacy to date. Still, there are significant obstacles to overcome in order to develop therapeutic agents. The dipeptide renin inhibitors lack good oral bioavailability, caused by either limited gastrointestinal absorption or high hepatic extraction, or both. These insufficiencies have provided the challenge for the improvement of the dipeptide renin inhibitors series and the development of nonpeptide renin inhibitors. Thus, the evolution of renin inhibitors continues. If solutions to poor gastrointestinal absorption and rapid hepatic extraction are found, then renin inhibitors will play a significant role in the treatment and control of hypertension and RAAS-related diseases.

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Nussberger, J., Fasanella d'Amore, T., Porchet, M., Waeber, B., Brunner, D. B.. Brunner, H. R., Kler, L., Brown. A. N . , and Francis, R. J . (1987). Repeated administration of the converting enzyme inhibitor cilazapril to normal volunteers. J . Cardiovasc. Pharmacol. 9, 39-44. Nussberger, J., Delabays, A., de Gasparo, M., Cumin, F., Waeber, B.. Brunner, H.. and MCnard. J. (1989). Hemodynamic and biochemical consequences of renin inhibition by infusion of CGP 38560A in normal volunteers. Hvperrension 13, 948-953. O'Connor, S. E. (1990). ACE inhibitors-new developments. D . N . P . 3, 133-141. Okunishi, H., Miyazaki, M., and Toda, N. (1984). Evidence for a putative new angiotensin IIgenerating enzyme in the vascular wall. J . Hvperfens. 2, 277-284. Ondetti, M . A , , and Cushman, D. W. (1981). Inhibition of the renin-angiotensin system. A new approach to the therapy of hypertension. J . Med. Chem. 24, 355-361. Painchard, C . , Ryan. M., Hicks, G., Fettia, M.. Rapundalo, S . , and Taylor, D. (1990). Effect of CGP-38560 on blood pressure (BP) and plasma renin activity (PRA) in high renin normotensive cynomolgus monkeys (CM). FASEE J . 4, A748. Pals, D. T., Thaisrivongs, S., Lawson, J. A., Kati, W. M., Turner, S . R., DeGraaf, G . L., Harris, D. W., and Johnson, G . A. (1986). An orally active inhibitor of renin. Hypertension 8, 1 105- I 112. Pals, D. T.. Ludens, J. H., and DeGrdaf, G . L. (1988). Systemic and renal hemodynamic effects of renin or angiotensin converting enzyme inhibition in non-human primates. J. Hyperrens. 7 , Suppl. 2, S43-S46. Pals, D. T., Lawson. J. A , , and Couch, S. J . (1990). Rat model for evaluating inhibitors of human renin. J . Pharmacol. Merhods 23, 239-245. Peach, M. J., and Dosldl, D. E. (1990). The angiotensin I1 receptor and the actions of angiotensin 11. J . Cardiovasc. Pharmacol. 16, Suppl. 4, S25-S30. Pellicer, A., Palumbo, A., DeCherney, A. H., and Naftolin, F. (1988). Blockage of ovulation by an angiotensin antagonist. Science 240, 1660- 1661. Plattner, J. J . . and Kleinert, H. D. (1987). Antihypertensive agents. Ann. Rep. Med. Chem. 22, 6372. Plattner, J. J . , Marcotte, P. A., Kleinert, H. D., Stein, H. H . , Greer, J . , Bolis, G . , Fung, A. K. L., Bopp, B. A., Luly, J. R., Sham, H. L., Kempf, D. J . , Rosenberg, S . H., Dellaria, J. F., De, B., Merits, I . , and Perun. T. J. (1988). Renin inhibitors. Dipeptide analogues of angiotensinogen utilizing a structurally modified phenylalanine residue to impart proteolytic stability. J. Med. Chem. 31, 2277-2288. Poe, M . , Wu, J. K . , Lin. T.-Y., Hoogsteen, K., Bull, H. G . , and Slater, E. E. (1984). Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu-Val-fle-His-Ser-OH, that is human renin specific and is resistant to cathepsin D. Anal. Biochem. 140, 459-467. Poulsen, K., and Jorgensen, J. (1974). An easy radioimmunological microassay of renin activity, concentration and substrate in human and animal plasma and tissues based on angiotensin 1 trapping by antibody. J . Cfin. Endocrinol. Metab. 39, 816-825. Roberts, D. A.. Bradbury, R. H., Brown, D., Faull. A , , Griffiths, D., Major, 1. S . , Oldham, A. A , , Pearce, R . J., Ratcliffe, A . H., Revill, J . , and Waterson. D. (1990). 1,2,4-Triazolo(4,3-I]pyrazine derivatives with human renin inhibitory activity. 1 . Synthesis and biological properties of alkyl alcohol and statine derivatives. J . Med. Chem. 33, 2326-2334. Rosenberg, S . H., Woods, K. W., Plattner. J. J.. Stein, H. H., Kleinert, H. D., and Cohen, J. (1988). Novel, subnanomolar renin inhibitors containing a postscissile site azide residue. Pepl., Chem. E i o l . , Proc. Am. Pepr. Symp.. 10th (G. R. Marshall, ed.), p. 500. ESCOM, Leiden. Rosenberg, S . H., Dellaria, J. F., Kempf, D. J . , Hutchins, C. W., Woods, K. W., Maki, R. G . , de Lara. E., Spina, K. P.. Stein, H. H., Cohen, I., Baker, W. R . , Plattner, J. J., Kleinert, H. D., and Perun. T. J. (1990). Potent. low molecular weight renin inhibitors containing a C-terminal heterocycle: hydrogen bonding at the active site. J . Med. Chem. 33, 1582-1590.

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Sassard, J . , Vincent, M., Annat. G . , and Bizollon, C. A. (1976). A kinetic study of plasma renin and aldosterone during changes in posture in man. J. Clin. Endocrinol. Merub. 42, 20-27. Schaffer, L. W., Winquist, R. J., and Siegl, P. K. S. (1988). Hyptensive effect of inhibition of renin and converting enzyme in normotensive African Green monkeys. FASEE J . 2, A605. Semple, P. F., Boyd, A. S., and Dawes, P. M. (1976). Angiotensin I1 and its heptapeptide (2-8). hexapeptide (3-8), and pentapeptide (4-8) metabolites in arterial and venous blood of man. Circ. Res. 39, 671-678. Sham, H. L., Rempel, C. A . , Stein, H., and Cohen, J. (1987a). Novel statine analogue synthesized from L-aspartic acid. J.C.S. Chem. Commun. pp. 683-684. Sham, H. L., Stein, H. H., Rempel, C. A,, Cohen, J . , and Plattner, J. J. (1987b). Highly potent and specific inhibitors of human renin. FEES Len. 220, 299-301. Siragy, H. M., Lamb, N. E., Rose, C. E., Jr., Peach, M. J., and Carey, R. M. (1988). Intrarenal renin inhibition increases renal function by an angiotensin 11-dependent mechanism. Am. J . Physiol. 255, F749-F754. Skeggs, L. T., Lentz, K. E., Kahn, J. R., Shumway, N. P., and Woods, K. R. (1956). The amino acid sequence of hypertensin 11. J . Exp. Med. 104, 193-197. Skeggs, L. T., Kahn, J. R., Lentz, K., and Shumway, N. P. (1957). Preparation, purification and amino acid sequence of a polypeptide renin substrate. J . Exp. Med. 106, 439-453. Skeggs, L. T., Lentz, K. E., Kahn, J. R., and Shumway, N. P. (1958). The synthesis of a tetradecapeptide renin substrate. J. Exp. Med. 108, 283-297. Skeggs, L. T., Lentz, K. E., Kahn, J. R., and Hochstrasser, H. (1968). Kinetics of the reaction of renin with nine synthetic peptide substrates. J. Exp. Med. 128, 13-34. Slater, E. E., and Strout, H. V., Jr. (1981). Pure human renin. Identification and characterization of two major molecular weight forms. J . Eiol. Chem. 256, 8164-8171. Slater, E., Memll, D., Guess, H., Roylance, P., Cooper, W., Inman, W., and Ewan, P. (1988). Clinical profile of angioedema associated with angiotensin converting enzyme inhibition. J . Am. Med. Assoc. 260, 967-970. Stammers, D. K., Dann, J. G., Harris, C. J., and Smith, D. R. (1987). Comparison of angiotensinogen and tetradecapeptide as substrates for human renin. Arch. Biochem. Eiophys. 258,413420. Starke, K. (1977). Regulation of noradrenaline release by the presynaptic receptor systems. Rev. Physid. Biochem. Pharmacol. 77, I- 124. Stein, H., Fung, A . , and Cohen, J. (1985). Purification of human renal renin by affinity chromatography. Fed. Proc. 44, 1363. Stein, H. H., Cohen, J., Tricarico, K. A., and Sham, H. L. (1988). Change in the kinetics of inhibition of human renin as a function of substrate. FASEE J. 2, A1479. Sweet, C. S., Ludden, C. T., Frederick, C. M., Bush, L. R., and Ribeiro, L. G. T. (1984). Comparative hemodynamic effects of MK-422, a converting enzyme inhibitor, and a renin inhibitor in dogs with acute left ventricular failure. J . Cardiovasc. Pharmacof. 6 , 1067-1075. Szelke, M., Leckie, B., Hallett, A., Jones, D. M . , Sueiras, J., Atrash, B.,and Lever, A. F. ( I 982a). Potent new inhibitors of human renin. Nature (London) 299, 555-557. Szelke, M., Leckie, B. J., Tree, M., Brown, A., Grant, J., Hallett, A., Hughes, M., Jones, D. M., and Lever, A. F. (l982b). H-77: A potent new renin inhibitor. Hypertension 4, Suppl. 11, I1-5911-69, Tewksbury, D. A., Premeau, M. R., Dumas, M. L., and Frome, W. L. (1977). Purification of human angiotensinogen. Circ. Res. 41, 11-29-11-33. Tewksbury, D. A., Dart, R. A., and Travis, J. (1981). The amino terminal amino acid sequence of human angiotensinogen. Biochem. Biophys. Res. Commun. 99, 1311-1315. Thaisrivongs, S., Pals, D. T., Kati, W. M., 'hrner, S. R., Thomasco, L. M., and Watt, W. (1986a). Design and synthesis of potent and specific renin inhibitors containing difluorostatine. difluorostatone, and related analogues. J . Med. Chem. 29, 2080-2087.

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Thaisrivongs, S.. Pals. D. T., Harris, D. W., Kati, W. M., and Turner, S. R. (1986b). Design and synthesis of a potent and specific renin inhibitor with a prolonged duration of action in vivo. J. Med. Chem. 29, 2088-2093. Thaisrivongs, S., Schostarez, H. J., Pals, D. T., and Turner, S. R. (1987a). a,a-Difluoro-paminodeoxystatine-containing renin inhibitory peptides. J. Med. Chem. 30, 1837- 1842. Thaisrivongs. S., Pals, D. T., Kroll, L. T., Turner, S. R . , and Han, F. S. (1987b). Renin inhibitors: design of angiotensinogen transition-state analogues containing novel (2R,3R,4R,5S)-5amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid. J. Med. Chem. 30, 976-982. Tree, M. Atrash, B., Donovan, B., Gamble, J.. Hallett, A , , Hughes, M., Jones, D. M., Leckie, B., Lever, A. F., Morton, J. J., and Szelke, M. (1983). New inhibitors of human renin tested in virro and in vivo in the anesthetized baboon. J. Hypertens. 1, 399-403. Tree, M.. MacArthur, K., Inglis, G . , Leckie, B., Lever, A. F., Morton, J. J., and Szelke, M. (1989). Effects of the renin inhibitor H 77 on angiotensin 11, arterial pressure and cardiac function in conscious dogs: Comparison with captopril. J. Hypertens. 7 , Suppl. 2, S51-S55. Umezawa, H., Aoyagi. T., Morishima, H., Matsuzaki, M . , Hamada, M., and Takeuchi, T. (1970). Pepstatin, a new pepsin inhibitor produced by actinomycetes. J. Antibiot. 23, 259-262. van den Meiracker, A. H., Admiraal. P. J. J.. Man in’t Veld, A. J., Derkx, F. H. M., van Eck, H. J. R . , Mulder, P.,van Brummelen, P.,and Schalekamp, M. A. D. H. (1990). Prolonged blood pressure reduction by orally active renin inhibitor RO 42-5892 in essential hypertension. Br. Med. J. 301, 205-210. Verburg, K. M . , Kleinert, H. D., Kadam, J. R. C., Wilkes, B., Mento, P., and Chekal, M. A. (1989). Effects of chronic infusion of the renin inhibitor A-64662 in sodium-depleted monkeys. Hypertension 13, 262-272. Verburg, K. M., Kadam, J. R. C . , Young, G. A., Rosenberg, S. H., and Kleinert, H. D. (1990a). Effect of intrarenal renin inhibition on renal hemodynamics and excretory function. Am. 1. Physiol. 259, R-7-R-14. Verburg, K. M . , Kleinert, H. D., Chekal, M. A,, Kadam, J. R. C., and Young, G. A. (1990b). Renal hernodynamic and excretory responses to renin inhibition induced by A-64662. J. Pharmacol. Exp. Ther. 252, 449-455. Waite, M. A. (1973). Measurement of concentrations of angiotensin I in human blood by radioimmunoassay. Clin. Sci. Mol. Med. 45, 51-64. Webb, D. J., Curnming, A. M. M., Leckie, B. J., Lever, A. F., Morton, J. J., Robertson, J. 1. S., Szelke, M., and Donovan, B. (1983). Reduction of blood pressure in man with H-142, a potent new renin inhibitor. Lancet ii, 1486- 1487. Weber, M. A., Neutel, J. M., Essinger, I., Boger, R. S., and Luther, R. (1990). Assessment of renin dependency of hypertension with a dipeptide renin inhibitor. Circulation 81, 17681774. Wilkin, J. K . , Hammond, J. J . , and Kirkendall, W. M. (1980). The captopril-induced eruption. A possible mechanism: cutaneous kinin potentiation. Arch. Dermatol. 116, 902-905. Wolfenden, R. (1972). Analog approaches to the structure of the transition state in enzyme reactions. Acc. Chem. Res. 5, 10-18. Wong, P. C., Price, W. A., Chin, A. T., Duncia, J. V., Carini, D. J., Wexler, R. R . , Johnson, A. L., and Timmermans, P. B. M. W. M. (1990). Nonpeptide angiotensin I1 receptor antagonists. VIII. Characterization of functional antagonism displayed by DuP 753, an orally active antihypertensive. J. Pharmacol. Exp. Ther. 252, 719-725. Wood, J. M., Gulati, N., Forgiarini, P.,Fuhrer, W., and Hofbauer, K. G. (1985). Effects of a specific and long-acting renin inhibitor in the marmoset. Hypertension 7 , 797-803. Wood, J. M., Baum, H.-P., Bews, 1. P. A , , Wachsmuth, E. D., Heusser, C., and Hofbauer, K. G. (1987a). Effects of chronic administration of a monoclonal antibody against human renin in the marmoset. Clin. Exp. Hypertens., Pan A A9, 1467-1478. Wood, J. M., Baum, H.-P., Jobber, R. A,, and Neisius, D. (1987b). Sustained reduction in blood

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pressure during chronic administration of a renin inhibitor to normotensive marmosets. J . Cardiovasc. Pharmacol. 10, S-96-S-98. Wood, J. M., Jobber, R. A . , Baum, H.-P., deGasparo, M., and Nussberger, J. (1989a). Biochemical effects of prolonged renin inhibition in marmosets. J. Hypertens. 7 , 615-618. Wood, J. M., Criscione, L., deGasparo, M . , Biihlmayer, P., Rueger, H., Stanton, J. L., Jupp, R. A., and Kay, J. (l989b). CGP 38 560: orally active, low-molecular-weight renin inhibitor with high potency and specificity. J. Cardiovasc. Pharmacol. 14, 221-226. Wood, S. M., Mann, R. D., and Rawlins, M. D. (1987). Angio-edema and urticaria associated with angiotensin converting enzyme inhibitors. Br. Med. J. 294, 91 -92. Yokosawa, H., Holladay, L. A., Inagami, T., Haas, E., and Murakami, K. (1980). Human renal renin. Complete purification and characterization. J. Biof. Chem. 255, 3498-3502. Zusman, R. M., Burton, J., Christensen. D., Nussberger, J., Dodds, A., and Haber, E. (1983). Hemodynamic effects of a competitive renin inhibitory peptide in humans: evidence for multiple mechanisms of action. Trans. Am. Assoc. Physicians 96, 365-374.

The Capacitative Model for ReceptorActivated Calcium Entry James W. Putney, Jr. Calcium Regulation Section Laboratory of Cellular and Molecular Pharmacology National Institute of Environmental Heulth Sciences Research Triangle Park, North Carolinu 2 7709

I. Introduction 11. Epithelial Cells: Models for Studying ReceptorActivated Ca2+ Signaling 111. Mechanisms of Ca2 Entry A. The Role of Inositol Phosphates 8.The Capacitative Model C. The Pathway for Ca2+ Entry D. Actions of Inositol I .3,4,5-Tetrakisphosphate and Relevance to the Capacitative Model IV. Conclusions References +

1. Introduction In eukaryotic cells, the calcium concentration in the cytoplasm is approximately four orders of magnitude less than in the extracellular space. This situation no doubt arose through evolution in an effort to protect the phosphate-based biochemistry of cells from concentrations of calcium which would cause calcium phosphate solubility products to be exceeded. Beyond this, however, modem organisms have also evolved various means by which this sizeable concentration gradient of calcium has been exploited as a rapid signaling system to initiate the diverse cellular responses to environmental stimuli. Historically, this role of calcium in cell function was first appreciated for cardiac muscle which, Ringer observed, could not continue to beat in vitro without a minimal level of extracellular Ca2+ (Ringer, 1883). It soon became apparent that all muscle cells utilized Ca2+ as a signal to initiate contraction, and work by Douglas (1974), Rasmussen (1 970), and others soon expanded the central role of this cation to a wide variety of cell types. It is now recognized that this signaling function of Ca2+ is achieved by the interaction of relatively low concentrations of Ca2+ with specific Ca2 -binding proteins which translate stimulus-induced increases in cytoplasmic Ca2 concentration into appropriate phosphorylations, enzyme +

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activations, etc., comprising the characteristic biochemical response of a panicular biological system (Blaustein, 1985; Klee and Newton, 1985). Despite the large gradient for Ca2 across the plasma membrane, studies on the role of Ca2 in muscle contraction revealed that activator calcium could also be recruited from intracellular stores (Bianchi, 1968). Perhaps the most specialized and well-characterized system in this regard is skeletal muscle; in these cells, virtually all of the Ca2+ recruited to initiate the rapid muscle twitch in response to a propagated action potential is derived from a highly specialized organelle of Ca2 metabolism, the sarcoplasmic reticulum. On the other end of the spectrum, cardiac muscle, as originally observed by Ringer, depends entirely on external Ca2+ to maintain contractility, although it is now clear that an internal calcium-induced calcium release also contributes activator Ca2 in these cells. Smooth muscle represents a system intermediate between these two. Here the situation is made more difficult by considerable functional variety among different smooth muscle types. Nonetheless, many examples have been noted of smooth muscles which apparently utilize both internal as well as external Ca2+ (Bohr, 1973; Van Breemen and Saida, 1989); since in some situations, smooth muscles are required to maintain persistent tone, finite internal stores cannot maintain a tonic response for prolonged periods of time. It is now widely recognized that most cells employ both intracellular and extracellular sources of Ca2+ in cellular signaling. This is especially true for receptor-activated Ca2 signaling, as distinguished from voltage-activated signaling which involves, at least in most instances, regulation of specific voltageregulated Ca2+ channels in the plasma membranes of excitable cells (Putney, 1987). (Skeletal muscle is a notable exception.) This review focuses on the former, receptor-activated Ca2 signaling processes, and specifically on the mechanism by which receptor activation leads to the entry of Ca2 into cells across the plasma membrane. The interested reader is referred to other recent reviews which have focused on physiological and pharmacological regulation of the voltage-activated Ca2+ channels (Fink and Kaczmarek, 1988; Armstrong et al., 1991). +

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II. Epithelial Cells: Models for Studying Receptor-Activated Ca2+ Signaling In excitable cells, there are numerous examples of interactions between receptoractivated and voltage-dependent processes. Thus, it has been argued that the analysis of the receptor-activated processes in their simplest state can best be accomplished by utilizing nonexcitable cells (or cell lines derived from them) as models. Among the various nonexcitable cell systems which have been utilized for such studies, blood cells and epithelial cells have provided especially useful models; among epithelial cells systems, studies on hepatocytes and the secretory

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acinar cells of exocrine glands, all of which express receptors for a wide variety of neurotransmitter and peptide hormones, have yielded valuable information of the regulation of Ca2+ pools and fluxes by surface membrane agonists. In the early studies, changes in intracellular calcium concentration ([Ca2 Ii) were inferred from changes in the magnitude of a cellular response or function believed to be quantitatively related to [Ca2+],. For epithelial cells, the activation of Ca2 -dependent potassium channels has often been used, and from such studies knowledge was obtained of the relationships between Ca2+ pools regulated by different receptor pathways, and between intracellular Ca2 release and the entry of Ca2 across the plasma membrane. With the advent of the fluorescent Ca2+ indicators that could be easily loaded into small eukaryotic cells (Tsien, 1989), it became clear that many of the end responses to cell activation were in fact poor reflections of changes in [Ca2 Ii; this was found to result from the roles of other, Ca2 -independent pathways which interacted with the Ca2+ signal and modified the end cell response at steps downstream of the mobilization of cellular Ca2 . A notable example in this regard is the secretory response of a variety of cells which in most instances is known to involve activation of protein kinase C as a signaling pathway operating in parallel to the Ca2+ pathway (Nishizuka, 1984). Fortunately, one Ca2 -dependent parameter for which this is apparently not a problem is the Ca2 -dependent K - conductance present in exocrine gland cells. Indeed, much of our knowledge of modes of Ca2+ regulation in nonexcitable cells by hormones has been derived from such studies and has since been confirmed by investigators using the more direct, fluorescent Ca2 indicators. Such studies have demonstrated rather conclusively that the initial response to receptor activation is release of Ca2+ from an intracellular pool and, on more prolonged stimulation, elevation in [Ca2 I i is maintained by increased influx of Ca2+ across the plasma membrane (Putney et al., 1981). Several kinds of observations have led to this view of two phases of Ca2+ mobilization following receptor activation: ( 1 ) Studies of kinetics of K permeability or with intracellular Ca2+ indicators demonstrate that the initial, rapid increase in [Ca2+ I i is largely independent of the presence of extracellular Ca2+, while the sustained, steady-state increase in [Ca2+Iidepends absolutely and continually on the presence of extracellular Ca2 (Fig. 1). (2) Similarly, the initial phase of the Ca2 signal is not blocked by the extracellular application of agents which interfere with the movements of Ca2+ through membranes (Ca2+ antagonists), while the second, sustained phase is blocked by such agents. (3) 45Ca2+ fluxes have indicated that, during the initial phase of Ca2 mobilization, enhanced unidirectional Ca2 efflux occurs, accompanied by net loss of Ca2 from the cell, while during the sustained, steady-state phase of Ca2 mobilization, Ca2 influx is increased, often accompanied by a net gain of Ca2+ by the cell. With the recent advent of intracellular Ca2 indicators, studies of 45Ca2 fluxes have become a less popular approach; however, isotopic fluxes still represent a useful strategy +

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Seconds Fig. 1 Biphasic Ca2+ signaling. In this experiment, AR4-21 pancreatoma cells (Womack er a/., 1985) were loaded with the fluorescent Ca2+ indicator fura-2 and changes in [Ca2+Ii assessed by monitoring the ratio of fluorescence intensities with alternating excitation at 340 and 380 nm (Grynkiewicz et a!., 1986). (A) Activation of muscarinic-cholinergic receptors with methacholine (MeCh) in the presence of extracellular Ca2+ causes a rapid rise in [Ca2+]i, and a sustained elevation of [Ca*+Ii above baseline until the stimulus is ended by application of the muscarinic antagonist atropine (Atro). (B)With no Caz+ present extracellularly, the rapid increase in [Ca2+ ] i is still seen, but the response rapidly returns to baseline. The sustained response can be restored by restoration of extracellular Ca2+, as indicated. (This figure was provided by Dr. Gary St. J . Bird.)

for studying cellular Ca2 metabolism when information on unidirectional movements of ions is needed. For example, the almost universal finding that initial rates of 45Ca2 influx and efflux are increased by Ca2 -mobilizing agonists leads to the conclusion that the steady-state elevation of [Ca2+Ii is due to increased permeability of the surface membrane of the cell to Ca2 , as opposed to a diminished rate of active Ca2 extrusion. In general, the findings with Ca2 indicators cannot readily discern between these alternatives. +

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111. Mechanisms of Ca2+ Entry A. The Role of Inositol Phosphates That inositol I ,4,5-trisphosphate [ ( I ,4,5)1P,]* acts as the primary signal for the initial, intracellular release phase of Ca’ mobilization is now widely accepted (Berridge and Irvine, 1989). However, the mechanisms regulating the second phase of calcium mobilization, that attributed to Ca2+ entry, are apparently much more complex than was originally envisaged, and are at present poorly understood at best (Putney, 1986). In at least one instance, specifically for the actions of extracellular adenine nucleotides, the direct regulation of a plasma membrane Ca2 channel by an activated receptor has been convincingly demonstrated (Benham and Tsien, 1987; Benham, 1989). On the other hand, intracellular application of (1,4,5)IP, to sea urchin eggs, lacrimal gland cells, and mast cells produces a response indicative of activation of both intracellular Ca2 release and entry of Ca2 from the extracellular space (Slack et al., 1986; Irvine and Moor, 1986, 1987; Morris et al., 1987; Llano et al., 1987; Changya et al., 1989b; Penner et al., 1988). In addition, the intracellular application of the (1,4,5)1P, antagonist, heparin, blocks both phases of the [Ca2+ t i signal in lacrimal cells (Changya et al., 1989a; G. St. J. Bird, unpublished data). These findings suggest that (l,4,5)IP3, or one of its metabolites, can activate both phases of cellular Ca2+ mobilization. In some (Irvine and Moor, 1986, 1987; Morris er al., 1987; Changya et al., 1989b) but not all (Slack et al., 1986; Penner et al., 1988; Llano et al., 1987) cases, the presence of inositol 1,3,4,5tetrakisphosphate [( 1,3,4,5)1P,], the product of (1,4,5)IP, phosphorylation, appears to be necessary for full expression of the response to internally applied (1,4,5)IP,, especially for the second Ca2+ entry phase, and this is discussed in more detail later. In most studies, (1,4,5)IP, applied directly to plasma membranes does not increase their Ca2 permeability (Delfert et al., 1986; Ueda et al., 1986; Dargemont et al., 1988). Furthermore, examination of the intracellular distribution of the ( I ,4,5)1P3receptor in Purkinje cells by immunohistochemistry revealed no receptors on the plasma membrane of these cells (Ross et al., 1989). However, (1,4,5)IP, did appear to increase the permeability of plasma membrane vesicles to Ca2+ in one study (Rengasamy and Feinberg, 1988) and, in B lymphocytes, (1,4,5)1P, was reported to increase a CaZ current in excised membrane patches (Kuno and Gardner, 1987). In mast cells, injection of ( 1,4,5)IP, increased Ca2 entry by a conductive (but not voltage-activated) pathway, but a ( I ,4,5)lP,-regulated Ca2+ channel (i.e., unitary conductance) was not identified (Penner et al., 1988). Thus, while it is clear that Ca2+ entry may be regulated by a variety of different mechanisms, depending on the cell +

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*Inositol polyphosphates are abbreviated according to the “Chilton Convention,” for example, ( 1 ,4,5)IP3 for D-m,VO-lnoSitol I .4,5-trisphosphate

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type and nature of the receptor, in those instances in which receptors are linked to activation of phospholipase C it appears that ( 1,4,5)IP3 may be responsible for the activation of calcium entry. However, in the majority of cases this does not appear to involve a direct action on a plasma membrane channel.

B. The Capacitative Model The capacitative model for Ca2+ entry suggests a mechanism whereby the binding of (1 ,4,5)IP3 to its intracellular receptor activates both phases of the Ca2+ signal; that is, the release of intracellular Ca2+ is proposed to be coupled in some way to the activation of Ca2+ entry (Putney, 1986). An early version of this idea appeared in a report in 1977, in which a model was proposed to explain the rapid replenishment of agonist-sensitive Ca2 stores from the extracellular space following their discharge by a muscarinic-cholinergic agonist (Putney, 1977). While this model depicted the surface membrane Ca2+ channels and the agonist-regulated intracellular Ca2 stores connected in series, its major flaw was the assignment of the intracellular pool to the plasma membrane, rather than as a discrete organelle within the cell. It is now clear that a discreet (1,4,5)IP,sensitive organelle exists in cells, although the precise nature of this organelle is controversial (Rossier and Putney, 1991). Subsequently, a model for the relationship in smooth muscle between Ca2+ entry and internal release was proposed by Casteels and Droogmans (1981), according to which Ca2 flows into the cytoplasm through a process of constant refilling and discharging of the internal pools in these cells, the sarcoplasmic reticulum. In a subsequent model in which the role of (1 ,4,5)IP3 was included in this process (Putney, 1986), the transfer of Ca2 from the extracellular space into the pool was proposed to occur by a pathway which did not traverse the bulk of the cytoplasm. The circumstantial evidence from which this idea evolved has been discussed in some detail previously (Putney, 1986). Much of the earlier evidence was derived from studies of the kinetics of refilling of the receptor-regulated calcium pool in parotid acinar cells. In parotid acinar cells under resting conditions, this intracellular calcium pool was found to be quite stable in the presence of extracellular chelating agents. However, after depletion by activation of surface receptors, this pool could be rapidly replenished from the extracellular milieu, even in the absence of agonists and (presumably) second messengers such as (1 ,4,5)IP3 (Aub et al., 1982). These results suggested that it was the decrease in the Ca2+ content of this pool which activated a pathway for refilling the pool from the extracellular space. Thus, in the sustained presence of an agonist, when the Ca2+-releasing messenger (1,4,5)IP3 is maintained continuously at an elevated level, the calcium content of this pool would presumably remain low, and this would then have the effect of maintaining an open pathway from the extracellular space. As +

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Ca2+ continuously flowed into the pool, it would rapidly enter the cytosol through the (1,4,5)IP,-activated channels. The conclusion that Ca2+ flows directly into the pool during this refilling process was based on observed behavior of intracellular Ca2 during this refilling process. In these earlier experiments, changes in cytosolic [Ca2+] were inferred from measurements of 86Rb+ efflux, an indicator of the activation of Ca2 -dependent K channels (Putney, 1976). Refilling of intracellular pools occurred when the cholinergic activation of cells was rapidly terminated by atropine. Since efflux of 86Rb+ did not appear to be increased during reloading of the pool, it was concluded that the pathway for Ca2+ entry from the extracellular space into the pool did not traverse the cytoplasm. (Aub er a l . , 1982). More recently, the intracellular Ca2 indicator fura-2 has been used to monitor changes in cytosolic [Ca2+] in a large number of cell types, including parotid acinar cells (Merritt and Rink, 1987a,b; Hughes ef al., 1988). With this more sensitive indicator, a transient increase in the intracellular [Ca2 ] was readily detected during the refilling process (Takemura and Putney, 1989). Thus, in a medium containing low [Ca2 1, the cytoplasmic [Ca2 ] of parotid acinar cells decreased from 100 to about 50 nM,and a corresponding restoration of cytosolic [Ca2+ ] occurred when extracellular Ca2 was increased to physiological (mM) concentrations. If extracellular Ca2 were added to cells whose intracellular Ca2 + pool had been previously depleted by the sequential addition and removal of an agonist, the initial increase in cytosolic [Ca*+] was transiently larger than in the control cells, i.e., those with the intracellular pool left intact (Fig. 2). This phenomenon of “Ca2 overshoot” has also been observed in endothelial cells (Hallam et al., 1989). In parotid acinar cells, the level of overshoot did not depend on the time interval between the removal of agonist and the addition of Ca2 , arguing that neither (1,4,5)IP, nor any of its metabolites was responsible for this effect. Rather, it was concluded that the Ca2 content of the intracellular pool serves as the sole determinant of this directly demonstrable, albeit transient, increase in membrane permeability to Ca2+. The phenomenon of Ca2+ overshoot not only provides strong evidence that depletion of the (1,4,5)1P,sensitive Ca2+ pool in the parotid acinar cell increases plasma membrane permeability to Ca2 +,but also argues that depletion of the intracellular pool triggers an increased flux of Ca2+ directly into the cytoplasm, rather than through a process of continuous reloading of the intracellular pool and subsequent release through (1,4,5)IP3-activated channels. Because of the transient nature of the Ca2+ overshoot phenomenon, it cannot be determined from experiments such as these whether such a mechanism can quantitatively account for the effects of agonists during the sustained phase of cell activation. That is, when Ca2 is restored extracellularly, the refilling of the intracellular pools rapidly terminates the activated entry. However, as shown by +

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Seconds Fig. 2 Calcium overshoot in parotid acinar cells. Rat parotid acinar cells loaded with the Ca2+ indicator fura-2 were incubated in medium containing no added Ca2+ and 0 . 2 mM EGTA. Ca2+ (10 mM) was added where indicated either to control cells (not previously stimulated, dotted line) or to cells whose intracellular Ca*+ pool had been discharged by addition of 0 . 2 mM methacholine followed by 10 pbf atropine (solid line). (For details see Takemura and Putney, 1989. Reproduced with permission.)

Hallam et af. (1989), this problem can be circumvented by utilizing Mn2+, which in many cell types enters through the same channels employed by Ca2+ (Anderson, 1983; Hallam and Rink, 1985). Mn2+ binds to fura-2 with high affinity and quenches its fluorescence, so that the rate of quench of fluorescence of intracellular fura-2 when Mn2+ is present outside of cells is taken as a measure of surface membrane divalent cation permeability. Thus, activation of endothelial cells with histamine was shown to cause a rapid quench of intracellular fura-2 when Mn2+ was present. When cells were treated with histamine in the absence of extracellular Ca2+ to deplete the intracellular pools, and the receptors were then blocked by application of an antagonist, still in the absence of extracellular CaZ-t , the addition of Mn2 again quenched the intracellular fura-2, as would be predicted from the Ca2+ overshoot demonstrated with parotid acinar cells. However, unlike the case for Ca2+ overshoot, the rate and extent of the Mn2+ quench were essentially identical to that seen in the cells activated by the agonist. This is presumably due to the inability of Mn2 to enter the agonist-regulated intracellular pool and/or its inability to shut off the capacitative entry channels. This indicates that, at least insofar as Mn2+ quench can be taken as an indicator of Ca2+ entry, the activation of divalent cation entry due to intracellular Ca2 pool depletion can quantitatively account for the effects seen with agonist activation. +

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A criticism of the evidence to this point is that it is only possible to demonstrate the phenomenon of Ca2 overshoot or Mn2 quench due to pool depletion in experiments carried out in the absence of extracellular Ca2+, and thus it could be argued that these effects are due to a combined effect of pool depletion and a low concentration of extracellular C a 2 + . Thus, it is important to examine the effects of depletion of this intracellular pool of Ca2+ in the presence of extracellular Ca2+ and in the absence of any changes in inositol phosphates. The nonphorbol ester tumor promoter thapsigargin (Fig. 3) was shown by Jackson et al. (1988) to release intracellular Ca2 without increasing the cellular levels of inositol phosphates. Thastrup et a / . ( 1990) have shown that this toxin depletes intracellular stores by a specific action on the Ca2 -ATPase on intracellular membranes; thapsigargin does not, however, inhibit active Ca2 transport by the plasma membrane of cells. In parotid acinar cells, thapsigargin did not affect inositol phosphate levels, but did induce a substantial elevation in [Ca2+],,which appeared to result from the sequential release of intracellular Ca2 , and sustained entry of Ca2 across the plasma membrane (Takemura et al., 1989). In the absence of external Ca2 , treatment of cells with thapsigargin caused a transient elevation of C a 2 + , and subsequent application of the muscarinic-cholinergic agonist methacholine failed to induce additional release; likewise, little additional Ca2 was released when the thapsigargin was applied to methacholine-treated cells. This indicates that the pools of Ca2 discharged by methacholine and thapsigargin are largely coincident, although the mechanisms by which these stimuli act on this Ca2+ pool are quite different. When cells were treated with a combination of thapsigargin and methacholine, there was a transient increase in cytosolic [Ca2+ I that was greater than that seen with either agonist alone (Takemura et al., 1989). This synergism is consistent with the idea that methacholine, through production +

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of (1,4,5)IP,, increases the permeability of the intracellular pool to Ca2 , while thapsigargin acts by inhibiting the pool's active Ca2+ uptake process (Thastrup, 1990; Thastrup et a/., 1990). However, in the second phase of the response, that is, during Ca2 entry, the combined addition of thapsigargin and methacholine did not increase [Ca2+Iito an extent greater than with either agent given alone (Takemura er al., 1989). This was most clearly apparent when methacholine was added during the Ca2 entry phase induced by thapsigargin (Fig. 4). Under these conditions, methacholine only transiently increased [Ca2+Ii,which subsequently returned to the level achieved by thapsigargin alone. Prior studies have demonstrated that, at an extracellular [Ca2+]of 1 mhf, Ca2+ entry is well below saturation (Marier et al., 1978). Thus, these results indicate that thapsigargin and methacholine must activate the same pathway for Ca2+ entry. This is the same line of reasoning previously used to argue that agonists acting on different receptors in the parotid activate a common Ca2+ entry pathway (Marier et al., 1978). When taken with the findings discussed above that emptying of the agonistregulated Ca2 pool can increase membrane permeability to Ca2 or Mn2 by a mechanism independent of inositol phosphates (Takemura and Putney, 1989), these actions of thapsigargin provide substantial support for the essential elements of the capacitative model for Ca2+ entry (Putney, 1986). First, it can be concluded that emptying of the agonist-sensitive intracellular Ca2 pool activates the receptor-regulated Ca2 entry mechanism; second, in agreement with the results of Mn2+ quench in endothelial cells (Hallam et al., 1989), it appears that this process can quantitatively account for the physiological actions of neu+

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Seconds Fig. 4 Failure of rnethacholine to further increase Ca2+ entry in thapsigargin-activated parotid acinar cells. Sustained Ca2+ entry was activated in fura-2-loaded parotid acinar cells by 2 p M thapsigargin. Addition of 100 methacholine causes a small transient increase in [Ca2+],, but does not elevate steady-state [Ca2+],. (Redrawn from Takemura et al., 1989, with permission.)

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Receptor-Activated Calcium Entry

rotransmitters and hormones in the parotid; third, the results with thapsigargin demonstrate that the depletion of intracellular Ca2 from the agonist-regulated pool activates the Ca2 entry mechanism equally well in the presence or absence of extracellular Ca2 . The mechanisms by which surface receptor agonists and thapsigargin are believed to activate Ca2+ entry are depicted schematically in Fig. 5. +

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C. The Pathway for Ca2+ Entry While findings discussed to this point provide strong support for the basic premise of the capacitative model, i.e., that the status of the agonist-sensitive intracellular Ca2 pool regulates the rate of Ca2 entry, these and other findings also demonstrate that certain aspects of the previous model are probably incorrect. Recall that, according to the original proposals of the capacitative Ca2+ entry model, the emptying of the intracellular pool would activate a pathway for its refilling that does not traverse the cytoplasm (Putney, 1986; Aub et al., 1982; Merritt and Rink, 1987a; Casteels and Droogmans, 1981), and it has been suggested that the pool and plasma membrane must interact in a region of close apposition (illustrated in Fig. 6). A number of more recent observations now argue against such a pathway for direct communication between extracellular Ca2+ and the intracellular Ca2+ pool. First, in contrast to conclusions drawn from earlier work on parotid cells, data obtained from fura-2-loaded parietal cells (Machen and Neglescu, 1988) and lacrimal cells (Kwan et al., 1990) indicate that, if sufficient Ca2+ is available, the intracellular Ca2+ stores can be completely refilled from the cytosol, rather than exclusively from the extracellular space as originally proposed. Second, as discussed above, it is now clear that a transient elevation of [Ca2 li occurs while the intracellular pools are refilled, again in contrast to earlier claims (Putney, 1986). Third, it has been recently demonstrated that, although agonists stimulate Ba2 entry in lacrimal cells, Ba2 is incapable of entering the agonist-regulated intracellular pool (Fig. 7) (Kwan and Putney, 1990). Finally, if the pathway for Ca2+ entry were through the ( 1 ,4,5)IP3-sensitive pool, then agonists [through (1,4,5)IP3 production] would increase the permeability of this organelle’s membrane to Ca2+, and should increase the rate of Ca2+ entry, even in the presence of thapsigargin (depicted in Fig. 8). However, the addition of rnethacholine to thapsigarginactivated cells did not further increase the rate of Ca2+ influx (Fig. 4) (Takemura et al., 1989). Thus, it appears that depletion of the agonist-sensitive intracellular Ca2 pool does activate Ca2+ influx at the plasma membrane, but apparently the pathway for Ca2 entry to the cytosol from the extracellular space is not obliged to traverse the (1,4,5)IP,-sensitive pool. This conclusion leaves the more difficult problem of trying to understand how the regulation of plasma membrane function by this intracellular organelle is +

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Fig. 5 The capacitative model for Ca2+ entry. (A) Agonists activate surface membrane receptors (R) which in turn activate a polyphosphoinositide-specific phospholipase C (Pu3); in many instances, a guanine nucleotide-dependent regulatory protein (Gp) is involved in coupling receptors to phospholipase C. This leads to the production of ( I ,4,5)IP3 which in turn activates the release of Ca2 via an inositol trisphosphate-regulated channel (IRC). The release of Ca2 is faster than the rate at which Ca2 is returned by the associated Ca2 -ATPase or pump, leading to depletion of the Ca2+ content of this internal organelle. The depletion of Ca2+ organelle causes, by an unknown mechanism (dotted arrow), the opening of a plasma membrane entry channel for Ca2+. (B) Thapsigargin also causes depletion of this pool and activation of the same entry channel but by a mechanism involving inhibition of the pump, allowing Ca2+ to exit the pool through a leak channel. +

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Fig. 6 Direct versus indirect routes of Ca2 entry. Early versions of the capacitative model held that Ca2+ entered the cell by directly entering the pool and then entering the cytosol via the ( I .4,5)IP3-activated channel (left). However, as argued herein, it now appears that the depletion of this intracellular pool activates a pathway for direct entry of Ca2+ to the cytosol (right). +

accomplished. Previously, it was suggested (Putney, 1986) that the rate of net uptake of Ca2 by the pool, especially in regions close to the plasma membrane, might affect plasma membrane permeability to Ca2 . The results with thapsigargin would argue against such a suggestion, since Ca2 entry can apparently be activated despite inhibition of intracellular Ca2+ transport. In fact, it can no longer be inferred that the organelle involved in regulation of the permeability of the plasma membrane to Ca2 is close to the surface of the cell. The possibility therefore exists that this communication could be accomplished through some kind of intracellular chemical signaling; however, to date there is no evidence for such a mechanism. Note, however, that immunohistochemical studies have localized at least a fraction of the (1,4,5)IP, receptors in Purkinje cells to small vesicles close to the plasma membrane (Ross et d., 1989). Also, there is recent evidence, based on subcellular fractionation data, to suggest that the (1,4,5)1P,sensitive organelle in hepatocytes may be linked to the plasma membrane through the cytoskeleton. However, it is not clear whether this indicates a means for functionally linking these organelles. Understanding the mechanism of interaction or communication between the (1,4,5)IP3-sensitive Ca2 pool and the plasma membrane will be a challenging topic for future investigations. Thapsigargin does not mimic the actions of agonists in inducing Ca2 entry in all cell types (Jackson et al., 1988), despite its ability to discharge the agonistsensitive intracellular Ca'+ pool. Also, experiments in chromaffin cells with the Ca2 + ionophore ionomycin clearly indicated that, for angiotensin 11, some signal other than, or in addition to depletion of the intracellular pool is required for +

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Fig. 8 Actions of thapsigargin and (1,4,5)IP3 in the context of direct coupling models. If Ca2+ were to Row in an obligatory fashion into the ( I ,4,5)IP3-sensitivepool before entering the cytosol, as originally envisaged, then thapsigargin would activate entry to the cytosol through the lead channel of the organelle. Addition of ( I ,4,5)IP3 in such circumstances would be expected to accelerate the rate of entry of C a 2 + . However, as shown in Fig. 4, this is not observed experimentally. indicating that the permeability of the limiting membrane of the ( 1 ,4,5)IP3sensitive organelle is not a rate-determining step i n Ca2 entry. +

activation of Ca2+ entry (Stauderman and Pruss, 1989). These results are consistent with the view that the mechanisms for regulating Ca2 entry in different cell types may vary (Hallam and Rink, 1989). Indeed, as suggested by Thastrup et al. (1989), thapsigargin may prove to be extremely useful in providing a simple pharmacological diagnosis for the capacitative entry mechanism. This reagent will no doubt prove to be a valuable tool in unraveling the mechanism of this important component of cellular Ca2 signaling. +

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D. Actions of Inositol 1,3,4,5-Tetrakisphosphate and Relevance to the Capacitative Model Irvine and collaborators have suggested that the phosphorylated product of

(1,4,5)IP,metabolism, (1,3,4,5)IP,,plays a significant role in the regulation of Ca2+ entry (Irvine et al., 1988; Irvine, 1989, 1990). Irvine and Moor (1986, 1987), in experiments on sea urchin eggs, provided the first evidence for a incubated a second time in the absence of divalent cations. As shown in B, these cells did not give a transient increase in fluorescence ratio in response to MeCh, indicating that the previous entry of Ba2+ was not associated with refilling of the intracellular pools. Ca2+ was then added as indicated, the cells washed a second time, and restored to Ca2+-deficient medium. As shown in C, the entry of Ca?+ was associated with replenishment of the agonist-sensitive pool. (For details see Kwan and Putney, 1990. Redrawn with permission.)

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biological role for ( I ,3,4,5)IP4when they reported that a full fertilization response to injected inositol polyphosphates depended on the presence of this product of (1,4,5)IP, metabolism. In these, and subsequent studies, (1 ,3,4,5)IP4 did not induce any apparent [Ca2+Ii signal on its own, but always depended on the simultaneous presence of (1 ,4,5)IP3 or one of its analogs [i.e., (2,4,5)IP3]. In patch-perfusion studies on lacrimal acinar cells (Morris et al., 1987; Changya et ul., 1989a,b), it appeared that it was the second, sustained phase of the [Ca2+Ii response which required the presence of ( 1 ,3,4,5)1P4 in the patch perfusate, again together with (1,4,5)1P3. However, in these same cells (Kwan et al., 1990) thapsigargin is capable of quantitatively reproducing the same level of Ca2 entry as phospholipase C-activating agonists, but without increasing the cellular levels of any inositol polyphosphates, and presumably solely by virtue of its ability to deplete critical intracellular stores of their Ca2+. A potential reconciliation of these apparently conflicting findings comes from the observation (Changya ez al., 1989b)that, following the release of a finite pool of Ca2 by perfusion of the patch pipette with (1,4,5)IP,, subsequent perfusion of the pipette with a combination of ( 1 ,4,5)IP3 and (1,3,4,5)IP, appeared to cause a second mobilization of intracellular Ca2 . This may indicate that there are two regulated pools of Ca2 in the lacrimal acinar cell, one which can be released by (1,4,5)IP,, and a second for which release requires (1 ,3,4,5)IP4 together with (1,4,5)IP,. Since it appeared that the combination of (1,4,5)1P, and (1 ,3,4,5)IP4 was also required for Ca2 entry in these cells, then, in the context of the capacitative model, it would be this second Ca2+ pool, which requires the two inositol polyphosphates for its release, that may be the critical pool regulating Ca2+ entry at the plasma membrane. However, it should be pointed out that clear evidence for a role of (1 ,3,4,5)IP4 in Ca2+ signaling has only been reported for a limited number of systems. Furthermore, in one cell type, activation of Ca2 entry has been demonstrated with the production of vanishingly small levels of ( 1 ,3,4,5)1P4. Thus, it remains to be seen whether this inositol polyphosphate plays a general role in Ca2+ signaling in hormone- and neurotransmitter-regulated cells. +

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IV. Conclusions This review has attempted to summarize a particular theory, termed a capacitative model, for the mechanism by which the entry of Ca2 is regulated by agonists acting through the phosphoinositide system. It is now quite well established that the initial, agonist-induced [Ca2+Iisignal in such cells is derived from the Ca2 -releasing actions of (1,4,5)IP, on an intracellular Ca2 sequestering organelle. However, in virtually all cells that utilize this mechanism, the initial release of intracellular Ca2+ is followed or accompanied by an accelerated entry of Ca2 into the cytoplasm across the plasma membrane. The +

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mechanism by which this process is regulated has been somewhat more elusive, and may vary in different cellular systems. According to the capacitative model, the depletion of the agonist-sensitive intracellular Ca2+ store by (1,4,5)IP, [or possibly (1,4,5)IP,and (1,3,4,5)1P,] generates a secondary signal of unknown nature that activates Ca2 entry (Fig. 5 ) . Further investigation of this interesting aspect of the Ca’ -phosphoinositide system should provide us with many additional discoveries and surprises in the near future. +

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References Anderson, A. (1983). Mn ions pass through calcium channels. J . Gen. Physiol. 81, 805-827. Armstrong, D., Rossier. M. F., Shcherbatko, A. D.. and White. R. (1991). Enzymatic gating of voltage-activated calcium channels. Ann. N . Y Acad. Sci. (in press). Aub, D. L.. McKinney, J . S . , and Putney, J. W., Jr. (I982). Nature of the receptor-regulated calcium pool in the rat parotid gland. J . Physial. (London) 331, 557-565. Benham, C. D. (1989). ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. J . Physiol. (London) 419, 689-701. Benham, C. D., and Tsien, R . W. (1987). A novel receptor-operated Ca’+-permeable channel activated by ATP in smooth muscle. Nature (London) 328, 275-278. Berridge, M. J . , and Irvine, R . F. (1989). lnositol phosphates and cell signalling. Nurure (London) 341, 197-205. Bianchi, C. P. (1968). “Cell Calcium.” Butterworth, London. Blaustein. M. P (1985). Intracellular calcium as a second messenger. What’s so special about calcium’?I n “Calcium in Biological Systems” (R. P. Rubin, G. B . Weiss. and J. W. Putney, Jr., eds.) pp. 23-33. Plenum, New York. Bohr, D. F. (1973). Vascular smooth muscle updated. Circ. Res. 32, 665-672. Casteels, R.,and Droogmans. G . (I981). Exchange characteristics of the noradrenaline-sensitive calcium store in vascular smooth muscle cells of rabbit ear artery. J . Physiol. (London) 317, 263279. Changya, L., Gallacher. D. V., Irvine, R . F., and Petersen, 0. H. (1989a). lnositol 1,3,4,5tetrakisphosphate and inositol I ,4.5-trisphosphate act by different mechanisms when controlling Ca2+ in mouse lacrimal cells. FEBS Let?. 251, 43-48. Changya. L., Gallacher, D. V.. Irvine. R . F.. Potter, B. V. L., and Petersen, 0. H. (1989b). lnositol I,3,4,5-trisphosphate is essential for sustained activation of the Ca? -dependent K + current in single internally perfused lacrimal cells. J. Metnbr. Biol. 109, 85-93. Dargemont, C . , Hilly, M . , Claret, M.. and Mauger. J.-P. (1988). Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles. Biochem. J. 256, 117- 124. Delfert, D. M., Hill, S . . Pershadsingh. H. A,. and Sherman. W. R. (1986). mvo-lnositol 1,4,5trisphosphate mobilizes Ca2 from isolated adipocyte endoplasmic reticulum but not from plasma membranes. Biochern. J. 236, 37-44. Douglas, W. W. (1974). Involvenient of calcium in exocytosis and the exocytosis vesiculation sequence. Biocheni. Soc. Symp. 39, 1-28. Fink, L. A., and Kaczmarek, L. K. (1988). Inositol polyphosphates regulate excitability. Trends Ncw-ol. Sci. 11, 338-339. Crynkiewicz., G., Poenie, M.. and Tsien, R. Y. (1986). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J . B i d . Chem. 260, 3440-3450. Hallam, T. J., and Rink. T. J. (1985). Agonists stimulate divalent cation channels in the plasma membrane of human platelets. FEBS Lerr. 186, 175-179. +

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Hallam, T. J., and Rink, T. J. (1989). Receptor-mediated Ca2+ entry: diversity of function and mechanism. Trends Pharmacol. Sci. 10, 8-10. Hallam, T. J., Jacob, R., and Merritt, J. E. (1989). Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells. Biochem. J. 259, 125-129. Hughes, A. R., Takemura, H.,and Putney, J. W., Jr. (1988). Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1:2,4,5-trisphosphate metabolism in intact rat parotid acinar cells: Relationship to calcium signalling. J. Biol. Chem. 263, 10314-10319. Irvine, R. F. ( I 989). How do inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate regulate intracellular Ca2+? Biochern. Soc. Trans. 17, 6-9. Irvine, R. F. (1990). “Quantal” Ca2+ release and the control of Ca2+ entry by inositol phosphatesa possible mechanism. FEES Lett. 263, 5-9. Irvine, R. F., and Moor, R. M. (1986). Micro-injection of inositol 1,3,4,5-tetrakisphosphateactivates sea urchin eggs by a mechanism dependent on external C a 2 + . Biochem. J. 240, 917-920. Irvine, R. F., and Moor, R. M. (1987). Inositol(l,3,4,5)tetrakisphosphate-induced activation of sea urchin eggs requires the presence of inositol trisphosphate. Biochem. Biophys. Res. Commun. 146, 284-290. Irvine, R. F., Moor, R. M., Pollock, W. K., Smith, P. M., and Wreggett, K. A. (1988). Inositol phosphates: proliferation, metabolism and function. Philos. Trans. R. Soc. London, Ser. B 320, 281-298. Jackson, T. R., Patterson, S. I., Thastrup, O., and Hanley, M. R. (1988). A novel tumor promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NGI 15-401L neuronal cells. Biochem. J. 253, 81-86. Klee, C. B., and Newton, D. L. (1985). Calmodulin: An overview. In “Control and Manipulation of Calcium Movement” (J. R. Parratt, ed.), pp. 131-145. Raven, New York. Kuno, M., and Gardner, P. (1987). Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature (London) 326, 301-304. Kwan, C. Y., and Putney, J. W., Jr. (1990). Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2 + and Ba2 of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool. J. Biol. Chem. 265, 678-684. Kwan, C. Y., Takemura, H., Obie, J. F., Thastrup, 0.. and Putney, I. W., Jr. (1990). Effects of methacholine, thapsigargin and La3 on plasmalemmal and intracellular Ca2 transport in lacrimal acinar cells. Am. J. Physiol. 258, C1006-CIO15. Llano, I., Marty, A . , and Tanguy, J. (1987). Dependence of intracellular effects of GTP(-y)S and inositol trisphosphate on cell membrane potential and on external Ca ions. Pfluegers Arch. 409, 499-506. Machen, T. E., and Neglescu, P. A. (1988). Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cell. Am. J. Physiol. 254, C498-C504. Marier, S. H., Putney, J. W., Jr., and van de Walle, C. M. (1978). Control of calcium channels by membrane receptors in the rat parotid gland. J. Physiol. (London) 279, 141-151. Merritt, J. E., and Rink, T.I. (1987a). Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells. J. B i d . Chem. 262, 17362-17369. Merritt, 1. E., and Rink, T. J. (1987b). The effects of substance P and carbachol on inositol tris- and tetrakisphosphate formation and cytosolic free calcium in rat parotid acinar cells. A correlation between inositol phosphate levels and calcium entry. J. Biol. Chem. 262, 14912-14916. Moms, A. P., Gallacher, D. V., Irvine, R. F., and Petersen, 0. H. (1987). Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K channels. Nature (London) 330, 653-655. Nishizuka, Y. (1984). ’hmover of inositol phospholipids and signal transduction. Science 225, 1365-1370. +

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Penner, R., Matthews, G . , and Neher, E. ( I 988). Regulation of calcium influx by second messengers in rat mast cells. Nature (London) 334, 499-504. Putney, J. W., Jr. (1976). Biphasic modulation of potassium release in rat parotid gland by carbachol and phenylephrine. J. Pharmacol. Exp. Ther. 198, 375-384. Putney, 1. W., Jr. (1977). Muscarinic, alpha-adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland. J . Physiol. (London) 268, 139-149. Putney, J. W., Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7 , 1-12. Putney, J. W., Jr. (1987). Calcium-mobilizing receptors. Trends Pharmacol. Sci. 8, 481-486. Putney, J. W., Jr., Poggioli, J., and Weiss, S. J. (1981). Receptor regulation of calcium release and calcium permeability in parotid gland cells. Philos. Trans. R . Soc. London, Ser. B 296, 37-45. Rasmussen, H. ( 1970). Cell communication, calcium ion and cyclic adenosine monophosphate. Science 170, 404-4 12. Rengasamy, A , , and Feinberg, H. (1988). Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles. Biochem. Biophys. Res. Commun. 150, 1021- 1026. Ringer, S. (1883). A further contribution regarding the influence of the different constituents of the blood on the contraction of the heart. J. Physird. (London) 4, 29-42. Ross, C. A,, Meldolesi, J., Milner, T. A , . Satoh, T., Supattapone, S., and Snyder, S. H. (1989). lnositol I ,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature (London) 339, 468-470. Rossier, M. F., and Putney, J. W.. Jr. (1991). The identity of the calcium storing inositol 1,4,5trisphosphate-sensitive organelle in non-muscle cells: Calciosome, endoplasmic reticulum, . . . or both'? Trends Neurol. Sci. (in press). Slack, B. E., Bell, J. E., and Benos, D. J. (1986). Inositol 1,4,5-trisphosphate injection mimics fertilization potentials in sea urchin eggs. Am. J . Physiol. 250, C340-C344. Stauderman, K. A., and PIUSS,R. M . (1989). Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin I1 in bovine adrenal chromaffin cells. J. B i d . Chem. 264, 18349-18355. Takemura, H., and Putney, I. W., Jr. (1989). Capacitative calcium entry in parotid acinar cells. Biochem. J . 258, 409-412. Takemura, H . , Hughes, A. R., Thastrup, O., and Putney. J. W., Jr. (1989). Activation of calcium entry by the tumor promoter, thapsigargin, in parotid acinar cells. Evidence that an intracellular calcium pool, and not an inositol phosphate, regulates calcium fluxes at the plasma membrane. J. Biol. Chem. 264, 12266-12271. Thastrup, 0. (1990). Role of Ca2 -ATPases in regulation of cellular Cali signalling, as studied with the selective microsomal Ca2 -ATPase inhibitor, thapsigargin. Agenls Actions 29, 8- 15. Thastrup, 0.. Dawson, A. P., Scharff, O., Foder, B . , Cullen. P. J., Drobak, B. K., Bjenum, P. J . , Christensen, S. B . , and Hanky, M. R. (1989). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Arrions 27, 17-23, Thastrup, 0..Cullen, P. J., Drobak, B. K.,Hanky. M. R., and Dawson, A. P. (1990). Thapsigargin, a tumor promoter, discharges intracellular Ca2 stores by specific inhibition of the endoplasmic reticulum Ca2+ -ATPase. Proc. Nar. Acad. Sci. U.S.A. 87, 2466-2470. Tsien, R. Y. (1989). Fluorescent probes of cell signalling. Annu. Rev. Neurosci. 12, 227-253. Ueda, T., Church, S. H., Noel, M. W., and Gill. D. L. (1986). Influence of inositol I ,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the NIE-I 15 neuronal cell line. J. Biol. Chem. 261, 3184-3192. Van Breemen, C.. and Saida. K. (1989). Cellular mechanisms regulating [Ca*+], in smooth muscle. Annu. Rev. Physiol. 51, 315-329. Womack. M. D., Hanky, M. R., and Jessel, T. M. (1985). Functional substance Preceptors on a rat pancreatic acinar cell line. J. Neurosci. 5, 3370-3378. +

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Calcium Channel Antagonists in the Prevention of Neurotoxicity Stuart A. Lipton Laboratory of Cellular and Molecular Neuroscience Department of Neurology Children's Hospital, Beth Israel Hospital Brigham and Women's Hospital, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School Boston, Massachusetts 02115

I . Introduction 11. Potential Problems in Interpreting the Effectiveness of Calcium Channel Antagonists in Preventing Neurotoxicity A. Use of in Vitro Models to Distinguish Direct Neuronal Effects from Systemic Effects of Calcium Channel Antagonists B . Multiple Sites of Action of Calcium Channel Antagonists 111. Types of Neurotoxicity Attenuated by Calcium Channel Antagonists IV. The Hypothesis of Calcium-Associated Neuronal Damage V. Potential Contribution of T, N. L, and P Types of Calcium Channels to Neurotoxicity VI. Types of Voltage-Dependent Calcium Channel Antagonists VII. I n Virro Models of Neurotoxicity and Effects of Calcium Channel Antagonists A. Cerebellar Cells B . Cortical and Hippocampal Neurons C. Retinal Ganglion Cell Neurons D. Possible Contribution of Intracellular Ca2+ Mobilization VIII. I n Vivo Animal Models of Ischemia and Effects of Calcium Channel Antagonists IX. Early Human Trials and Prospects for Calcium Channel Antagonists in Preventing Neurotoxicity A . For Acute Ischemic (Nonhemorrhagic) Stroke B . Alzheimer's Disease and Senile Dementia C. Calcium Channel Antagonists for the AIDS Dementia Complex? X . Conclusions References Advon' YS m t ' h o r m u d o g \ , Volurnu 22 Copyright 6')1441 hy Academic P r e s . Inc All nghn 01 reproducuon In any fomi reserved

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1. Introduction This review presents evidence that, at least under particular sets of circumstances, calcium channel antagonists can be neuroprotective agents. We distinguish between neuronal and systemic effects of calcium channel antagonists. The types of neurotoxicity to be considered are delineated. Next, the various types of calcium channels that are voltage-dependent (also known as voltageactivated, -stimulated, or -gated) are briefly discussed in order to determine which calcium channels are important in neurotoxicity. This knowledge is necessary in order to assess the effectiveness of the various types of pharmacological antagonists that are available against each of the channel types. With this information as background, we then consider the effects of calcium channel antagonists on various in vitro models of primary neurons, followed by in vivo models of neurotoxicity. Finally, the prospects and results of preliminary human studies are discussed.

II. Potential Problems in Interpreting the Effectiveness of Calcium Channel Antagonists in Preventing Neurotoxicity

A. Use of in Vitro Models to Distinguish Direct Neuronal Effects from Systemic Effects of Calcium Channel Antagonists There are confounding problems in deciphering the action of the several types of voltage-dependent calcium channel antagonists. One major obstacle to determining their site of action occurs in studies performed in whole animals. In this case, it is difficult if not impossible to determine if the calcium channel antagonists are working, at least in part, directly on neurons or if their actions are exclusively mediated by systemic effects, e.g., on blood pressure, or via blood vessel dilatation and subsequent increases in blood flow to ischemic areas. For this reason, tissue culture models of primary central neurons are useful to determine if direct toxicity-preventing effects on nerve cells can be observed, at least in vitro. Therefore, in this review we consider first the effects of calcium channel antagonists on in vitro models of neurotoxicity, followed by a discussion of animal models. Prior to this presentation, however, we must first consider the types of neurotoxicity and the types of calcium channels that we are dealing with.

B. Multiple Sites of Action of Calcium Channel Antagonists Another potential problem in the interpretation of these kinds of experiments is the fact that many of the so-called calcium channel antagonists, like other phar-

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macological agents, exhibit multiple actions. Thus, these drugs not only affect calcium channels but also, in some cases at only slightly higher (micromolar) concentrations, exhibit other effects that may reduce Ca2 in central neurons. For example, they may antagonize Na -Ca2 exchange or inhibit calmodulindependent enzyme activity. Other mechanisms of action cannot be excluded (for a more complete list see Glossmann et al., 1989; Zernig, 1990). +

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111. Types of Neurotoxicity Attenuated

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Calcium Channel Antagonists

Considerable evidence has been gathered in recent years that, in some cases, antagonists of voltage-dependent calcium channels can prevent or at least attenuate the lethal effects of certain neurotoxins. Chief among these deleterious agents are the excitotoxins that act at the various subtypes of glutamate receptors found in the mammalian central nervous system. Excitotoxins are currently thought to be responsible at least in part for the neuronal injury observed after hypoxia/ischemia, trauma, hypoglycemia, epilepsy, and several types of neurodegenerative maladies, including Huntington’s disease and the acquired immune deficiency syndrome (AIDS) dementia complex (for reviews see Choi, 1988a,b; Meldrum and Garthwaite, 1990; Rothman and Olney, 1986, 1987). The present review focuses on the possible effects of calcium channel antagonists in preventing neuronal injury due to glutamate-like toxins. Since it is currently thought that much of the damage occurring after ischemia to the brain is mediated in this manner, this type of tissue injury will also be considered in some detail. The endogenous glutamate-like agonist that is responsible for neuronal cell injury may in many cases be glutamate itself; however, other candidates for this role include aspartate, quinolinate, homocysteic acid, small peptides containing glutamate, and others. Similarly complex are the receptor types that bind glutamate and glutamatelike agonists. The three main subtypes that directly trigger receptor-operated ion channels are named after their preferred agonists: N-Methyl-D-aspartate (NMDA), kainate, and quisqualate (or AMPA). A fourth subtype, the aminophosphonobutyrate (APB) receptor, exists but may not be directly involved in excitotoxicity. One obvious difference among the main subtypes is that the NMDA receptor-operated channels are permeable not only to monovalent cations but also to Ca2+. With some exceptions that may be regionally determined within the brain (Iino et al., 1990), the receptor-operated channels that are activated by the other subtypes are not permeable to Ca2+. However, the depolarization of neurons triggered by stimulation of kainate and quisqualate receptors may indirectly produce Ca2 entry by activating voltage-gated calcium channels. In addition, quisqualate may activate another, “metabotropic” or Gprotein-coupled receptor that triggers intracellular Ca2 mobilization via the +

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phosphoinositide pathway rather than via a plasma membrane ion channel. Increases in intracellular Ca2 have been associated with subsequent cellular injury, and therefore calcium homeostasis is hypothesized to be critical for cellular integrity (see below). In several areas of the brain, the predominant form of neuronal injury following brief exposure to giutamate or ischemia appears to be mediated by activation of the NMDA receptor. Prevention of this NMDA receptor-mediated form of neurotoxicity has therefore been the object of many studies in the past few years (for reviews see Choi, 1988a,b; Meldrum and Garthwaite, 1990; Rothman and Olney, 1986, 1987). Non-NMDA receptors may also play a role in neuronal damage, as demonstrated by several groups (Frandsen et al., 1989). However, the fact that the NMDA receptor may play a major part in physiological processes (such as long-term potentiation) as well as in pathological events (ischemia, trauma, etc.) has led to intense study of this receptor’s properties. One important concept that may not be evident in reviewing individual papers on the subject of excitotoxicity, is the importance of a particular type of glutamate receptor in neuronal damage in a given area of the brain. The relatively different distributions of the receptor subtypes in disparate brain regions, as well as in a single area during varying developmental stages, may account for this differential vulnerability. For example, after 5 days in culture, embryonic hippocampal neurons (previously harvested from 17-day-old fetuses) are relatively resistant to NMDA receptor-mediated neurotoxicity but sensitive to kainateinduced damage (Mattson et al., 1989); patch-clamp and binding studies suggest that, unlike kainate receptors, NMDA receptors have not yet developed in these neurons at this stage of development. Similarly, the contribution of voltageactivated calcium channels to increasing intracellular Ca2 levels in different areas of the brain may vary with the distribution of the voltage-gated calcium channels, of NMDA receptors, and of non-NMDA (kainate/quisqualate) receptors. In the presence of many NMDA receptors, Ca2 entry via NMDA receptoroperated channels may outweigh entry via the voltage-dependent channels. In contrast, when non-NMDA receptors are mainly present, monovalent ion flux depolarizes the neuron; under these conditions, voltage-activated calcium channels may be relatively more important in determining Ca2+ entry since these channels are activated by depolarization of the neuronal cell membrane. This scenario raises the possibility that antagonists of voltage-dependent calcium channels may be able to attenuate damage associated with Ca2+ entry in some areas of the brain but not in others. However, there are further caveats in the interpretation of experiments purporting to show that either voltage-gated calcium channels or NMDA receptoroperated channels are important in a particular form of neurotoxicity. For example, one group of antagonists, represented by dextrorphan, that blocks NMDA receptor-mediated neurotoxicity and possibly NMDA receptor-operated channels +

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(Choi et a f . , 1987) has also been recently found to block voltage-gated calcium channels (Carpenter et a/., 1988). Thus, some drugs thought to be specific inhibitors for NMDA-mediated events might in addition reflect blockade of calcium channel activity.

IV. The Hypothesis of Calcium-Associated Neuronal Damage Several recent reviews have dealt with the concept of delayed neurotoxicity that is triggered by an early rise in intracellular [Ca2+] (Choi, 1988a; Lipton and Kater, 1989; Siesjo, 1981, 1988). It should be emphasized, however, that calcium-induced neuronal damage is still an hypothesis, albeit one with considerable and continually mounting evidence. Since this evidence is presented in these other reviews, it is not elaborated here in detail. The chain of events associated with a massive increase in intracellular [Ca2+] has also been emphasized in recent reviews (Boobis et al.. 1989; Choi, 1988a; Meldrum and Garthwaite, 1990; Orrenius ef af., 1989). Briefly, pathological increases in intracellular [Ca2 ] can lead to several delayed events associated with neuronal damage, including calcium-activated enzyme activation (calpains I and 11, phospholipases, protein kinase C, xanthine oxidase, endonucleases, Ca2 -calmodulindependent protein kinase 11, nitric oxide synthase), triggering lipid peroxidation, free radical formation, and DNA fragmentation. In light of the increasingly convincing evidence for calcium-associated neuronal damage, several therapeutic maneuvers to attenuate or prevent excitotoxicity have been aimed at decreasing or stabilizing intracellular [Ca2+]. It is in this setting that calcium channel antagonists have been suggested to be one possibly useful pharmacological agent. +

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V. Potential Contribution of T, N, L, and P Types of Calcium Channels to Neurotoxicity In the mammalian central nervous system at least four types of voltage-gated calcium channels have been described based on single-channel properties and/or pharmacological sensitivities. The properties of the various types of calcium channels have been reviewed in detail elsewhere (Fisher et al., 1990; Fox et al., 1987a,b; Lin e t a / . , 1990; Plummer et al., 1989). For the purposes of the current review, it is necessary to discuss several of these properties briefly. First, not all central neurons display each of these various types; even the exact characteristics of any one type may vary somewhat among different regions of the brain (Miller,

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1987). A major distinction between the various types of channels is drawn based on their kinetics, e.g., the duration of calcium current in response to a sustained depolarization of the membrane such as that observed following exposure to glutamate. For example, the T or transient type of calcium channel undergoes rapid desensitization, whereas the N or neuronal type and also the P (for Purkinje cell) type display somewhat slower decay. In contrast, the current of the L or long-duration type calcium channel generally has a sustained component, i.e., exhibiting very little decay with time. Therefore, even in brain areas in which the Ltype current is less prominent than the other types, it may still make the major contribution to sustained Ca2+ influx such as that needed for neurotoxicity since its channels remain active in response to depolarization for a substantially longer period of time than the other channel types. Along these lines, even though relatively specific antagonists exist for the various types of calcium channels, antagonists of the Ltype channel may contribute the most, at least in theory, to pharmacological blockade of the component of excitotoxicity mediated by Ca2 flux through the voltage-gated calcium channels. For this reason and because these types of antagonists are already clinically available, this review focuses mainly on the effects of the antagonists of L type calcium channels. +

VI. Types of Voltage-Dependent Calcium Channel Antagonists Tables I through IV present menus of the various types of antagonists that inhibit the voltage-gated calcium channels. The various antagonists are currently used to distinguish the contribution to total voltage-activated calcium current of T-, N-, L,and P-type channels in a particular preparation. As mentioned in the previous section, the Ltype channels may contribute substantially to neurotoxicity in some preparations because they are activated for relatively long periods of time. Of course, this does not preclude the other channel types from contributing to deleterious accumulations of intracellular [Ca2 ] as well. Nevertheless, for the aforementioned reasons, we will concentrate on Ltype antagonists and their potential effects on neurotoxicity. L Q p e channels exist in many tissues, including both the heart and brain. However, there is some indication that in the central nervous system not all L type (also known as one type of high-threshold) calcium channels are equally affected by antagonists that inhibit heart Ltype channels (Miller, 1987; Yaari et al., 1987). Therefore, differential susceptibility among various neuronal cell types may obfuscate the mechanism of action of these drugs. Thus, to prevent neurotoxicity in some preparations (e.g., fetal cortical neurons in vitro, see section VII,B), micromolar concentrations are necessary. These concentrations are higher than predicted by binding studies but might be consistent with concentra+

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Table I General Classes of Voltage-Dependent Calcium Channel Antagonists Antagonist

References

Specific antagonists against h y p e channels A. Dihydropyridines (e.g., nimodipine and others, see Table 11) B. Phenylalkylamines [e.g., verapamil, (S)-emopamil, D-600, D-8881 C. Benzothiazepines (e.g., diltiazem) D. Diphenylpiperazines or diphenylalkylamines (e.g., tlunarizine/cinnarizine series and related drugs) E. Bepridil and related drugs F. Diphenylbutylpiperidines G . HOE 166 and related drugs H. Fluspirilene and related drugs

Boddeke ef a/. (1989), Greenberg (1987), Hosey and Lasdunski (1988), Ohtsuka ef nl. (1989), Triggle and Rampe (1989)

Specific antagonists against N-type calcium channels Snail toxins: o-conotoxin GVIA and GVIlA

Cruz and Olivera (1986). Jones er a/. (1989)

111.

Antagonists against T-type calcium channels A. Low concentrations of Ni2+ B. Amiloride (has other effects as well)

Tang ef al. ( 1988)

IV.

Antagonists against P-type calcium channels Spider web funnel toxin (but specific fractions also block N- and Ltype channels)

Lin er a/. (1990)

1.

11.

Table I / Dihydropyridine Calcium Channel Antagonistsu Nifedipine Nimodipine Niludipine PY 108-068 (darodipine) Mesudipine GX 1048 Floridine Nitrendipine Nisoldipine Nicardipine Felodipine PN220-110 (isradipine) CV4093 UFrom Ohtsuka er a/. (1989).

KW3049 Oxodipine CD349 TC8 1 YM-09730-5 or (4S)DHP MDL72567 Ro18-3981 DHP-2 18 Nilvadipine Amlodipine 8363-S Iodipine Azidopine

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Table 111 New Calcium Channel Antagonistso Diclofurime Pimozide Prenylamine kndiline Perhexiline Mioflazine Lidoflazine CERM- 1 1956 R-58735 Ranolazine R-56865 %om Boddeke el a / . (1989).

tions that block Ltype calcium channels. However, at micromolar concentrations, other mechanisms of action of the antagonists cannot be excluded. There are four major classes of antagonists of the Ltype voltage-gated calcium channels (entries A-D in Table I). These classes are named for their chemical derivations and each has examples (listed in parentheses) that are already clinically utilized for other conditions: dihydropyridines (nifedipine and nimodipine), phenylalkylamines (verapamil), benzothiazepines (diltiazem), and diphenylpiperazines, also termed diphenylalkylamines by some authors (flunarizine). These as well as the other types of antagonists (Table I) bind to distinct but interacting sites on voltage-activated calcium channels (Miller, 1987). Table IV Other Common Drugs that also Affect Calcium Channels to Some Degree but Are Less Potent and/or Less Specifica ~~~~

Phenytoin Thioridazine Tricyclic antidepressants Local anesthetics Diphenylbutylpiperidines Ethanol Reserpine Phencyclidine Cyproheptadine Loperamine Diphenoxylate Tamoxifen Anthracyclines aFor references see Greenberg ( 1987)

Calcium Channel Antagonists

279

In an attempt to prevent neurotoxicity directly, calcium channel antagonists that are CNS-permeable would have the greatest potential benefit. Thus, drugs that have been proven to cross the blood-brain barrier would be of most interest. Such calcium channel antagonists include the dihydropyridines nimodipine, to some extent nicardipine, and, potentially, several of the newer agents in this category. In addition, the phenylalkylamine (9-emopamil (which also possess antiserotonergic properties) has substantial CNS permeability, as does the diphenylpiperazine flunarizine and related agents.

VII. In Vitro Models of Neurotoxicity and Effects of Calcium Channel Antagonists As discussed above, in order to distinguish systemic effects of calcium channel antagonists from direct actions on neurons, tissue culture model systems of neurotoxicity have been used. Direct protective effects of calcium channel antagonists have been observed in vitro using primary, central neurons obtained from cerebellum, cortex, and retina. However, the results do vary considerably from preparation to preparation.

A. Cerebellar Cells Organotypic cultures of rat cerebellum can be protected from anoxia by nimodipine if this dihydropyridine is added to the cultures immediately after the insult (Renkawek and Lazarewicz, 1989a,b). Since anoxic damage is thought to be mediated by an excitotoxic mechanism, predominantly via NMDA receptor activation (Goldberg el af.,1987), these studies suggest that at least under certain conditions calcium channel antagonists can prevent this type of neuronal damage. In contrast, when cerebellar slices from 7-day-old rats were incubated directly with NMDA (100 pM), the resulting acute swelling and subsequent persistent toxicity could not be eliminated with flunarizine, a diphenylpiperazine calcium channel antagonist (Lehman, 1987). However, the failure to protect from NMDA receptor-mediated insults can be a relative one, as demonstrated in the next section on cortical neurons.

B. Cortical and Hippocampal Neurons In cultures of rat embryonic hippocampus, Abele el al. (1990) reported that short exposures to potent, endogenous excitotoxins produced delayed-onset neuronal injury; this form of neurotoxicity could be blocked by 10 pA4 of the dihydropyridine nitrendipine as well as by NMDA receptor antagonists. Similarly, in cultures of fetal murine cortex, Weiss et al. ( 1990) found that exposure to NMDA for only a few minutes can induce delayed-onset neuronal degeneration. In contrast to hippocampal cultures, this lethal effect of potent NMDA agonists could

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not be blocked by calcium channel antagonists such as nifedipine. However, these workers did find that nifedipine could attenuate the delayed neurotoxicity produced by non-NMDA agonists or low levels of less potent NMDA agonists that require several hours of exposure to exert deleterious effects. The conclusion of this study was that voltage-gated calcium channels contributed substantially to the toxic effects of “slow” excitatory amino acid neurotoxicity in these cultures since nifedipine could prevent a lethal outcome. Nevertheless, the relatively high concentrations (micromolar) of nifedipine required to attenuate the neurotoxic effects raised the possibility that the calcium channel antagonist might exert its protective action at sites other than calcium channels (Weiss et al., 1990). In the rat hippocampal slice, nimodipine has been reported not to protect against anoxic damage (Kass et al., 1988). This poor physiological outcome with a calcium channel antagonist, following 5 to 10 minutes of anoxia, is in some ways analogous to the lack of effect observed by Weiss et al. (1990) in cortical cultures exposed to potent NMDA agonists for several minutes. On the other hand, one whole-animal study has shown that the destructive effects of the weak NMDA agonist quinolinate (30 pg) could be prevented by the phenylalkylamine type of calcium channel antagonist, verapamil (25 Kg), given concurrently with quinolinate by central cannulae injection. The diphenylpiperazine calcium channel antagonist cinnarizine (25 pg given orally for 8 days) was also effective (Kozlovskii et al., 1990). As discussed below, focal hypoxic/ischemic lesions are currently thought to be mediated by NMDA receptor activation. Therefore, further results of calcium channel antagonists in experiments performed in vivo with ischemia models are considered in Section VIII.

C. Retinal Ganglion Cell Neurons 1 . Glutamate Neurotoxicity Unlike fetal cortical neurons, postnatal rodent retinal ganglion cells in culture are relatively resistant to NMDA-induced death (Hahn et al., 1988; Levy and Lipton, 1990; Levy et al., 1990). Not only are several hours of exposure to NMDA required to produce neurotoxicity, but also the extracellular [Ca2 ] must be elevated or glucose must be depleted from the medium, possibly to enhance oxidative stress and disrupt calcium homeostasis. Hence, to some extent, NMDA receptor-mediated toxicity in retinal ganglion cells in vitro resembles the less potent or even the non-NMDA induced toxicity observed in cortical neurons with respect to length of exposure required for neuronal cell death. Moreover, patchclamp electrophysiology studies have revealed only very small NMDA-evoked currents in cultured retinal ganglion cells (Aizenman et al., 1988; Karschin et al., 1988). Under these conditions therefore it might be expected that the voltagegated calcium channels, in addition to the NMDA receptor-operated channels, could make a substantial contribution to increasing intracellular [Ca2+ ] and +

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Calcium Channel Antagonists

subsequent neuronal injury. In fact, neurotoxicity studies on cultured retinal ganglion cells have demonstrated that glutamate-induced toxicity can be completely prevented not only by NMDA antagonists (Hahn er al., 1988; Levy and Lipton, 1990; Levy et al., 1990) but also by calcium channel antagonists such as nimodipine or nifedipine (Offermann et al., 1990; Sucher et al., 1991).

2. HIV Coat Protein gp120 Induces Neurotoxicity Recently, it has been shown that the coat protein gp120 of the human immunodeficiency virus type I (HIV-1) produces a dramatic increase in intracellular [CaZ+]in both rodent retinal ganglion cells and hippocampal neurons with subsequent neuronal cell death (Brenneman er al., 1988; Dreyer et al., 1990). The rise in [Caz+liand the neurotoxic effect on retinal ganglion cells can be abrogated with dihydropyridine calcium channel antagonists such as nimodipine and nifedipine. The effective concentration of nimodipine was I 0 0 nM, but, accounting for protein binding of the drug because of the presence of serum in the culture medium, the free concentration of nimodipine was only about 4 nM. As predicted from binding studies, this level of dihydropyridine is in accordance with a specific inhibitory effect on voltage-dependent, L-type calcium channels. Interestingly, the increase in both intracellular [Ca2 ] and gpl20-induced neurotoxicity can also be blocked by NMDA antagonists. However, purified, recombinant preparations of gp 120do not directly activate NMDA-evoked currents in whole-cell patch-clamp recordings (Lipton et al., 1990, 1991). In addition, complete protection from gpl20-induced [Ca2 Ii changes and neurotoxicity of retinal ganglion cells is also afforded by incubation of the cultures with glutamate-pyruvate transaminase, an enzyme that breaks down endogenous glutamate as verified by HPLC analysis (Lipton et al,, 1990, 1991). Taken together, these results indicate that concurrent activation of NMDA receptors by endogenous glutamate is necessary for gp I20 neurotoxicity. Furthermore, since neither glutamate nor gp120 is deleterious on its own under these conditions, the findings suggest that they are synergistic effectors of neurotoxicity. Finally, in whole-cell patch-clamp recordings, glutamate- and NMDA-activated membrane currents are not affected by gp120. Thus, the synergism between gp120 and glutamate may possibly be mediated downstream to events at the neuronal cell membrane, perhaps at the level of intracellular [Ca2+ I . Other mechanisms, however, cannot yet be excluded, such as indirect effects of gp 120 on neurons mediated by macrophages or astrocytes. Preliminary data suggest that other classes of voltage-dependent calcium channel antagonists may also be effective in preventing gpl20-induced neurotoxicity, at least to some extent. At the maximal dose (1 to 100 fl)that did not cause neuronal damage by itself, a diphenylpiperazine derivative (flunarizine) was the most effective, a phenylalkylamine (verapamil) was possibly effective, while a benzothiazepine (diltiazem) was ineffectual in protecting rat retinal ganglion +

+

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Stuart A. Lipton

cells from gpl20-induced toxicity in vitro (Lipton, 1991). Unlike the nanomolar concentrations of dihydropyridines, micromolar amounts of these other classes of calcium channel antagonists are necessary for effectiveness. Micromolar levels are higher than predicted by binding studies but might be consistent with concentrations that block Ltype calcium channels; however, as discussed in a previous section, other mechanisms of action cannot be excluded. Another important point with respect to efficacy in preventing neurotoxicity is the dose-response curve of the calcium channel antagonists. In general for each calcium channel antagonist used in the retinal ganglion cell preparation, the dose-response curve can be described as an “inverted U.” Thus, too little drug is, not surprisingly, ineffective in producing protection, but also too high a concentration can be lethal by itself (>1 pkl in the case of nimodipine). Similarly shaped dose-response curves have been observed in some early human studies (see below). In summary, the finding that calcium channel antagonists are more effective in preventing NMDA receptor-mediated neurotoxicity in retinal ganglion cells and hippocampal neurons than in cortical neurons in culture may simply reflect the fact that intense stimulation of NMDA receptors in cortical neurons produces a more prominent influx of Ca2+ via receptor-operated channels. Thus, the relative distribution of the various channel types (NMDA receptor-operated channels versus voltage-gated calcium channels) and hence their contribution to intracellular [Ca2 1 may predict the effectiveness of specific channel antagonists in preventing neurotoxicity in a particular neuronal cell type. +

D. Possible Contribution of Intracellular Ca2 + Mobilization One question that naturally arises from these reports is why should either NMDA antagonists or calcium channel antagonists completely prevent the neurotoxicity of retinal ganglion cells in these cultures? Since the lethal effects of the HIV-1 coat protein gp120 appear to be dependent on concurrent activation of NMDA receptors, both gp120 and glutamate toxicity will be considered together in this vein. One answer to this question may lie in the following: the fact that calcium antagonists prevent the neurotoxic effects of glutamate or gp120 does not prove that the pathophysiology of these conditions is mediated solely by voltage-gated calcium channels. For example, consider the hypothesis: If neurons have a total cellular burden of [Ca2 J that they can tolerate before triggering delayed-onset cellular death, then any maneuver that lowers the total [Ca2+] may prove to be an effective protective agent. Thus, a drug may be neuroprotective even if this effect has no direct action on the mechanism causing the greatest increase in [Ca2+]. Moreover, there are many mechanisms for calcium homeostasis [for a review see Wong and Haley (1990, Fig. 1) or Hallenbeck and Dutka (1990, Fig. 2 ) ] .These include not only the ionic channels that allow Ca2 entry, but also Na -Ca2 exchange, intracellular sequestration of Ca2 , and others. +

+

+

+

+

Calcium Channel Antagonists

283

Along these lines, evidence has already been presented that both extracellular and intracellular Ca2+ stores are necessary for the increase in internal [Ca2+] and subsequent neurotoxicity caused by gpl20 in retinal ganglion cells (Dreyer et al., 1990). This finding raises the possibility that for neurotoxicity a small influx of extracellular Ca2+ triggers a more massive mobilization of intracellular Ca2 from internal stores, such as that observed previously in sympathetic neurons (Lipscombe et al., 1988). If this is indeed the case, then a reduced Ca2+ influx below some critical threshold, whether produced by NMDA antagonists or voltagegated calcium channel antagonists, might effectively prevent intracellular Ca2+ mobilization. In turn, this would prevent the ensuing dramatic and sustained rise in internal [Ca2+] and the subsequent death of the cell (Dreyer et al., 1990). Although this section is quite speculative, it is included because it represents an area of intense current research. The major point here is that the effectiveness of calcium channel antagonists in protecting neurons from lethal damage may not reflect the fact that the voltage-gated calcium channels are primarily involved in the toxic action. Stated another way, if calcium-associated injury is a final common pathway for the multiple etiologies of anoxia/ischemia, trauma, hypoglycemia, and several neurodegenerative diseases, then prevention from death may not point the way to the initial pathophysiology of each insult. +

VIII. In Vivo Animal Models of Ischemia and Effects of Calcium Channel Antagonists Although the in vitro studies suggest that under certain conditions calcium channel antagonists may offer protection to neurons from excitotoxic and related insults, it appears that the duration of the insults, the exact location, and the age or stage of development of the neurons may all be important parameters. It is not surprising therefore that in vivo experiments have yielded somewhat conflicting results with regard to neuronal protection by calcium channel antagonists. A compilation of representative works can be found in Table V (for recent reviews see also Grotta, 1987; Scriabine et al., 1989, 1991; Scriabine and van den Kerckhoff, 1988; Wong and Haley, 1990). Overall, many studies have shown that several calcium channel antagonists may be at least partially effective in protecting neurons from damage under certain conditions. Part of the confusion with regard to the effectiveness of calcium channel antagonists may relate to the consideration of global versus focal ischemia models. Overall, calcium channel antagonists appear to be more often effective in studies using focal rather than global ischemia models, similar to reports for NMDA antagonists (Albers er al., 1989). For example, four times as many experimental paradigms revealed beneficial rather than negative neurological outcomes in the

Table V Calcium Channel Antagonists in Animal Models of Cerebral Ischemia

g P

Drug. Nimodipine

SpecieslModelb

Nimodipine

Doglglobal: 4VO Doglglobal: 4VO

Nimodipine

Mkylglobal: 4VO

Nimodipine

Ratlglobal: 4VO

Nimodipine

Ratlglobal: 4VO + .U. BP

Verapamil Nicardipine

Ratlglobal: 4VO Ratlglobal: 4VO

Dosec

CBFd

Outcomee

3

6 pglkg, iv, after reperfusion

3

10 pglkg, iv, 2 rnin postischemia, then 1 pglkglmin X 2-3 hr or

fi

fiv

given preischemia 10 pglkg, iv, 5 min postischemia + Ipglmin, iv, x 10 hr

11.

0

5-20 pglkg, iv, given 3 min postresuscitation; then 1 pglkglmin X 2-6 hr 10 pg/kg, iv, 15 min prior to ischemia; then 10 rnin after ischemia I pglkglmin, iv X 2 hr 2 mg/kg, iv prior to ischemia 0.05 mglkglhr, sc x 96 hr

Reference Newberg et al. (1984) Steen et a/. (1984)

,I?.

Steen et al. (1985)

*

Vibulsresth er a / . (1987)

w

Lazarewicz et al. ( 1989)

* .Tr

Berger et a/. (1984) Grotta et al. (1986, 1988)

behavior but 3 histology Flunarizine

Dog/global: CA

Nimodipine

Catlglobal: CA

Nimodipine Flunarizine

Rat/global: CA Ratlglobal: 2VO + .U. BP

Emopamil

Ger/global: 2VO

0.1 mglkg, iv X 10 min at onset of resuscitation 10 pglkg, iv X 2 min, then 2 pglkg X 10 hr beginning 5 min postresuscitation 1-5 pg/kg/min, iv 40 mglkg, PO 4 and 24 hr prior to insult, or 0.1 mglkg, iv after insult, then 40 mglkgldose at 8 and 24 hr 31.6 mglkg, iv 30 min before insult

fi

fig

Kumar er a/. (1987)

3

Tateishi e t a [ . (1989)

3

Calk er af. (1990)

.Tr

Deshpande and Wieloch (1986)

.Tr

Szabo and Hoffman (1989)

Emopamil Emopamil KB-2796 > Flunarizine Nimodipine

6 mglkg, iv begun 5 min postischemia 2-6 mglkg, iv begun 5 min preischemia 10 mgikg, ip after resuscitation 3-10 mgikg, ip after resuscitation 0.3 rngikg, po I hr prior

fi

Szabo and Hoffman (1989) Bielenberg et al. (1989)

fih

fih

Yoshidomi et a/. (1989)

3

.Tr 3 fi

j

fi

Nuglisch et a/. (1990)

0.1 mg/kg, ip, or 0.01 mg/kg, ip each just prior or 10-30 min postocclusion

fi V

Fujisawa et a / . (1986)

fi

lzumiyama and Kogure (1988)

Nimodipine

Ratiglobal: 2VO + .U. BP Ratiglobal: 2VO + .U. BP Geriglobal: 2VO

Piperazinyl-ethanol derivative

Geriglobal: 2VO

30 mg/kg, ip immediately postischemia

Nilvadipine > Nimodipine Nicardipine

Geriglobal: 2VO

0.01-10 mgikg, sc, each drug 1 hr prior to insult, then X 4 days

Kuwaki et al. (1989)

Nicardipine > Flunarizine = Lidoflazine > Nimodipine

Geriglobal: 2VO

500 F g i k g , ip, each drug given 15 min prior to occlusion, then twice daily

Alps et a / . (1988)

Flunarizine Nimodipine

Geriglobal: 2VO

400 W k g , ip

Bunnel et a / . (1987)

Isradipine

Ratifocal: MCA

Nimodipine

% VI

Ratiglobal: 2VO + JJ BP Ratiglobal: 2VO + bd. BP Geriglobal: 2VO

0.1-1.0 mglkg,

PO,

prior to insult

Krieglstein et al. (1989)

400 W k g , ip each drug given 45 min postocclusion

0.3 X 3 mg/kg, sc administered daily

fi

fi

Sauter et a/. (1988) (continued)

Table V (Continued) h,

m

m

SpecieslModelb Isradipine > Nimodipine > Nitrendipine > Darodipine > Nicardipine

Ratifocd: MCA

Isradipine > Nicardipine

Ratlfocal: MCA

Nimodipine

Rat/focd: MCA

Nimodipine

Ratlfocal: MCA

Nimodipine

Rat/focal: MCA

Emopamil

Rat/focal: MCA

Nimodipine Lidoflazine Nicardipine

Rat/focal: ME

Nimodipine

Catlfocal: MCA

Dosec

CBFd

2.5 mg/kg,sc 5 mglkg, sc 10 mglkg, sc 10 mg/kg, sc 10 mg/kg, sc 0.3 mglkg x 3 per day, sc starting 45 min prior, or 15 min postinsult (both regimens effective) 1 pg/kg/min, iv x 30 min before and after occlusion

Outcomee

fi fi fi fi fi fi fi

Sauter e? al. (1989)

fif

Mohamed er a!. (1 985)

1 pglkglmin, iv begun 5 min

postocclusion. or prior to occlusion 2 pglkglmin, ivx 10 rnin at 1, 4, or 6 hr after occlusion (each regimen was effective) 10 mg/kg, ip before, during, or 1 hr after occlusion, followed by doses at 2.5 hr and twice daily for 2 days (each regimen was effective) 5-50 pglkg, iv 1.0 mglkg, iv 0.2-1 .O mg/kg, iv each drug given 5 min postinsult 5 pglkglmin, iv x 3 min, then 1 pglkglmin to begin 5 min after occlusion

Reference

Sauter and Rudin (1986)

Gotoh et al. (1986)

fi fi

German0 et al. (1987)

Nakayama et 01. (1988)

Lyden et a/.(1988)

fi

fi

Uematsu et al. (1989)

Nimodipine

Babifocal: MCA

PY 108-068 Flunarizine

Ratifocal: ME Dogifocal: ME

Flunarizine

Ratlfocal: CO + hypoxia YRatlfocal: CO + 2 hr hypoxia YRatifocal: CO + 2 hr hypoxia Ratifocal: MCA

Flunarizine Flunarizine KB-2796 (a piperazine derivative) KB-2796 > Nicardipine

Ratifocal: MCA

2 pglkglmin, iv X 50 min before and then 1 pglkglmin, iv X 96 hr after a 6-hr occlusion 300 pgikg, PO x 3 days 0.1 mg/kg, iv immediately after insult + 2 hr later 10-40 mgikg, PO pretreatment

30 mglkg, ip beginning immediately before hypoxia 15 mgikg, ip at onset and 2 hr later 10 mgikg, ip before or 1 hr after insult (each regimen effective) 10 mgikg, ip 1 mg/kg, ip each drug administered immediately after occlusion

fi'

Hadley et al. (1989)

Wiernsperger et a/. (1984) De Ley er a / . (1989) Van Reemps er al. (1983) Silverstein er al. (1986) Gunn et a/.(1989) Harada et a/.(1989) Shiino (1989)

Q>,=, had a significantly greater effect than the next listed drug, or equivalent effect, respectively.

@nly representative studies with some indication of the effect on neuronal survival have been included. Y, Young or immature animals; Bab, baboon; Mky, monkey; Ger, gerbil; 2V0, bilateral carotid artery occlusion; 4V0, bilateral carotid and vertebral artery occlusion (or complete cerebral ischemia, e.g., by aorta and vena cava ligation), MCA, middle cerebral artery occlusion; CO, carotid occlusion; ME, microembolization with microspheres or autologous clot; CA, cardiac arrest for 14 min. Gv, intravenous; sc, subcutaneous; PO, oral; ip, intraperitoneal administration of drug. dCBF (cerebral blood flow) during reperfusion in the treatment group compared to control animals that had undergone a similar ischemia paradigm. 3 ,CBF does not change; fi. CBF increased. Poutcome was assessed by morphological neuronal injury at least 24 hours after the insult, by hemisphere weight, or by neurological examination (behavioral). fAssessment of outcome in this case was histological but only 3 hours after occlusion. KMorphological assessment in this case was only at 8 hours and showed increased neuronal survival in hippocampus but not in cortex or cerebellum, 'CBF was increased in cortex, but only during the first 2 minutes of reperfusion, and was not increased at all in hippocampus; morphologically, the most neurons were spared in the hippocampus with the lowest dose of drug. "Zlinical neurological scores were improved but the histological area of infarction was not significantly smaller at days 7 and 14.

-

2

I.

288

Stuart A. Lipton

focal studies; but only about twice as many paradigms had successful rather than negative outcomes in the global models (see Table V). Reviews of ischemia studies using the phenylalkylamine (S)-emopamil (Defeudis, 1989) and the dihydropyridine nimodipine (Scriabine et al., 1989, 1991; Scriabine and van den Kerckhoff, 1988) have been most encouraging. In addition, as alluded to in previous sections, calcium channel antagonists affect not only neurons but also systemic factors such as blood pressure and cerebral blood flow. Since cerebral blood flow directly impinges on the wellbeing of neurons, these data are also included in Table V. However, increased blood flow can be a two-edged sword since it can paradoxically increase reperfusion injury, apparently by leading to an increase in intracellular calcium levels (for a review see Hallenbeck and Dutka, 1990). In some cases the beneficial effect of calcium channel antagonists on neuronal survival has been distinguished from an action on cerebral blood flow, e.g., in studies on nimodipine in which cerebral blood flow was not significantly affected but increased neuronal survival was observed (Nuglisch et al., 1990). Another important feature of some calcium channel antagonists is their voltage dependence of action. For example, the dihydropyridine class of drugs is much more effective in binding when the neuronal membrane is depolarized to activate or subsequently inactivate the channels (Bean, 1984; Kass and Sanguinetti, 1984). Thus, injured neurons, which become depolarized since they cannot maintain their normally negative resting membrane potential, are more susceptible to the action of dihydropyridines. Actually, this is exactly what one would want in a protective agent, i.e., to work differentially and selectively on the most vulnerable neurons. On the other hand, the early rise in intracellular [Ca2+] apparently triggers a delayed-onset neurotoxicity that may be hard to curb once set in motion. These mechanisms of action indicate that the timing of administration of calcium channel antagonists is crucial. Attempts at treatment too early or too late would produce ineffectual therapy. Finally, the effectiveness of the voltage-dependent calcium channel antagonists in protection from ischemia or excitotoxicity may vary with the region of the brain or the age of the animal since disparate populations of neurons are differentially susceptible to the actions of calcium channel antagonists. This topic was raised earlier (Section VII) in this review.

IX. Early Human Trials and Prospects for Calcium Channel Antagonists in Preventing Neurotoxicity

A. For Acute Ischemic (Nonhemorrhagic) Stroke Several small studies have suggested that calcium channel antagonists might be beneficial if given early enough in the course of events following an acute focal cerebral infarct. Two studies using nimodipine from the same group have been

Cokium Channel Antogonisk

289

encouraging (Gelmers, 1984; Gelmers et al., 1988). The first study suggested that oral nimodipine (120 mg orally in three divided doses) significantly improved neurological outcome (e.g., Matthew scale) in 60 patients enrolled in a single-blind, randomized trial. The second study reported a prospective, doubleblind, randomized, placebo-controlled trial of nimodipine (120 mg in four divided doses for 28 days), resulting in a 58% decrease in mortality and improved neurological outcome in patients with moderate-to-severe deficits at entry. The data of larger, multicenter studies using multiple doses of nimodipine are currently under analysis. Possible benefit may exist in a subgroup of patients treated within 12 hours of the onset of stroke with low-dose rather than high-dose drug (Mohr, 1991). The fact that a low rather than a high dose of nimodipine may be more effective may reflect an “inverted U” shape of the dose-response curve, similar to that observed in the in vitro experiments (Section VII,C,2, above; but see below for preliminary data in dementia studies). Nimodipine may prove to be a particularly good choice since it appears to have relatively few systemic side effects, including only minimal hypotension. The only FDA approved use of nimodipine at the present time is for improvement of neurological outcome following subarachnoid hemorrhage (Allen et al., 1983; Pickard et al., 1989; for review see Wong and Haley, 1990). In these cases, nimodipine appears to decrease vascular spasm and its known morbidity/ mortality but may also increase neuronal survival. Another trial for ischemic stroke used the calcium channel antagonist PY 108068 (Oczkowski et al., 1989). In this pilot study, 19 patients received oral PY 108-068 and experienced improved neurological outcomes compared to the control group. Recently, a multicenter, prospective, double-blind, randomized, placebo-controlled trial of PY 108-068 was completed and the data are being analyzed. Thus, no clear-cut evidence has yet been presented showing that calcium channel antagonists are an effective form of therapy for nonhemomhagic cerebral ischemia producing neurotoxicity in man, but further data are being gathered and analyzed in this regard.

B. Alzheimer’s Disease and Senile Dementia Neurons die in many neurodegenerative diseases, and, as suggested above, a rise in intracellular calcium triggering neurodestructive events may be a common final pathophysiology in cell death. Following this reasoning, binding sites for the dihydropyridine and phenylalkylamine type of calcium channel antagonists have been measured in normal and Alzheimer-diseased human brain. No striking differences were found (Quirion and Nair, 1989). This result may be of clinical interest since recent evidence has suggested that the dihydropyridine nimodipine can promote learning in aged rabbits (Deyo et al., 1989) and in man (Bono et al., 1985). For this reason, clinical trials of nimodipine are underway in patients suffering from dementia such as the type observed in Alzheimer’s disease. In this

290

Stuart A. Lipton

case, interim analysis may suggest some small benefit to patients on higher doses of nimodipine, but further patients must be studied to prove a substantial and statistical effect. This work is currently in progress, as are trials of calcium channel antagonists in other neurodegenerative diseases. Further studies will be necessary to determine if these drugs are of true benefit to patients with these maladies.

C. Calcium Channel Antagonists for the AIDS Dementia Complex? Approximately two-thirds of AIDS patients eventually suffer from a subcortical type of dementia that has been termed the AIDS dementia complex. The neurological syndrome comprises abnormalities in cognition, motor performance, and behavior (Price ef al., 1988). Gross neuropathological inspection of such brains shows mainly white matter (glial) injury. However, recent morphometric analysis of the frontal cortex of AIDS patients has in addition demonstrated an 18% loss of neuronal density and a 3 1% reduction of neuronal perikaryon volume (Ketzler et al., 1990). Given that few if any human neurons are infected by HIV- 1, it has remained a puzzle how neurons might be injured. As discussed above, however, recent in vitro results have suggested one pathogenesis that could be at least partly responsible for neuronal damage in AIDS involves neurotoxicity from the coat protein gp120 (Brenneman et al., 1988; Dreyer et al., 1990). Nevertheless, these results were obtained in rodent neurons in a tissue culture dish instead of in human neurons in the intact brain. Thus, these experiments must be interpreted with caution. The enticing possibility exists, however, that calcium channel antagonists might ameliorate gpl20-induced damage in AIDS patients since these drugs block neurotoxicity from the coat protein in the culture dish. In the absence of an adequate animal model of dementia and given the apparent safety of drugs such as nimodipine, a clinical trial for the AIDS dementia complex is currently being organized after lengthy discussions (Gibbons, 1990). Of course, it will be important to first prove in vitro that calcium channel antagonists such as nimodipine do not interfere with other therapies used in AIDS, such as zidovudine (ZDV, formerly called AZT).

X. Conclusions Tissue culture experiments on neurons from the mammalian brain suggest that under certain conditions calcium channel antagonists can be neuroprotective against excitotoxicity. The action of the calcium channel antagonists in these in vitro paradigms is probably mediated directly at the level of the neurons to improve intracellular calcium homeostasis. Although an interaction with Ltype calcium channels is probable, other mechanisms of action cannot be excluded in

Calcium Channel Antagonists

29 1

some cases such as fetal cortical neurons, especially given the relatively high (micromolar) levels of calcium channel antagonist that are required to ameliorate neurotoxicity. It is possible, however, that regional differences within the brain may account for differences in potency and efficacy of calcium channel antagonists. For example, in retinal ganglion cells low (nanomolar) concentrations of nimodipine are effective in preventing neurotoxicity from glutamate or the HIV coat protein gp120. This result suggests that, at least in this preparation, the calcium channel antagonist is most probably acting specifically on the Ltype of voltage-gated calcium channel. Animal studies also suggest that calcium channel antagonists can prevent excitotoxic neuronal damage, although the results have to some degree been variable among different laboratories. The site of action (e.g., systemic, vasculature, neurons) is often less clear in whole-animal studies than in tissue culture models, but the improved outcome suggests that CNS-permeable calcium channel antagonists may be neurologically beneficial in conditions such as focal ischemia. However, treatment must begin in a timely fashion relative to the insult. Recently, the AIDS viral coat protein has been suggested to act synergistically with excitotoxins (Lipton et al., 1991). This form of neuronal injury can be prevented in vitro by calcium channel antagonists, so clinical studies are being planned to test their use for AIDS-related dementia. Although not addressed here, calcium channel antagonists may also prove useful after traumatic injury, and this is an area of active investigation (Scriabine et a l ., 1991; Rich and Hollowell, 1990). In summary, calcium channel antagonists may prove to be at least partially useful neuroprotective agents for one or more neurological disorders mediated by excitotoxic mechanisms. However, further clinical studies will be necessary to determine if such a benefit can be realized in man.

Acknowledgments I wish to thank current and former members of my laboratory whose work made this review possible, including E. B. Dreyer. N. Sucher, P. K . Kaiser, D. Leifer, S . Lei, V. H . 4 . Chen, L. Wong, E. Aizenman, A. Karschin, M. P. Frosch, D. I . Levy, J . T. Offermann, M. Oyola, B . Cahoon, D. L. Tauck, K. Uchida, T. P. 0. Cheng, J. S. Hahn. P. G . Harcourt, M . Marceillo, M . P. Phillips, R. Campo, J. Arroyo, E. Friedman. K . Weber, K. Rothe, K . Upchurch, and J. Pelligrini. This work was supported in part by NIH grants EY05477, EY06087, NS00879, and NS07264, and by the American Heart Association, the Sunny von Bulow Coma and Head Trauma Research Foundation, and the American Foundation for AIDS Research.

References Aizenman. E.. Frosch. M. P.. and Lipton. S. A. (1988). Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells from rat. J . Physio[. (London) 396, 75-91. Albers, G . W., Goldberg, M . P., and Choi. D. W. (1989).N-Methyl-o-aspartate antagonists: ready for clinical trial in brain ischemia? Ann. Neurol. 25. 398-403.

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New Directions in the Delivery of Drugs and Other Substances to the Central Nervous System Yvette Madrid,* Laura Feigenbaum Langer,t Henry Brem,* and Robert Langer*pt *Department of Chemical Engineering Massachusetts Institute of Technology Cambridge, Massachusetts 02139 rHarvard-MIT Division of Health Sciences and Technology Massachusetts institute of Technology Cambridge, Massachusetts 02139 *Department of Neurosurgery Johns Hopkins University School of Medicine Baltimore, Maryland 21205

1. Introduction 11. Altering the Barrier 111. Altering Agents

A. Latentiation B. Carrier Systems 1V. Circumventing the Barrier A. Pumps B . Implantable Polymer Systems C. Implantation of Biological Tissues V. Conclusion References

1. Introduction The morphological and physiological features that restrict the transport of materials from the blood to the central nervous system (CNS) are known as the bloodbrain barrier (BBB). The barrier is useful in creating and maintaining the environment required by the brain by limiting the transport of certain substances and by promoting the transport of needed substances. Difficulties ensue, however, when the brain, due to illness, requires therapeutic substances which are not normally transported through the BBB. In this case, the BBB is self-defeating because it does not permit the uptake of the very substances the brain needs. There is a variety of disorders which afflict the CNS, including epilepsy, Advances zn Phurntacoloy,, Volurnr 22 Copyng,hi 0 1991 by Academic Press. Inc All righir of rcprcduclion in any form reserved

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Parkinson’s disease, Alzheimer’s disease, primary and metastatic brain tumors, some states of hormonal deficiencies, and viral infections. Occasionally, the therapeutically active agents necessary for the treatment of these diseases pass through the BBB; many times they do not. Herein lies the motivation for and the difficulty in finding ways to deliver these substances to the CNS. If one considers that the median age of the population in industrial countries is increasing and that many CNS disorders occur more frequently in those of advanced age, one begins to understand why it is that the treatment of CNS disorders is rapidly becoming a health-care priority. To understand the complexity of this problem better, it is necessary to comprehend something more of what the BBB comprises. The barrier which protects the central nervous system is not one single system but rather a combination of systems that acts to maintain the desired environment of the brain. A major contribution to this barrier can be found in the endothelial cells of the brain capillaries. They are morphologically different from those of other capillaries of the body because there are tight junctions between the cells which restrict the transport of many agents from the blood to the brain. Another part of the barrier consists of the circumferential junctions between the cells of the choroid epithelium in the lateral ventricle which prevent the free diffusion of substances from the blood to the cerebrospinal fluid (CSF). This fluid can be found in the ventricles within the brain and in the subarachnoid space which surrounds the outer surface of the brain. This is often referred to as the blood-CSF barrier. The term “blood-brain barrier” is often taken to refer to both barriers, although strictly speaking it is actually describing the bamer at the capillary endothelium. It is not conceptually incorrect to generalize them into one barrier because the extracellular space of the brain and the CSF are in equilibrium. For the purposes of simplicity, the term BBB will be now taken to refer to this more general form. Yet, the barrier, for all its protective functions, does not exclude all substances from entry into the CNS. Several mechanisms exist by which substances can enter the brain. Some substances enter by simple transport systems, mostly by diffusion through the cells (Fenstermacher, 1983). It is also thought that some vesicular transport introduces proteins and other substances to the CSF (Rapoport, 1982). Certain physiochemical features determine which substances pass into the brain by these mechanisms. Generally, lipophilic substances can penetrate the barrier more easily because these agents pass through the membranes of cells which consist mostly of lipids (Greig, 1989). An associated factor affecting entry into the CNS is the degree of ionization. Substances which are highly ionized generally do not pass through cellular membranes easily because they are repelled by or attracted to and bound by charged portions of the membranes. There

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is also a small pH difference between the blood (pH 7.4) and the CSF (pH 7.3) which favors the entry of slightly basic compounds over slightly acidic compounds because the former would be in a less ionized state in the blood (Baggot, 1974). The molecular size of the solute is another important characteristic; the larger the solute is, the more difficult it is for it to penetrate the barrier. Substances which bind significantly to proteins in the blood may have difficulty in entering the CNS because the bound agent is not considered available to distribute into the brain. Finally, the transport through the BBB is also regulated through physical features such as cerebral flow rate (Fenstermacher, 1983). There also exist active or facilitative mechanisms that transport substances into the CNS. such as D-glucose, the brain’s main source of energy. These substances do not diffuse in because they generally are not very lipophilic, yet because they are needed, special mechanisms exist to allow them to enter. A facilitative mechanism uses transporters, which are usually proteins within the membrane, to enhance diffusion. Active transport requires energy which may involve Na+ K pumps and is characterized by three steps. First, a solute binds to a carrier, then the solute and the camer move across the membrane, and finally, the solute is freed on the other side. These carrier-mediated systems are stereospecific, selfsaturated, and are competitively inhibited; they can be modeled by MichaelisMenten kinetics (Bodor and Brewster, 1983). Substances other than glucose which are known to be transported in this fashion are monocarboxylic acids, certain amino acids which the brain cannot produce, and amino acid precursors. The brain can also eliminate some of its waste products through carrier-mediated transport. To summarize, the BBB is characterized by a sheet of endothelial cells with tight junctions. There is generally a low permeability to hydrophilic compounds. Passive solute permeability occurs intercellularly and there exist carrier-mediated transport mechanisms for certain organic compounds. Until recently, the useful clinical approaches for improving the distribution of agents into the CNS involved changing the route of administration from oral to intravenous or administering larger doses of the agent (Greig, 1989). Obviously, these modes of treatment may be adequate for some agents but are less effective for those compounds which are restricted by the BBB. Currently, methods are available to address the problem of BBB penetration more directly by either altering the barrier itself, altering the agents, or circumventing the barrier altogether, In many cases, it is possible to enhance the penetration of necessary agents into the CNS but the cost, side effects, and other difficulties associated with these methods do not always make them worthwhile treatments. These methods are discussed in some detail here, in light of what has been accomplished to date, as well as where the technology might lead in the future. Furthermore, as we learn more about brain diseases themselves, and of the functioning of the CNS, it is likely that many therapeutic agents will require +

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special delivery methods. Neuronal peptides, for example, vasopressin, leutinizing hormone/releasing hormone, thyrotropic releasing hormone, and adrenocorticotrophic hormone (ACTH), play an integral role in the maintenance of the CNS function (Kreiger, 1983). The delivery of proteins and peptides can present special problems, not only because of stability or diffusion difficulties, but also because the maintenance of proper and consistent levels of these agents is critical. The trends of the future in terms of the delivery of therapeutic agents to the CNS will center on minimizing the problems associated with the methods currently available. Furthermore, new methods or modifications of the current methods will be needed to deliver potentially effective agents of the future. We therefore examine recently developed methods in view of their current effectiveness and future utility.

II. Altering the Barrier Because changing doses or routes of administration has not been effective in enhancing CNS delivery, new methods of delivery have been developed, some of which involve the manipulation of the tight junctions of the BBB so as to make the barrier more permeable to therapeutic agents. These techniques are invasive and may be subject to problems of control and selectivity, but nonetheless have proved useful in some cases. Some of the first attempts that were made in this area involved the use of chemical solvents; the most widely used were ethanol and dimethyl sulfoxide (DMSO). It was hoped that these agents would enhance the movement of hydrophilic drugs across the BBB. It was determined, however, that ethanol does not effectively open the barrier unless administered at toxic levels (Philips, 1981; Philips and Cragg, 1982). Initially, DMSO was reported to induce a reversible opening of the BBB (Broadwell et al., 1981), but further studies indicate that it has no beneficial effect in enhancing the penetration of a variety of proteins and anticancer agents; in fact, it has neurotoxic properties (Greig et al., 1985; Neuwelt et al., 1983). A more successful technique involves the intravenous administration of high doses of Metrazol, an analeptic agent. Two anticancer agents which do not normally pass the BBB, razoxane and melphalan, showed greater brain uptake in rats when administered with Metrazol than when administered without. Furthermore, the opening effect was shown to be reversible (Greig et al., 1984). Although it is not known exactly how Metrazol acts, it is thought to increase brain uptake through neuronal excitation, vasodilation, and increased blood flow (Greig and Hellmann, 1983). Another method which has met with some success is the hyperosmotic open-

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ing of the BBB. This method, as developed by Neuwelt, involves the infusion of a hyperosmotic sugar solution (mannitol or L-arabinose) into the carotid artery. Methotrexate, a water-soluble agent which is widely used to treat a variety of carcinomas, can be administered intraarterially as part of a chemotherapy regime just immediately following the disruption of the barrier induced by the sugar infusion. Following this osmotic treatment, methotrexate levels show a sevenfold increase over its levels in the brain following conventional intraarterial chemotherapy. Furthermore, the treatment is reversible within 4 hours, thereby limiting the uptake into the brain of unwanted substances and also “trapping” the methotrexate in the CNS. Morphological studies indicate that the increased permeability results from the widening of the endothelial cell tight junctions due to the osmotic shrinkage of the cells. The process is reversed when the cells are rehydrated (Greig et al., 1990; Neuwelt et al., 1984). To date, this method appears to have the most success in the treatment of brain lymphomas but in the future it might be used to treat other disorders. Many neurodegenerative disorders are caused by genetic enzyme deficiencies which might be effectively treated if the exogenous gene can be introduced into the CNS. Studies have been conducted which indicate that viral particles may be delivered across the BBB by use of osmotic disruption. Therefore, it is conceivable that viral vectors carrying the necessary genetic material might be used for the treatment of these neurodegenerative disorders if they can be effectively introduced into the CNS via osmotic disruptions of the BBB (Neuwelt et al., 1991). There are some complications associated with osmotic BBB modification, such as seizures and stroke as well as other nonneurological problems, but the incidence of these difficulties is low (Neuwelt and Dahlborg, 1989). Recent studies have shown that this method is more effective in extending the survival of patients with lymphoma than is the traditional cranial radiotherapy (Neuwelt et al., 1991). In addition, patients treated through osmotic BBB disruptions maintained or improved their neuropsychological functions and thereby avoided the known cognitive risks associated with radiotherapy (Crossen et al., 1991). This method of BBB disruption may not be appropriate for the delivery of proteins and other large molecules because the size of the opening between cells created by the osmotic effect is still not large enough to allow these highmolecular-weight compounds to pass (Segal and Zlovick, 1990). Nonetheless, this drawback can be viewed as an advantage in some cases. It might be used to lower systemic drug toxicities while simultaneously targeting drugs to the CNS. More specifically, it might be possible to administer to a patient antibodies to a therapeutic agent sometime after the agent is administered (with or even without osmotic manipulation), thereby reducing the concentration of unbound or active drug in the bloodstream. Since the antibodies cannot pass the BBB, however, the activity of the drug in the CNS would remain unaffected (Nazarro et al., 1991).

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Because it is desirable to have a controllable method of opening the BBB, attempts to regulate the opening and closing of the tight junctions by modulating certain molecules on the surface of endothelial cells have been made. Scientists at Athena Neurosciences in the San Francisco Bay area claim that in preliminary studies a method of BBB opening which employs this concept provides more control over the barrier opening than does infusion with hyperosmotic solutions. Furthermore, it is hoped that this technique can be used to introduce a variety of lower molecular weight therapeutic agents, such as opiates and anticancer drugs, into the CNS (L. Rubin, personal communication). Nonetheless, it is not likely to be useful in the delivery of proteins unless the gap between cell junctions can be enlarged. In a recent study, the BBB surrounding brain tumors has been opened without affecting the entire BBB following an intracarotid infusion of leukotriene C4, a naturally occurring fatty acid. Normally, an enzyme, glutamyl transpeptidase, is found in capillaries which inactivates leukotrienes, but this enzyme is not present in the brain-tumor barrier, thereby allowing leukotriene to open that barrier selectively (Black et al., 1990). This is a potentially effective way to deal with brain tumors because it can target drugs not just to the brain, but to the specific region within the brain where it is needed.

111. Altering Agents As an alternative to altering the barrier itself, one can change the agents or the way that these agents are “viewed” by the barrier. These methods tend to be less invasive and therefore are more useful for non-life-threatening disorders which require long-term therapy.

A. Latentiation Lipid-soluble compounds have a better chance of crossing the BBB. Therefore, if one encapsulated a water-soluble compound in a lipid sphere, thereby creating what are called liposomes, one could deliver these substances to the brain. Delivery of liposomes to brain tissue, however, has not been very successful because they selectively enter the liver and spleen and do not exhibit significant uptake in the brain (Patel, 1984; Segal and Zlovick, 1990). This was one attempt at latentiation; other ways exist to make a compound more lipophilic. It is recognized that both hydroxyl groups and amide linkages can decrease the lipophilicity of a molecule. Therefore, to create a useful agent with a potentially greater ability of passing the BBB, it is desirable to modify or replace these groups without reducing the therapeutic effectiveness of the drug. For example, one can couple the terminal carboxy and amino groups of peptides to, in effect, cyclize the molecule and make it more lipophilic. Pardridge has

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investigated the latentiation of small neuropeptides. He has chosen cyclosporin as a prototype; this is a very lipid-soluble peptide with 1 1 aliphatic amino acids. Nonetheless, the uptake of this agent by the brain is surprisingly slow. It is not clear why this is the case, but it is possible that the molecule undergoes some conformational changes in vivo, possibly as a result of protein binding in the blood (Pardridge, 1985). The method of cyclizing a protein is one of several methods aimed at creating a more BBB-permeable analog of a particular agent. In general, analogs are created from a parent compound by irreversibly altering one or more of the chemical groups so as to make the compound more lipophilic while still maintaining activity. Studies have been performed on a series of nitrosourea analogs and it has been shown that it is not desirable to make the compound too lipophilic because of the increase in protein binding which also ensues. This binding not only decreases the amount of free drug in the bloodstream but also may result in alkylation of the compound, thereby rendering it inactive (Hansch, 1969).The key to making the right analog lies in increasing the lipophilicity of the parent compound, but only to a certain level. This optimization may be very difficult to obtain. Heroin, which is formed by acetylation of morphine’s two hydroxyl groups, is two orders of magnitude more permeable through the BBB than is morphine. In the brain heroin is metabolized back to morphine (Pardridge, 1985).This method of delivery is called the prodrug approach. Prodrugs and analogs both involve the chemical conversion of agents to allow for their passage through the BBB, but prodrugs, unlike analogs, revert back to the parent compound. For CNS delivery, a prodrug should be cleaved within the brain, thereby leaving the active agent “trapped” in the brain. Ideally, one would want this rate of conversion back to the parent drug to occur rapidly enough to ensure effective drug concentrations at the site of action, but not so rapidly that the prodrug destabilizes before it reaches the site. Furthermore, the conversion from prodrug to parent compound should not release toxic moieties. The prodrug technique has been applied to delivery of several different kinds of agents, including antibiotics, hypnotics, and vitamins, to a variety of organs. Directing prodrug agents to the brain can be difficult because of enzyme catalysis in the blood and first-pass metabolism in the liver and is one of the disadvantages of this technique. Dibenzoyl-ADTN (2-amino-6,7-dihydroxytetrahydronaphthalene) is an example of a hydroxyl-linked prodrug which has had some success in entering the brain. ADTN is adopaminergic agonist which does not normally pass through the BBB; it has been shown that intraperitoneal administration of dibenzoyl-ADTN to rats can lead to significant and steady levels of ADTN in their brains (Horn et al., 1979). Not all prodrugs have been used successfully. Monobutyl methotrexate, which is a lipophilic, carboxyl-linked prodrug of methotrexate, is an example. This compound exhibited such high protein binding in vivo that a significant uptake of the drug to the CSF was not observed (Rosowsky rt al., 1982).

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y-Aminobutyric acid (GABA) is a neurotransmitter which may be useful in the treatment of Huntington’s disease and epilepsy but which does not pass well through the BBB. Some GABA prodrugs have been developed by acetylating the amino group. Use of these prodrugs resulted in a significant improvement of GABA brain concentrations in rats, but the results were not as successful in humans due to problems with the cleavage of the prodrug (Galzigna el al., 1978). Shashoua has created aliphatic and steroid esters of GABA which act as prodrugs. When tested in rodents, all the prodrugs entered the brain to a greater extent than did GABA alone, but not all of them showed effective neuropharmacological activity. Such activity is not only a function of the lipid solubility of the agent but also of the rate of hydrolysis of the GABA prodrugs and of the GABA receptor binding capabilities (Shashoua et al., 1984). Recently, a GABA prodrug has been created which readily enters the CNS following administration to mice. Once in the brain, it releases by hydrolysis both GABA and a GABA transaminase inhibitor. This prodrug introduced significant amounts of GABA to the brain tissue and, additionally, demonstrated effective neuropharmacological activity (Jacob et al., 1990). Over the years, GABA prodrugs have become both more sophisticated and more effective; their evolution demonstrates that not all prodrugs are initially useful, but that with improvements and modifications many can demonstrate therapeutic utility. Bodor has done some creative work in latentiations using prodrug type approaches called “redox chemical delivery systems.” Whereas a prodrug is converted back into its parent compound through a single enzymatic or chemical step, the redox carrier is converted back to the original therapeutic agent through a series of steps. N-methylpyridinium-2-carbaldoxime chloride (2-PAM) is a drug that is used in cases of organophosphate poisoning. It activates the enzyme acetylcholinesterase which is deactivated in these cases. This drug, unfortunately, is also a highly polar, quarternary ammonium salt which does not normally pass the BBB. Bodor has found that reducing 2-PAM to a tertiary dihydropyridine produces a tertiary amine which is more lipophilic. The amine is also, conveniently, readily converted back to the parent compound. The use of pr0-2PAM showed dramatically elevated 2-PAM levels in the brain tissues of mice, as compared to the brain tissue levels found following administration of only the parent compound. Despite this, however, it was shown that most of the pr0-2PAM actually went to the peripheral tissues (Bodor et al., 1975, 1978). This technique is the basis of other prodrugs created by Bodor. He has created a redox delivery system for dopamine involving a dihydropyridine carrier of dopamine. This carrier underwent several steps in vivo, including hydrolysis and oxidation, before it actually formed the precursor which was locked into the brain and released dopamine. The prodrug provided sustained and brain-specific release of dopamine when administered to rats (Bodor and Simpkins, 1983). In general, the latentiation methods are not useful for all compounds but can

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be effective in some cases. Unfortunately, liposomes, which could have been used as a general method to make hydrophilic compounds appear more lipophilic to the BBB, are not taken up well by the brain. Analogs prove to be difficult to make because they must not alter the drug activity and yet they must achieve an optimal lipophilicity. Prodrugs and redox carriers can be used to create more lipophilic derivatives and offer an advantage over analogs in that they have a greater chance of being trapped in the CNS due to the reversible nature of their bond. The key to prodrugs is that they must be made stable enough to exist as a complex when administered, but unstable enough to convert back to the parent compound. All of these methods, furthermore, suffer from an inability to target the agent exclusively to the CNS, resulting usually in a higher distribution of the agent throughout the body.

B. Carrier Systems All of the previous approaches have involved making the compound appear more lipophilic to the BBB. There are, however, other methods by which compounds may enter the CNS which do not involve passive diffusion; some can be transported via specific carrier systems. It is known that the CNS has specific mechanisms for the uptake of nutrients and certain amino acids. Certain drugs such as levodopa use these transport systems to enter the CNS. Levodopa is a precursor for dopamine, a neurotransmitter found in the brain but which cannot usually pass the BBB. In Parkinson’s disease there is a substantial loss of neurons containing dopamine in the substantia nigra. Therefore, levodopa is administered as a dopamine replacement therapy because it enters the CNS through active transport and is enzymatically cleaved in the brain to release dopamine. In f a g , levodopa is actually a prodrug for dopamine, but in this case, the prodrug is created to take advantage of active transport mechanisms in the brain rather than to increase passive diffusion. Pardridge has used this concept to develop a physiologically based method for the delivery of peptides to the brain called the “chimeric peptide” approach. A transportable protein, such as insulin or cationized albumin, is covalently bonded to a nontransportable protein and this bond is then cleaved enzymatically in the tissue. An example of this kind of a system is the P-endorphin/cationized albumin complex which has been shown to enhance the transport of p-endorphin in isolated bovine brain capillaries (Kumagai el al., 1987). An advantage of using a prodrug designed to function with an active transport system is that the compound may be targeted to the brain because the affinity of the brain transport system for the compound is likely to be higher than the affinities of other transport systems for the same compound elsewhere in the body. Therefore, this may become a useful method for delivering some peptides and proteins to the brain. Researchers at Alkermes (Cambridge, MA) are attempting to develop recep-

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Fig. 1 Chemical alterations of agents for CNS delivery.

tor-mediated drug carriers based on a similar concept: One attaches a drug of interest to a carrier which normally binds to the endothelial cell surface and is transported across the barrier. It is expected that the carrier conjugate will also pass through the barrier and thereafter release the drug into the CNS (S. Davidson, personal communication). The active carrier methods help in part to resolve the issue of targeting drugs to the CNS, and they may be useful for the delivery of peptides and proteins. However, this approach may raise other problems because these carriers compete for sites with naturally occurring substances, and therefore, there is the risk that they may saturate these sites. Furthermore, active carrier prodrugs face the same stability/destabilization problems as latentiated prodrugs and their toxicity has yet to be tested. Figure 1 summarizes the methods of CNS delivery that involve chemical alteration of therapeutic agents.

IV. Circumventing the Barrier Other methods also exist for introducing agents into the CNS. These methods are used when systemic delivery of a therapeutic agent by one of the other means

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cannot be achieved efficiently, but the need to introduce the agent is great. These methods, because of their invasiveness, are not indicated for improving the delivery of agents that normally penetrate the BBB or for which less heroic delivery methods exist. On the other hand, there are some advantages to circumventing the barrier entirely: There are fewer side effects, because the drug is targeted to where it is needed, and protein-binding effects become less of a problem. Traditionally, circumventing the barrier has meant injecting or infusing an agent into the CNS. There are several places where this can be done. One can superficially apply a chemical to the cortical surface of the brain by placing drugsoaked filter paper on the surface or by intracisternal injections into the CSF. One can also direct the agent into the lateral, third, or fourth ventricles of the brain by injection or chronic infusion. If an agent difTuses very slowly, however, it may take a significant time for it to reach the desired site and it may never reach therapeutic concentrations. Crystalline substances or small volumes of solution can also be deposited or injected directly into the brain tissue. These methods offer the advantage of placing an agent directly where it is wanted but they are invasive and are not always effective; they are warranted only in specific situations. However, advances in the development of pumps, polymers, and the use of implanted biological tissues to deliver drugs may make these methods more powerful therapeutic tools.

A. Pumps To minimize risks and to maximize the quality of life for the patients involved, all the devices for the long-term delivery of therapeutic substances to the CNS are fully implantable. In the early 1960s a reservoir device called the Ommaya reservoir was developed to allow access to the CSF. The device consists of a silastic catheter connected to a depressible capsule. The capsule is placed subcutaneously under the scalp and it allows repeated punctures so that a drug can be injected into the capsule. By manual compression the drug is sent to the site at the end of the catheter, usually one of the ventricles of the brain. Different variations of this device are available and are used in therapy today. These are simple devices with relatively low cost, but they do not provide accurate control over the dosage administered and do not have a bacteriostatic filter (Ommaya, 1984). Other types of manual pumps, with similar limitations, also exist. Other than the manual pump, there are two other types of pump devices which are used for CNS drug delivery. These are the vapor-pressure-powered and the electronically controlled pumps. The vapor-pressure-powered pump (Fig. 2), manufactured by Infusaid, is an implantable device that was developed by Blackshear and others (Blackshear el

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al., 1972). It is disk-shaped and contains two chambers which are separated by a flexible wall. One of the chambers contains a fluorocarbon propellant which normally exists as a vapor at room temperature and which is compressed when the other chamber containing the drug is filled. The vapor pressure of the propellant in one chamber then slowly forces the drug out of the other chamber at a constant rate. The drug solution moves through a bacteriostatic filter, through a catheter, and is fed to wherever the tip of the catheter is placed. When the drug solution is refilled, the pump is recharged. This is the most commonly used device for CNS studies (Rohde et al., 1988).

Fig. 2 (A) An lnfusaid model 400 pump. (Photograph courtesy of Infusaid, Inc.) (B) Crosssection of an Infusaid model 400 pump. (Courtesy of Infusaid, Inc.)

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Fig. 2 (Continued)

Infusaid also manufactures a programmable pump which is based on the vapor-pressure technology but which contains an electronically controlled accumulator valve. Therefore, this pump can be used to deliver therapeutic agents by constant flow, periodic flow, or multiple flow rates. There are also two other types of electronically controlled pumps. The peristaltic pump functions through the action of rollers, which are rotated on a motor. These rollers then press against a flexible tube which is housed in a Ushaped chamber, and this causes the fluid in the tubing to move out. These pumps are powered by a battery (life span about 5 years), are programmed externally to provide complicated input profiles, and contain monitoring devices and alarm systems. Peristaltic pumps have been developed by groups at Sandia Laboratories (New Mexico), by Siemens AG (Germany), and by Medtronic Inc. (Minnesota) (Rohde et d . , 1988). They differ in size and programmable drug delivery capabilities. Solenoid pumps have been developed by Fischell and colleagues (Rhode et al.. 1988). These pumps are also battery operated and are programmable. They deliver the drug in small pulses by the filling and discharging action of a solenoid pushing against a flexible chamber. Catheters with check valves are connected to the chamber to allow for loading and unloading of the chamber. Programming of this unit can even be done by telephone. Unfortunately, the electronic pumps are significantly more costly than other pump systems.

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Pumps have been used to deliver both heparin and insulin systemically (Rhode et al., 1977; Spencer, 1978). The delivery of insulin was particularly difficult because of the accumulation of denatured protein which resulted in the contamination of the interior of the reservoir and of the connecting catheter (Rhode et al., 1988). Therefore, it appears that the use of these pumps for the delivery of other proteins to the CNS may pose similar problems. More recently, pumps have been used for the intraspinal administration of morphine to patients with chronic pain. Therapeutic effects were achieved for patients with chronic malignancies but the usefulness of these devices for patients with chronic nonmalignant pain was not demonstrated, perhaps due to the development of tolerance for the drug (Coombs et al., 1983). The amount of literature on this subject is quite extensive and indicates that, for certain applications, drug administration intraspinally via pumps can be quite useful (Brazenor, 1987; Penn et al., 1984; Penn and Paice, 1987). Pumps have also been used for the administration of baclofen in patients with spasticity. Unfortunately, the oral delivery of this agent frequently produces drowsiness and confusion and can result in memory loss. To reduce these undesirable side effects, the vapor-pressure-powered pump has been used to deliver baclofen intraspinally and it has been demonstrated that spasticity could be controlled (Muller et al., 1987; Penn and Kroin, 1985). Another drug which has been administered by a constant infusion pump to the CNS is bethanechol, an acetylcholine agonist which is also resistant to acetylcholinesterase. It is used in the treatment of Alzheimer’s disease. In this disease, deficits in learning and memory tasks are believed to result from inadequate cortical levels of acetylcholine. It was hoped that replacement therapy involving the introduction of an acetylcholine agonist to the CNS would alleviate some of the symptoms of this disease. In an initial study, subjective but not objective improvement in patients was observed (Harbaugh, 1986). A subsequent multicenter double-blind study indicated a small improvement in patients, but it was determined that the improvement was not great enough to warrant its continued use (Harbaugh, 1987). Implantable pumps have also been used to deliver methotrexate and other chemotherapeutic agents directly to the brain. Studies with methotrexate indicate high concentrations of the drug were achieved in the tumor as well as in the whole brain, relative to those concentrations which were obtainable following systemic delivery alone. Despite this, large decreases in tumor size were not observed (Harbaugh et al., 1988a). Nonetheless, further studies involving the delivery of chemotherapeutic agents via this route seem warranted (Harbaugh et a l . , 1988b). Pumps show potential for the delivery of certain key substances, including neurotransmitters, hormones, and anticancer agents, to the CNS . The advantage of using pumps is that complex and prolonged delivery schemes can be created as needed and that the drug is targeted to the site where it is needed. The concerns

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with this method include pump failures, kinks, surgical risks, and high cost. Furthermore, not all drugs can be delivered by this route. The agents must be stable at physiological temperatures and for the length of time they will remain in the reservoir. Furthermore, they must be compatible with pump materials and must be soluble enough in water to achieve reasonably high concentrations. For example, 5-fluorouracil is a common anti-cancer agent, but it is not usually used in pumps because its low solubility would involve refilling the pump too frequently to be practical. Pumps are not generally an ideal method for the delivery of proteins due to problems with denaturation and aggregation which lead to contamination of the devices. There is, furthermore, the concern that the diffusion of proteins into the brain tissue would not be great enough to produce the necessary therapeutic effect if they are delivered into the CSF.

B. Implantable Polymer Systems Sustained release systems are aimed at slowing the delivery of drugs so that the therapeutic effects can be maintained over longer times. Controlled release formulations, which usually involve embedding a drug in a polymer, can provide sustained release, but more importantly they provide a controllable, predictable, release pattern as well as a way of protecting the drug from the physiological environment until it is needed. Diffusion is the most common method of release, but other methods, such as release due to the chemical degradation of the polymer from which the device is made, are also possible (Langer, 1990). In recent years, polymer systems have begun to be investigated for drug delivery to the brain (Tamargo et a l . , 1989). In one study, dopamine, which may be useful in the treatment of Parkinson’s disease, was embedded in a matrix made from an ethylene vinyl acetate (EVA) copolymer. The system was designed to allow for the constant release of dopamine for several months. Following implantation into rat brains, high brain tissue concentrations of dopamine were achieved relative to the concentrations that were obtained following no implantation or the implantation of non-drug-loaded devices. Release was stabilized on the twentieth day and was maintained stable until the end of the experiment at day 65 (Freese et al., 1989; During et al., 1989). These studies suggest that controlled dopamine release from polymer devices could be useful when less invasive methods, such as oral administration of levodopa, are no longer adequate. Ethylene vinyl acetate polymers have also been used to obtain sustained release within the brain of dexamethasone, a corticosteroid commonly used in the treatment of cerebral edema. This agent is normally administered systemically but because its movement into the CNS is restricted by the BBB, high doses are required to achieve therapeutic CNS levels. Subsequently, undesirable systemic side effects such as skin atrophy, osteoporosis, diabetes, and psychosis may develop. EVA-dexamethasone devices were created by incorporating the drug

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into the EVA matrices through solvent evaporation. Studies involving the implantation of these devices into rat brains indicated that biologically active dexamethasone was released into the brains, that peritumoral brain edema was effectively treated, and that lower plasma concentrations were observed (compared to those obtained following systemic administration) when these controlled release polymers were used for interstitial drug delivery (Reinhard et al., 1991; Tamargo et al., 1991). Polymers may also be useful in the treatment of Alzheimer’s disease. For this application, microspheres of a copolymer were impregnated with bethanechol, to be used as acetylcholine replacement therapy, and were implanted into rat brains. Polyanhydride polymers were used: They are biodegradable and exhibit surface rather than bulk erosion because hydrolytic degradation of the surface bonds occurs more quickly than the rate of water penetration into the polymer matrix. The microspheres (3-5 pm) were a 50 : 50 formulation of a copolymer of 1,3bis( p-carboxyphenoxy) propane : sebacic acid anhydride and provided a nearly linear release of the active agent for up to 20 weeks. These microspheres were formed by placing the drug and the polymer solution into an organic solvent and dispersing this in silicone oil, thereby creating the spheres. Subsequently, they are solidified and removed (Howard et al., 1989) and implanted in rats with lesion-induced memory deficits. Lesions in the bilateral fimbia-fornix had been previously created in these rats, resulting in the cholinergic denervation of the hippocampus and a loss of spatial memory. The microcapsule implants were placed in the hippocampus and were well tolerated by the rats. The rats were divided into groups, all with lesions; one group was implanted with microcapsules impregnated with bethanechol, another with microcapsules without bethanechol, and the third with no implants whatsoever. The rats were tested for spatial memory using an eight-arm radial maze. Although there were individual differences, the group of rats treated with the drug-impregnated polymer implants showed a significant improvement over the other two groups. For the duration of the experiment, beneficial effects in spatial memory were observed. It was concluded that this therapy successfully improved the spatial memory of rats with lesion-induced cholinergic denervation of the hippocampus and might be of therapeutic value in Alzheimer’s disease (Howard et al., 1989). Polyanhydride polymers have recently been used clinically to treat brain tumors. N,N-Bis(2-chloroethyl)-N-nitrosourea(BCNU), also known as carmustine, is commonly used to treat malignant primary brain tumors. Unfortunately, it has a half-life of 12 minutes in vivo and provokes very undesirable side effects when it is delivered systemically. A method of releasing BCNU locally and directly where it is needed is clearly called for. For this application, the BCNU was incorporated into polyanhydride disks by either pressing weighed aliquots of dry powdered BCNU with dry powdered polymer or by codissolving

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the drug and the polymer in methylene chloride, evaporating the solvent, and then pressing the mixture (Fig. 3). Initially, BCNU/polyanhydride disks (prepared by both methods) were implanted into rabbit brains. At various times following the implantation the animals were sacrificed and the concentration of BCNU in the tissue was determined by quantitative autoradiography. In animals implanted with the polyanhydride disks, significant quantities of BCNU were found even after several weeks, whereas animals that received the same total dose as a bolus injection had almost no BCNU in their brains after the third day. Devices that were formed by codissolving the drug and polymer and evaporating the solvent yielded release times that lasted nearly twice as long as disks formed by the other method both in vivo and in vitro (Grossman et a / ., 1988). Four sets of safety and preclinical studies showed the polymer to be safe and effective in animals ranging from rats to monkeys (Brem et al., 1989). Studies were then performed with patients with glioblastoma who were undergoing reoperation for the removal of the tumor. After removal, the cavity was lined with BCNU/polyanhydride disks so that the BCNU would be released directly onto any remaining malignant cells. The safety of the polyanhydride device in humans has been demonstrated. In addition, the toxic side effects of BCNU usually observed in patients treated systemically have been avoided. This method ap-

Fig. 3

BCNU/Polyanhydride devices for cerebral implantation

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pears to be an effective way of delivering chemotherapy and has the advantage of being localized where it is most needed (Brem, 1991; Brem er al., 1991; Chasin er al., 1990). Phase 111 clinical trials examining this system in over 32 hospitals in the United States and Canada and involving approximately 200 patients are currently under way. Controlled release devices for the delivery of drugs to the CNS are beginning to emerge as a promising technology. Perhaps their greatest advantage is their versatility. They can be used to deliver a variety of agents, including anticancer drugs, neurotransmitters, and proteins, and, potentially, several agents could be released simultaneously. The release time can last from a few days to several years, depending on the chemistry of the polymer. Release rates can be controlled, very often by modifying the shape of the device or by changing the polymer chemistry slightly (Pitt, 1990). Furthermore, with the use of ultrasound or by applying magnetic forces to magnetic beads implanted into some devices, periodic release of drugs can be obtained (Edelman etal., 1985; Kost eraf., 1989). As with pumps, the delivery of the drug can be localized, but the invasiveness of this drug delivery method limits its applicability to cases of severe illness.

C. Implantation of Biological Tissues Another potential method of drug delivery involves the use of cell transplantation techniques. A great amount of research in the past two decades has focused on the concept that viable neural tissue could be transplanted to damaged or diseased areas within the brain in order to reestablish some normal connections or to release neural substances or trophic factors that might prove beneficial in treating a disease. The most extensive work in this area has focused on a treatment for Parkinson’s disease, due mostly to the fact that the progressive degeneration of dopamine-containing neurons of the substantia nigra is largely responsible for the development of the debilitating symptoms of Parkinson’s disease and the fact that embryonic dopamine-containing neurons show excellent survival and growth characteristics following transplantation in animals (Bjorklund and Stenevi, 1979; Sladek and Gash, 1988). In studies of animal models of Parkinson’s disease, neural tissue from fetal substantia nigra is transplanted into the striaturn where the dopamine neurons normally contact. Several studies have shown that fetal nigral neurons are able to reestablish contacts within the striatum (Bjorklund and Stenevi, 1985; Bolam et al., 1987). In some behavioral studies of animal models of Parkinson’s disease, significant behavioral improvements have been seen (Bjorklund et al., 1980; Sladek and Gash, 1984). For example, Sladek and colleagues have shown substantial functional recovery in monkeys with experimental PD induced by the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) after receiv-

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ing bilateral grafts of tissue from fetal substantia nigra (Sladek et al., 1986). Even the most severely affected animals showed a reversal of such symptoms as tremor and rigidity. Further study has shown that their improvements coincided with successful graft survival, the ability of the transplanted neurons to grow into the host striatum, and the subsequent elevation of dopamine levels in striatal regions even 7 months later (Sladek and Shoulson, 1988). Clinical trials in Parkinsonian patients are now underway. However, placebo effects, adjustments of medication prior to and after surgery, and even stimulatory effects of the grafting procedure itself can contribute to changes in Parkinsonian symptoms. Until more is understood about the growth characteristics of human neural transplants and even potential practical and immunological problems, clinicians are cautious about drawing strong conclusions about the success or failure of neural tissue grafting in humans (Backlund et al., 1985; W. Freed et a/., 1985, 1990; C. Freed et a/., 1989; Madrazo et al., 1987; Hitchcock et al., 1989; Lindvall et al., 1989; Sladek and Shoulson, 1988). However, the dramatic success of fetal cell grafting in animal models indicates clearly that repairing a diseased brain with transplanted tissue could lead to a practical and successful treatment regimen. The key to this type of therapy, however, will be in using (or creating) cells that survive transplantation, secrete the necessary neurotransmitters or neurotrophic factors, and that will, perhaps, even be able to establish new contacts with the appropriate target tissue. New studies conducted on growing distinct cell types in culture for later use in transplantation may be able to circumvent some of the problems related to the source of transplant material. Many cultured human neuroblastoma (tumorogenic) cell lines have been studied and these cells can synthesize a variety of neurotransmitters, and, when transplanted, do not appear to exhibit any type of neoplastic growth (Bottenstein, 1981; Gupta et al., 1985; Gash et al., 1986). Recently, Snyder and colleagues have even cultured a nontumorogenic, human neuronal cell line capable of displaying mature neuronal morphology and which also appears to contain a variety of neurotransmitters and other neuron-specific markers (Ronnett et a / . , 1990). In addition to using cells which may release neurotransmitters, implanting cells which have a neurotrophic effect on other cells may also play a greater role in transplant therapy. For example, cografts of fetal nigral tissue and fetal striatal tissue have been placed in rat striatum, and it appears that the embryonic striatal cells may stimulate the development of the fetal nigral cells (Yurek et al., 1990). These and other studies (DeBeaurepaire and Freed, 1987) indicate that trophic cografts may not only stimulate development but may help to maintain the synthesis of neurotransmitters and even enhance fiber outgrowth of the fetal nigral transplant. The use of multiple-polymer drug delivery systems may also be employed to enhance the survival and growth of transplants. For example, one could implant

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polymer matrices that slowly release growth factors or other substances in combination with neural cells to improve the viability or efficacy of the neural transplant. Similarly, if the polymers incorporated biosensors or other methods to release substances “on demand” or in a given temporal sequence, they could then affect specific aspects of the transplants’ growth or release. For example, one could design a polymer delivery system to release factors that might enhance the elongation of transplanted neurons and would then subsequently release another factor more effective in enhancing the establishment of contacts with other neurons. Some interesting approaches for delivering agents to the brain also involve novel transplantation techniques. For example, Aebischer and colleagues recently encapsulated neural cells in polymer fibers and placed them in the brain. They used a cross-species procedure of implanting embryonic cells from mouse mesencephalon (the region containing and surrounding the substantia nigra) into the brains of rats and monkeys. They reported that the permselective polymer membrane allowed for the diffusion of nutrients and metabolites without immunorejection of the neural transplant. The transplant survived for at least 12 weeks. In this case, implanting neurotransmitter-secreting cells that have been encapsulated inside a polymer into the brain created a long-term, viable source of neurotransmitter, and at the same time eliminated the problem of immune rejection (Aebischer et al., 1988). Finally, one might be able to insert vectors and genes into cells that could be transplanted directly into the brain and use this as a method for developing new types of transplantable cells that could secrete desired agents. For example, Gage and collaborators have infected rat fibroblasts with a retroviral vector carrying the gene for tyrosine hydroxylase (TH) [an enzyme that converts tyrosine to dihydroxyphenylalanine (DOPA) in neurons synthesizing catecholamines such as dopamine] and transplanted these cells into the brains of rats possessing unilateral 6-hydroxydopamine lesions, a common model of Parkinson’s disease. After transplantation, the genetically altered fibroblasts produce DOPA which, in turn, reduces behavioral abnormalities (Wolff er al., 1988). It may be possible to use similar methods involving transfecting specific brain cells with vectors in vivo to impart new functions to these cells, as Mulligan and co-workers have done in certain animal models for liver cells (Wilson et al., 1988). Although there have been significant advances in neural transplantation in the past 20 years, the use of transplanted cells as a treatment for certain brain diseases is still at a very early stage. Compared to more conventional drug delivery techniques, the eventual use of such technology will certainly depend on developing the ability to control both cellular growth and the release of putative therapeutic substances carefully. However, due to the seriously debilitating effects of brain disease, transplantation technology either alone or in combination with other drug delivery techniques will continue to provide hope for a viable

Table l Partial Summary of Methods for the Delivery of Agents Across the Blood-Brain Barrier ~

Aller bamer Hypermmotic opening

Latentiated prodrug*

Invasive

Administration lnvasiveness Maintenance of therapeutic levels o f drug over time

Nut suitable for long-term

Effectiveness Desirable neurophmacological effects

Yes. with anticancer aeenls

Targeted to CNS

Trapped in CNS Agents

Problems

therapy

To some extent. because

~

Alter agents

Ttisue Carrien

Pump\

Polymers

uansplanlauon

Relatively noninvasive Possible to obmn s u s ~ mined release

Relatively noninvasive Possible. hut may require frequent administration

Very invasive

Very invasive Easy: controlled release

Very invasive Possible; cell viability needs to be maintained

Yes. in some cases

Yes. in some cases

Yes. in many cases

Yes. in many c a m

Possible

Yes

Yes

Yer. in specific studies only Yes

Yes. if it is cleaved within CNS Protems. neurotransmitten

Yes. if agent normally does not pass BBB Agents which satisfy solubility and stability

Yes. i i agent normally does not pass BBB Anricancer. neurutranrmitters. proteins

Yes. if agent normally does not pass BBB Naturally occurring neurochemicals only

Cornpound instability: competition fur sites

criteria Surgical risks: pump failures: battery replacement

Surgical risks

Surgical risks: cell viability: immunological issues

carotid a e r y supplies

Poscihle: more frequently exhibit \ys-

blood to brain Yes, because vpening IS reversible Anticancer and other lowM W agents; not proteins

ternic dirtrihution Yes, if i t i s cleaved within CNS Opiates. anticancer. neurochemicals: not

Risk o f introducing un-

t e s t for proteins Compound instability:

wanted agents

Circumvent bamcr

toxic metabolites

Easy. especially with programmable modcls

pasrible

4Jnavoidably, this table presents an oversimplified discussion of the technology available for CNS drug delivery. It gives an overview of some of the available methods but it is not intended to provide a comprehensive evaluation of them.

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neurotransmitter replacement therapy. It may even be possible to use this technology to enhance the viability of otherwise damaged neurons and thereby ultimately obtain functional recovery.

V. Conclusion The development of techniques aimed at the successful delivery of drugs to the CNS has most recently focused directly on the problem of penetrating the BBB. Three main approaches have been used: modifying the barrier, modifying the agents, and circumventing the barrier. Ideally, the best method for CNS delivery should be noninvasive, and yet allow for the drug to be localized to specific parts of the brain. It should be applicable for a broad range of compounds, and it should also be appropriate for both long-term and short-term use. Obviously, no one form of therapy existing today satisfies all these requirements. Table I contains a partial summary of the technologies available and some of their advantages and disadvantages. In the next few decades there will be an increased need for the effective delivery of therapeutic agents to the CNS. It is the goal of research in this area to meet these intensifying demands by improving the present technology, combining current drug delivery methods, or developing altogether new methods.

Acknowledgments The authors express their appreciation to Dr. Edward Neuwelt for helpful discussions concerning BBB manipulation and to Dr. James Johnston for kindly providing photographs for this manuscript.

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Hormonal Regulation of Cytochrome P-450 Gene Expression Johan Lund, Peter G. Zaphiropoulos, Agneta Mode, Margaret Warner, and Jan-Ake Gustafsson Department of Medical Nutrition Karolinska Institutet Huddinge University Hospital S-141 86 Huddinge, Sweden

1. General Introduction 11. Cytochrome P-450 Nomenclature

and Gene Structure 111. Transcriptional Regulation of Cytochrome P-450 Genes: Involvement of &-Acting DNA Elements IV. Hormonal Regulation of Cytochrome P-450 Involved in the Biosynthesis of Hormones A. Cytochrome P-450 in the Adrenals and Gonads B. Regulation of Renal Mitochondria1 Vitamin D, la-Hydroxylase V. Regulation of Liver Cytochrome P-450 by Sex Steroids and Growth Hormone VI. Regulation of Cytochrome P-450 in the Prostate, Pituitary, and Brain VII. General Conclusions VIII. Future Perspectives-Novel Endocrine Systems? References

1. General Introduction From a pharmacological perspective, the most significant aspect of the cytochrome P-450 (abbreviated to P-450) family of proteins is its role in the hepatic metabolism of therapeutic agents. These enzymes, because they activate, inactivate, and facilitate the excretion of many drugs, have a major influence on the duration of action as well as on the toxicity of therapeutic agents. In addition, the level of specific forms of P-450 can be induced or diminished by drugs. A knowledge of the forms of P-450, their substrate specificities, and their regulation is, therefore, important for the effective therapeutic use of drugs and for understanding and perhaps predicting drug interactions. In this article, we discuss another aspect of P-450 function and regulation which should also be of pharmacological relevance, namely, the endocrine regulation of P-450. Advuncer in Pharmucoliw, Volvme 22 Copyright 0 1991 by Academic Press. Inc All rights of reproduction in any form reserved

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The expression of P-450 genes in the liver, lung, kidney, adrenals, gonads, prostate, and brain is regulated by hormones. Although the contribution of extrahepatic P-450 to the overall metabolism of drugs in the body is probably low, the in situ formation of metabolites in specific tissues may influence target organ sensitivity, responsiveness, and toxicity. This fact in itself motivates studies to characterize the forms of P-450 in these tissues and to understand how their levels change under physiological conditions. Equally interesting are the mechanisms involved in these hormonal regulations. Not only will such studies shed light on mechanisms of hormone action but, as the individual steps in the signal transduction pathways are elucidated, new target sites for pharmacological intervention may be discovered. If one considers the forms of P-450 in the various tissues of the body, a major tissue-specific influence of hormones on P-450 is apparent. In the adrenals, gonads, and renal mitochondria, which are the sites of synthesis of the glucocorticoids, mineralocorticoids, sex hormones, and 1cw,25-dihydroxyvitamin D,, there is strict regulation by the peptide hormones adrenocorticotropic hormone, luteinizing hormone, follicle-stimulating hormone, and parathyroid hormone (ACTH, LH, FSH, and PTH, respectively). In the prostate, a sex accessory tissue, both the organ and its P-450 content are dependent on androgens. The overall levels of P-450 in the brain and pituitary are increased in pregnancy, during lactation, and by dihydrotesterone. Some lung P-450 forms are induced by progesterone, whereas in the liver the major forms of P-450 which are involved in the disposal of steroids and xenobiotics are regulated by growth hormone. For the sake of brevity and focus, this review primarily concerns itself with mechanisms by which hormones regulate P-450 gene expression. This review is in no way comprehensive and, in some instances, only recent mechanistic studies are discussed, at the expense of earlier pioneering work, and we would like to acknowledge the many contributions to the field by investigators not mentioned in this text. Before considering what is known about the mechanisms of hormonal regulation of P -450, the nomenclature and gene structure of the members of this super family of enzymes are summarized.

II. Cytochrome P-450 Nomenclature and Gene Structure Today, over 140 cytochrorne P-450s have been isolated from various mammalian and nonmammalian species (Nebert et al., 1991). In general, it has been very difficult to categorize these cytochromes according to function because there are many examples of P-450s which have overlapping substrate specificities and many whose physiological functions are unknown. Evolutionarily, P-450s are related to each other and constitute a group (superfamily) composed of 25 fami-

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lies and 21 subfamilies. As a rule, P-450s with an amino acid identity of over 40% are considered to be members of the same family. Within each subfamily mammalian P-450s are over 60% identical. This is the basis for a new nomenclature of P-450s which was initiated in an effort to avoid the confusion caused by the use of different names for the same species of P -450 as they were purified and cloned by various laboratories. For example, P-450 2C 12, the twelfth member of subfamily C within family 2, was previously named i, 2d, UT-1, or 15p. This assignment of P-450s into families and subfamilies appears arbitrary, but it has evolutionary significance. The genes for P-450s within the same subfamily have thus far always been found to lie within the same gene cluster (Higashi et a / ., 1986; Rampersaud and Walz, 1987; Matsunaga et al., 1990). Furthermore, the intron-exon organization is unique to each family. For example, family 1 has seven exons, family 2 nine, family 21 ten. In addition, the intron-exon junctions occur at the same sites for members of each family (Gonzalez et al., 1985; Morishima et al., 1987; Umeno et a / ., 1988; Kimura et a/., 1989; Zaphiropoulos et a l. , 1990a). Although P-450s of the same subfamily are structurally very similar, their catalytic profiles as well as their regulation of expression can vary significantly. A classical example is provided by the two mouse P -450s, 2A4 and 2A5 (Squires and Negishi, 1988). These have only I I amino acid differences within their 494 residues, yet, 2A4 is a testosterone 15a-hydroxylase while 2A5 is a coumarin hydroxylase (Lindberg and Negishi, 1989). Furthermore, 2A4 predominates in the male kidney, whereas 2A5 is more abundant in the female kidney. Other examples include the rat P-450s 3A1 and 3A2 (90% identity), with only 3A1 being induced by dexamethasone (Gonzalez e f a l . , 1986a); and the rat 2C 12 and 2C13 (88% identity), with the former being induced and the latter suppressed by growth hormone (Zaphiropoulos e f u l . , I990b).

111. Transcriptional Regulation of Cytochrome P-450 Genes: Involvement of cis-Acting DNA Elements Transcriptional regulation of gene expression has been suggested to occur through the interaction of specific transcription factors with the general transcriptional machinery. This consists of the multicomponent enzyme RNA polymerase I1 and five thus far characterized general transcription factors, TFIIA, TFIlB, TFllD (the TATA factor), TFIIE, and TFIIF (for review see Sawadogo and Sentenac, 1990). The specific transcription factors are considered to be composed of distinct functional domains, e.g., the DNA binding region, which interacts with a well-specified DNA sequence motif located within or flanking the responsible gene, and the transcriptional activation region, which is thought to be involved in protein-protein interactions with components of the general

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transcriptional apparatus either directly, or, most likely, through some as yet not completely characterized adaptor/coactivator molecules (for review see Lewin, 1990). The first P-450 gene to be isolated and sequenced was the 14-kb long and phenobarbital-inducible 2B2 gene (Mizukami et al., 1983). Since then, many genes for P-450s have been characterized and, by the use of computer search programs, DNA sequences identical to or related to consensus DNA sequences for known regulatory factors have been identified. This is true for almost all known P-450 genes. In general, though, such claims have not been substantiated by gel retardation analysis to show that binding of nuclear factors actually occurs or by deletion analysis to prove their regulatory function. In some instances, however, functional data are starting to appear (see below). A novel cis-acting DNA element designated BTE for basic transcription element has been identified in the region proximal to the TATA box of the 1Al gene (Yanagida et al., 1990). This sequence has some similarities to the sequence with which the DNA binding factors SPl and NF-I interact. BTE binds nuclear proteins and is involved in basal transcription of 1Al. Since this sequence is highly conserved among the 5' flanks of other P-450s, including the 1B 1, 1B2, 2E1, 1 l A l , and 21A2 genes, it appears that its function is not restricted only to IAl. In addition to the BTE element, XRE sequence (xenobiotic responsive element) conferring dioxin inducibility on the 1Al gene have been well characterized (see, e.g., Fujisawa-Sehara et af., 1988). Within the genes of the mouse 2D subfamily, two sequences important for P-450expression as indicated by in vitro transcription have been characterized: a sequence termed SDI (sex difference information) located between -84 and -102 from the transcription start site of the male-specific 2D9 gene and a sequence termed CTE (common transcription element) located between -44 and -68 from the transcription start site of both the 2D9 and the sexually nondifferentiated 2D10 gene (Yoshioka et al., 1990). Two distinct nuclear factors appear to interact with these elements and their functional importance has been demonstrated by the reduced transcriptional activity of 5' flanking constructs lacking these sequences. The CTE but not the SDl element is also conserved in the rat 2D3 and 2D5 genes, which are expressed equally in both male and female animals. It is therefore implied that the SDl element has a necessary role for the sex-specific expression of the 2D9 gene. The fact that this element alone is not sufficient for sex-dependent expression (transcriptional activation of the 2D9 promoter was equal with both male and female liver extracts) indicates that other as yet uncharacterized factors are required for the male-specific expression of the 2D9 gene. In addition to the functional cis elements of the liver P-450s mentioned above, a number of DNA sequences involved in hormonal regulation of steroidogenic P-450s are starting to be characterized and are described in the following sections.

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Regulation through DNA binding factors is not the only means for transcriptional activation of P-450 genes. For example, developmental expression of the 2E 1, the 2D3, and the 2D5 P-450 genes has been correlated with demethylation of specific cytosine residues within the first exons or the 5' region of these genes (Umeno et al., 1988; Matsunaga and Gonzalez, 1990). Although a rigorous proof that this observed demethylation is a cause and not a result of gene activation remains to be obtained, it is certain that the role of methylation patterns and chromatin structure on the transcriptional regulation of of P-450 genes merits further studies. The following sections provide examples of hormonal regulation of P-450s that are relatively well characterized (CAMPeffects on steroidogenic P-450s), are starting to be characterized (growth hormone regulation of hepatic P-450s), or remain to be characterized (hormonal regulation of novel brain P-450s).

IV. Hormonal Regulation of Cytochrome P-450 Involved in the Biosynthesis of Hormones

A. Cytochrome P-450in the Adrenals and Gonads In the adrenals and gonads, the levels of cytochrome P-450 enzymes responsible for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones are controlled by pituitary peptide hormones circulating in the blood. The steroid hormones thus produced exert their biological effects through cognate intracellular soluble receptors which act as ligand-activated transcription factors modulating the activity of target genes. Presumably through such receptor-dependent modulation of target genes in the central nervous system, the peptide hormones are in turn synthesized and secreted under feedback control of the steroid hormones produced by the P-450-catalyzed reactions. In this way, the controlled release of ACTH, FSH, and LH serves to optimize the output of steroid hormones from the adrenals and gonads in response to various physiological stimuli and developmental cues. Through work in a number of laboratories, the primary structures of most steroidogenic P -450 enzymes and the structures of their corresponding genes are known (for refs. and nomenclature see Nebert et al., 1991). In the adrenal cortex of most species except rodents the genes for four distinct forms of P-450 are expressed: P-450 1 lA1 (P-450,,,), P-450 17A (P-450,,,), P-450 1 1A2 (P-450,la), and P-450 21 A (P-450,,,). In the gonads, P-450,,, and P-450,,, are expressed as well as P-450 19A (P-450,,,,,,), the enzyme responsible for estrogen formation. It has become apparent that much of the hormonal fine-tuning of steroidogenesis occurs through the control of the rate of initiation by RNA polymerase I1 at these genes and several reviews of this area already exist (Simpson and Waterman, 1988; Waterman et al., 1989; Miller, 1988; Simpson et

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al., 1990). In the following section, we review some of the recent experiments aimed at the elucidation of the mechanisms by which the transcriptional activities of the genes for steroidogenic P-450s are controlled by hormones.

1. Hormonal Regulation via the cAMP Second Messenger System The trophic hormones regulate adrenal and gonadal steroidogenesis via their cellsurface receptors. These peptide-hormone receptors are coupled to adenylate cyclase and the binding of hormone results in an increase in intracellular cAMP and a subsequent activation of the CAMP-dependent protein kinase pathway (PKA; for review see Taylor et al., 1990). In the adrenal it is clear that cAMP acutely increases the steroid hormone secretion within seconds and that it rnobilizes cholesterol to the inner mitochondria1 membrane (Privalle et al., 1983). In addition, a long-term effect of the rise in intracellar cAMP is a modulation of the transcriptional activity of the genes encoding the steroidogenic P-450s (John el al., 1986). For some of these genes it has been shown that the transcriptional activation requires a functional PKA. Schimmer, Parker, and colleagues have, through the use of mutant cell lines, convincingly shown that mutants in the regulatory subunit of PKA (so-called KIN mutants of the mouse adrenal tumor cell line Yl) have a diminished expression of P-450,,, and P-450,,, in response to ACTH and cAMP (Wong et al., 1989). The CAMP-responsive state could be restored by transfection with genes encoding normal subunits of PKA. These phenotypic revertants recovered CAMP-inducible expression of P-450,,, and P-450,,, (Wong et al., 1989). Thus, a central role for PKA in the transcriptional activation of the genes for steroidogenic P-450s in response to trophic hormones has been demonstrated. important questions then become, how is the activating signal transduced from PKA to the individual genes, what are the structural determinants within the genes that specify responsiveness, and how is the increase in transcriptional initiation by RNA polymerase I1 brought about? The usual strategy for addressing these questions has been first to define the structural elements within the steroid hydroxylase genes which specify responsiveness. In general, much of the regulation of transcriptional activity is thought to occur as the consequence of protein-DNA and protein-protein interactions somehow anchored at regulatory DNA sequences within and flanking structural genes (Johnson and McKnight, 1989; Mitchell and Tjian, 1989). It is therefore the hope that, by delineating regulatory sequences in steroid hydroxylase genes, these can then in turn be used to find proteins involved in transcriptional activation or repression. Once such factors are identified, it should be possible to determine whether the activity of these proteins is influenced by the activation of PKA. It is thus the aim of such studies to bridge the gap in the signal transduction

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pathway from the hormone-receptor interaction on the cell surface and the increased initiation of RNA polymerase 11 at the appropriate target genes. This general strategy has been very successful in the case of some other CAMP-regulated genes (for review see Roessler et al.. 1988). Thus, an 8-bp palindromic CAMP-response element (CRE) has been defined in a large number of genes and a family of transcription factors, the members of which interact with these sequences to activate transcription, has been identified (reviewed in Habener, 1990; Ziff, 1990). Perhaps the best characterized member of these factors is the 43-kDa CREB (CRE-binding protein). It has been shown to be phosphorylated by PKA and this is accompanied by a concomitant increase in the ability of CREB to activate transcription from a CRE-containing gene (Yamamoto et a / . , 1988; Gonzalez and Montiminy, 1989). These data suggest that CREB may be a direct target for the catalytic subunit of PKA in the cell and indicate a relatively simple, direct signal transduction pathway. So, to the issues to be addressed regarding CAMP-dependent regulation of steroidogenic P-450s we an add the question of to what extent the CREiCREB pathway of transcriptional regulation is involved. The remainder of this section briefly reviews some results from the general strategy outlined above applied to the individual steroid hydroxylase genes. a. P-450,,, (1 1Al) In all steroidogenic pathways, the initial and rate-limiting reaction is the cleavage of the side chain from cholesterol in the mitochondrion. This reaction produces pregnenolone and is catalyzed by P-450,,,. Due to the central role of this enzyme in steroidogenesis, much attention has been focused on agents that affect its levels. Trophic hormones such as ACTH and gonadotropins increase the levels of P-450,,, mRNA in their respective target tissues, adrenals and gonads. In primary bovine adrenal cells this increase has been shown to result primarily from an increased transcription rate via the cAMP second messenger system (John et al., 1986) and, to a lesser extent, from an effect on the half-life of the message (Boggaram et al., 1989). The molecular mechanisms underlying the mRNA stabilization are completely unknown, whereas data are beginning to accumulate on the transcriptional regulation of the P-450,,, gene by cAMP (Inoue et a/., 1988; Chung et al., 1989; Ahlgren et al., 1990a; Rice et a / . , 1990b; Moore et al., 1990). DNA sequences able to confer basal expression and cAMP regulation have been identified through deletion mutant analysis of the promoteriregulatory region of the P-450,,, gene isolated from different species (Ahlgren et al., 1990a; Rice et al., 1990b; Moore et a/., 1990). The interaction of such DNA sequences with putative regulatory proteins has been described (Rice et al., 1990b). No distinct consensus sequences for basal expression and cAMP induction of the P450,,, gene have emerged from these studies; rather, they suggest that multiple c-is-acting elements may be involved. In two of the studies, the functional significance of sequences identified by 5' deletions were corroborated by additional

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data. Thus, Ahlgren et al. (1990b) showed that a sequence of the bovine P-450,,, gene extending between - 183 and -86 will direct both basal and CAMP-enhanced transcription when fused to a heterologous promoter and that this sequence is equally active in either the correct or reverse orientation. Through site-directed mutagenesis, Rice et af., (1990b) showed that mutation of elements centered at -40, -70, and - 120 of the mouse P-450,,, gene significantly reduced the basal levels of expression. In studies of the human P-450,,, gene, the authors conclude that a CAMP-responsive region is located between -2327 and -605 (Moore et al., 1990). Clearly, a more detailed analysis of the various regions and their overall contribution to the transcriptional activity of the P-450,,, gene will be required before common and distinct features of regulation between the genes from different species can be evaluated. In no case has a consensus CRE sequence been shown to mediate the cAMP responsiveness of the P-450,,, gene in any of the species studied but, rather, novel CAMPresponsive sequences appear to be involved. gene appears mainly regulated by changes b. P-450,,, (17A) The P-45Ol7, in intracellular levels of CAMP, as evidenced by the inability to detect P-450,,, mRNA in bovine adrenocortical cells cultured in the absence of ACTH (Zuber et al., 1986). Furthermore, P-450,,, mRNA levels are essentially undetectable during a period of bovine fetal development when ACTH levels in the fetal circulation are very low (Lund et al., 1988). The CAMP-dependent increase in P-450,,, mRNA has been shown to require on-going protein synthesis in primary cultures of bovine adrenocortical cells and to involve an increased transcriptional rate (John et al., 1986). Initial studies have located cis-acting DNA sequences in the promoter/regulatory region of the bovine P-450,,, gene involved in cAMP regulation and nuclear proteins interacting with DNA in these regions have begun to be characterized (Lund et al., 1990). Thus, the bovine P-4501,, gene appears to contain at least two regions able to confer cAMP regulation on a heterologous promoter when transfected into the mouse adrenal tumor cell line Y 1 (Lund et al., 1990). This transient, CAMP-dependent reporter gene expression was unaffected by the presence of an inhibitor of protein synthesis, cycloheximide, regardless of whether Y 1 cells or primary bovine adrenal cells were used. It therefore appears that the transient transfection model is unable to mimic all aspects of CAMP-dependent regulation of the endogenous P-450,,, gene. The reasons for the difference in sensitivity to cycloheximide are not understood. One of the two sequences shown to confer cAMP responsiveness (CAMP responsive sequence 1; crsl, located between -243 and -225 with respect to the star site of transcription) interacted with nuclear proteins, as shown by gel retardation analysis. The protein-DNA interaction was not only competed for by unlabeled oligonucleotides containing the crsl sequence but also by oligonucleotides containing a CRE, suggesting that a functional relationship between the two

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sequences and their cognate factors may exist, despite the lack of obvious sequence similarities. As mentioned above, the CRE interacts with a family of transcription factors (CREB/ATF factors) of which the 43-kDa CREB initially characterized by Montminy and co-workers is the best studied. Recent data (U. Zanger, M. Waterman, and J. Lund, unpublished observations) suggest that it is unlikely that the factor(s) interacting with crsl is the 43-kDa CREB or factors recognized by antibodies to CREB. This in turn may suggest that the crsl sequence represents a novel prototype CAMP-responsive element. Interestingly, a sequence unrelated to CRE but similar to crsl in the human interleukin-6 promoter was recently shown to confer cAMP responsiveness as well as phorbol ester responsiveness (Ray et al., 1990). Whether crsl mediates responsiveness to phorbol esters is not known. c. P-450,,, (21A) In all species so far examined, there are two closely related genes encoding P -450,, I , one functional gene and one pseudogene. Both the mouse and the human functional genes have been studied with respect to sequences conferring cAMP regulation (Parker et al., 1986; Handler et al., 1988; Rice et ul., 1989; Kagawa and Waterman, 1990). In the mouse gene, a region between -330 and - 156 participates in ACTH regulation (Handler et al., 1988). In the human gene, a 34-nucleotide sequence has been found to be required for CAMP-dependent transcription (Kagawa and Waterman, 1990). Neither sequence contains a consensus CRE and oligonucleotides containing a CRE do not compete for nuclear proteins shown to interact with these regions (Kagawa and Waterman, 1990; Rice et al., 1990a). d. P-450,,, (1 IA2) In the case of the mouse P-45O1,, gene, it has been shown that a CRE-like sequence localized between -56 and -49 is a major determinant of cAMP induction (Rice et al., 1989). DNase I footprinting studies and Southwestern blotting further suggested that the element interacts with CREB or CREB-like protein factors. (19A) The mRNA levels for P -450,,,,, are regulated by intrae. P-450,,,, cellular cAMP levels both in the ovary and in adipose cells (Steinkampf et al., 1987; Evans et af., 1987). To date, no results are available regarding functional elements for CAMP-dependent regulation of the P -450,,,, gene. A CRE-like sequence in the 5' flank of the human gene has been observed (Means et al., 1989). In summary, the initial characterizations of regions conferring cAMP regulation to the genes for steroidogenic P -450s point to a perhaps surprising degree of complexity. Both CRE/CREB-dependent regulation and novel CAMP-responsive elements and factors are of importance. However, until the factors interacting with these varying response elements have been characterized in some functional, biochemical, and molecular detail, it is difficult to judge to what extent common features do exist in the CAMP-dependent regulation of the individual P -450 genes for the steroid hydroxylases.

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Finally, it should be pointed out that any influences on the intracellular levels of cAMP may influence the levels of the steroidogenic P-450s. Thus, a number of autocrine, paracrine, and pharmacological agents have been suggested to influence steroidogenesis in this fashion (see, e.g., Trezciak et al.; 1987; Dufau et al., 1989). Presumably, the signal transduction pathways for these agents share common features distal to cAMP production.

2. Non-CAMP-Dependent Hormonal Regulation In addition to the trophic hormones, many other hormones and growth factors modulate the expression of the genes for the steroidogenic P-450s. Unfortunately, the molecular mechanisms involved in the regulation by these agents have not been studied to the extent that the CAMP-dependent regulation has. However, since without such studies our understanding of the hormonal regulation of steroidogenic P-450s would be incomplete, we give some examples where hormones or growth factors have been shown to influence the steady-state levels of the mRNAs for these enzymes. A number of agents have been shown to influence the levels of P-450,,,. In cultured Leydig cells, glucocorticoids decrease P-450,,, mRNA levels via a glucocorticoid receptor-dependent mechanism (Hales and Payne, 1989). Interestingly, glucocorticoids have the opposite effect in MA- 10 tumor Leydig cells, i.e., they induce P-45OsCcmRNA (Hales et al., 1990). The phorbol ester tetradecanoyl phorbol acetate (TPA) suppresses the FSH-induced increase in P-450,,, mRNA in rat ovarian granulosa cells (Trezciak et al., 1987). Also, yinterferon has been shown to decrease the P-450,,, mRNA levels in porcine Leydig cells (Orava et al., 1989). In the ovine placenta, I7a-hydroxylase activity and P-450,,, protein increase prepartum, and this increase appears to be naturally induced by fetal glucocorticoids and can be artificially induced by infusion of synthetic glucocorticoids into the fetus (Mason et al., 1989). This would suggest that the P-450,,, gene can be cell-specifically regulated by glucocorticoids since presumably the gene would not be induced in the adrenocortical cells that continuously synthesize these hormones. So far, no glucocorticoid-responsive elements in the P-450,,, gene have been described. In mouse Leydig cells, testosterone has been shown to inhibit the CAMP-induced de novo synthesis of P-450,,, by an androgenreceptor-mediated mechanism (Hales et al., 1987), and in ovine adrenocortical cells transforming growth factor p decreases the basal level and completely blocks the stimulatory effect of ACTH on P-450,,, mRNA levels (Rainey et al., 1990). The inhibitory effect of the growth factor is not overcome by CAMP. Ample evidence exists to show that the P-450,,,, gene expression is regulated by diverse hormonal mechanisms, some of which presumably do not act via the cAMP signal transduction pathway (see, e.g., Hickey et a / . , 1989; Krasnow et

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al., 1990). In corpora lutea of pregnant rats, aromatase mRNA appears to be maintained by prolactin at a low level of expression during the first half of pregnancy, may be modulated by LH at midgestation, and is subsequently induced to high levels in the second half of gestation by placental factors and by estradiol in the corpus luteurn. Thus, it is clear that the major regulatory pathway for steroidogenic P-450s, involving tissue-specific trophic hormones acting via CAMP, can be modulated by other agents. It is, however, essentially unknown at which level this modulatory regulation occurs and, in many instances, to what degree this occurs via distinct, converging, or cross-talking signal transduction pathways.

B. Regulation of the Renal Mitochondria1 Vitamin D, la-Hydroxylase The P-450 which catalyzes the la-hydroxylation of 25-hydroxyvitamin D, (laOHase) is the only P-450 involved in the biosynthesis of a known hormone which remains to be purified and cloned. The importance of this enzyme to the maintenance of calcium and phosphorus homeostasis can be appreciated from the disease vitamin D-resistant rickets type I , which results from a deficiency of the enzyme (Marx e l a/., 1983). The la-OHase is located in the renal mitochondria where the P-450 content is approximately 5- 10 pmol mg - * mitochondria1 protein (Warner, 1982). This means that if the 1 a-OHase were the only form of P-450 in the mitochondria, a 2000- to 5000-fold purification would be required to achieve a homogeneous preparation. This low concentration, together with the presence of at least two other forms of P-450 in renal mitochondria, 25hydroxyvitamin D, 24-hydroxylase (24-OHase) (Tanaka and DeLuca, 1984) and sterol 26-hydroxylase (Anderson et a l . , 1989a) may account for the fact that this important P-450 has not yet been cloned. Several animal models are used to study the regulation of the catalytic activity of the enzyme in the kidney and usually the plasma levels of the hormone 1,25dihydroxy vitamin D, [ 1,25(OH),D,] confirm a similar pattern of regulation in man (Gray et al., 1977; Rosen and Chesney, 1983; Kaplan et a / . , 1977; Bilezikian et ul., 1978). Catalytic activity can be induced by making animals vitamin D-deficient, hypocalcemic, or hypophosphatemic (for review see DeLuca and Schnoes, 1983). Under physiological conditions, the enzyme is induced 2- to 3fold during lactation (Robinson et al., 1982). There are also changes with age. During development, activity is highest around puberty and declines after 2 months of age in rats to a level of about 20% of maximum by 12 months (Ishida et a / ., 1987). A loss of la-OHase activity with age is thought to be responsible for the decrease in calcium absorption from the gut which is common in elderly human beings (Bullamore et al., 1970). In postmenopausal osteoporosis, a disease characterized by accelerated loss of bone, there is evidence, in some pa-

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tients, of an inappropriately low plasma level of 1,25(OH),D, (Lore et al., 1984) and in some of a defect in the regulation of the enzyme by parathyroid hormone (PTH) (Slovik et al., 1981). Several hormones are involved in the regulation of the renal enzyme in response to the body’s need for calcium and phosphorus. These are PTH, which is secreted in response to hypocalcemia (Garabedian et al., 1972); growth hormone, which mediates the response to hypophosphatemia (Gray and Garthwaite, 1985); prolactin, which is necessary for the induction which occurs during pregnancy (Robinson et al., 1982); 1,25(OH),D,, which functions in a feedback loop to decrease the levels of la-OHase in the kidney (Tanaka and DeLuca, 1984); and sex hormones, whose importance in the regulation of the enzyme in mammals is not yet completely clear. Estrogens have been shown to stimulate the enzyme in birds (Castillo et al., 1977), but no direct effects have been demonstrated in mammals. In guinea pigs, the la-OHase activity is higher in males than females. Ovariectomy has no significant effect on enzyme activity, but castration of male guinea pigs reduces the level of activity to that of a female and male levels are restored by androgen replacement (Hagenfeldt et al., 1989). The kidney is responsible for maintenance of the plasma levels of 1,25(OH),D,, and is the source of hormone for its subsequent action in the small intestine and bone. The placenta synthesizes 1,25(OH),D, and this extra source of la-OHase accounts for the increased plasma levels of the hormone during pregnancy (Paulson and DeLuca, 1986; Weisman et a l . , 1979). 1,25(OH),D, is is also synthesized in other tissues such as keratinocytes (Bikle et al., 1986), melanoma cell lines (Frankel et al., 1983), transformed lymphocytes (Reichel et al., 1987), and bone cells (lhmer et al., 1980b), where it probably serves autocrine or paracrine functions. Most studies of the regulation of the la-OHase are based on measurement of either catalytic activities in kidney mitochondria or changes in plasma levels of 1,25(OH),D, in response to hormonal manipulations. Quantitation of the level of the protein and its mRNA awaits the purification of the enzyme and cloning of the corresponding cDNA. Primary kidney cells in culture have also been used to identify those hormones which act directly on the kidney to affect vitamin D metabolism (Trechsel et al., 1979; Turner et al., 1980a; Bar et al., 1981; Henry, 1981; Henry and Luntao, 1989). These studies showed that kidney cells from rats or chicks in primary culture retain the characteristic vitamin D metabolic profiles which are observed in vivo, i.e., high levels of la-OHase in vitamin D deficiency and high 25-hydroxyvitamin D, 24-hydroxylase in animals treated with 1,25(OH),D3. The physiological role of the 24-OHase, which is also a P-450, is not known. It may simply participate in the inactivation of 1,25(OH),D3 by the formation of 1,24,25(OH),D,, or in the excretion of 25-hydroxyvitamin D, (25-OH-D3) through the formation of 24,25(OH),D,. PTH and 1,25(OH),D,,

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when added to the primary cell cultures, elicit the expected effects on 25-OH-D, metabolism, but there was no response to estradiol. Efforts have been made to study the mechanisms through which these hormones exert their effects on 25-OH-D, metabolism (Henry and Luntao, 1989). PTH was shown to act directly on kidney cells to stimulate la-OHase activity by a mechanism which requires protein synthesis, and its action could be mimicked by forskolin and dibutyryl cyclic AMP (Bu,cAMP). 1 ,25(OH),D, increased the level of the 24OHase and reduced that of the la-OHase. The action of 1 ,25(OH),D, could be mimicked by the tumor promotor TPA. However, these two agents appear to act by different pathways since their actions are additive. Interestingly, as has been observed with the P-450,,, in ovarian granulosa cells (see above, Section IV,A,2), there is some interaction between the phorbol esters and the CAMP cascade since the presence of Bu,cAMP prevents the action of TPA on 25-OHD, metabolism. The use of kidney cells in culture has provided some useful information about the regulation of the enzyme, but in these experiments the duration of hormone exposure has been short, usually 4 hours, and the changes in catalytic activity are small after such a brief time interval. The system will be much more useful when specific antibodies and cDNA probes are available in order that changes in the level of protein and its mRNA can be quantitated.

V. Regulation of liver Cytochrome P-450 by Sex Steroids and Growth Hormone In rat liver, more than 20 distinct forms of P -450 have been isolated and characterized to varying extents. Endogenous compounds, such as steroid hormones and fatty acids, as well as exogenous compounds such as drugs and environmental pollutants are substrates for these P-450s (for review see Whitlock, 1986). In the case of steroid hormones, several P-450s do exhibit high substrate and product specificity (Wood et al., 1983). These metabolites are thought to be products of degradative pathways but, in view of the precise age- and sexdependent regulation of many steroid-metabolizing hepatic P -450s, it is conceivable that some hepatic metabolites of gonadal steroid hormones may yet be found to exhibit physiological functions. Long before distinct P -450 proteins were purified, specific antibodies raised, and cDNAs and genes cloned for liver P-450s, the hormonal regulation by androgens and estrogens was observed by measuring specific catalytic activities (for reviews see Gustafsson, 1978; Colby, 1980). Most studies have been carried out in rats, mainly because of the pronounced sex differences in hepatic metabolism of endogenous compounds as well as of xenobiotics in this species. There-

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fore, we focus on studies using the rat as an experimental model. It should be emphasized that observations in rats are not necessarily relevant for all mammalian species. The large sex difference in the hepatic metabolism of both steroids and drugs could be attributed to androgenic exposure during both the neonatal period (imprinting) and later on in life (Einarsson et al., 1973; Gustafsson et a / . , 1975). The active metabolite of testosterone in the neonatal brain which is responsible for this imprinting turned out to be estradiol. In adult life, estrogens administered exogenously were found to be of less importance, although they suppressed male and induced female metabolic characteristics. Following a variety of endocrine manipulations, it was concluded that gonadal hormones exerted their actions on liver steroid metabolism via the hypothalamo-pituitary axis (for review see Gustafsson et al., 1980). Several lines of evidence indicated that growth hormone (GH) could be the pituitary factor mediating the gonadal hormone effects (Gustafsson et al., 1976; Kramer and Colby, 1976; Gustafsson and Skett, 1978; Skett et a / ., 1978; Rumbaugh and Colby, 1980; Mode et al., 1981; Norstedt et al., 1983). Around this time, it was shown that GH was secreted in a sexually differentiated fashion (Ed& 1979). Male rats secrete GH in regular surges every 3-4 hours, with low, essentially undetectable levels in between, whereas in females the GH secretion is characterized by more frequent pulses of lower amplitudes and with higher trough levels than in males. Subsequently, gonadal hormonal modulation of GH secretion was demonstrated and found to be related to the regulation of hepatic steroid metabolism (Jansson et al., 1985). Thus, when GH is administered to hypophysectomized rats in a sex characteristic mode, the pattern of P-450 forms responsible for the sexually differentiated steroid metabolism seen in normal rats is essentially reestablished (Morgan et al., 1985; MacGeoch et al., 1985; Mode et al., 1989a). Accordingly, recent studies concerning regulation of constitutively expressed P-450s in rat liver have to a large extent been focused on GH effects. The majority of these P-450s belong to the P-450 2 family (Nerbert et al., 1991). The present discussion is restricted to the P-450 2C subfamily. It should, however, be mentioned that the expressions of P-450 2A2 (Waxman et al., 1988), 2B1 and 2B2 (Yamazoe et al., 1987), 2E1 (Williams and Simonet, 1987), as well as 3A2 expression (Waxman et al., 1988) are also modulated by GH, indicating that GH regulatory effects are not confined to a specific P-450 subfamily or even family. For all these gene products, GH has been shown to exert its effects pretranslationally and it would seem mainly at the transcriptional level. P-450 2C12 and 2C7 are predominantly female forms, whereas 2C11 and 2C13 are male characteristic forms (Zaphiropoulos et al., 1988; Gonzalez et a / ., 1986b; Yoshioka et al., 1987; McClellan-Green et al., 1989a). They are all developmentally regulated in a manner largely coinciding with the maturation of

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the GH secretory pattern in the respective sex. By comparison of the steady-state mRNA levels of these forms in normal, hypophysectomized, or GH-treated hypophysectomized animals, several modes of GH action have been discovered (MacGeoch et al., 1987; Mode et (11.. 1989a,b; McClellan-Green et al., 1989b; Janeczko et a l . , 1990; Sasamura et ul., 1990; Westin et al., 1990). For example, one and the same gene, 2C11, may be regulated in completely opposite modes by the same hormone (GH), depending on the way it is presented to the liver. Continuous administration of GH via osmotic minipumps, mimicking the female pattern, efficiently downregulates 2C11 both in normal male rats and hypophysectomized rats of both sexes. On the other hand, intermittent injections of GH (mimicking the male pulse-like pattern) upregulate 2C11 in hypophysectomized rats of both sexes. Another level of complexity is added by the fact that continuous GH administration downregulates certain genes (2C 1 I and 2C 13) but upregulates others (2C7 and 2C12). As an illustration, the different effects of continuous and discontinuous GH administration on 2Cll and 2C12 gene expression are presented in Fig. I . Obviously, it is an intriguing task to attempt to define the molecular mechanisms behind this complex control of liver P-450 by GH. The results from the hormonal manipulations in vivo referred to above show the importance of GH in the regulation of hepatic forms of P-450. However, a definite proof for a direct effect of the hormone on the liver awaited experiments with isolated hepatocytes. It has now been firmly established that GH has a direct effect on the liver since primary rat hepatocytes in culture, maintained in a completely hormone-free medium on a biomatrix, respond to GH with an increased expression of P-450 2C12 (Guzelian et af., 1988). Some studies indicate that the expression of certain P-450 genes which are mainly under the control of GH are also affected by thyroid and glucocorticoid hormones (Mode et af., 1989a; Yamazoe et al., 1990; Waxman et al., 1990; Ram and Waxman, 1990). In this context, it may be mentioned that the P-450 2CI 1 gene (CYP2C I 1) contains an upstream hexameric sequence which has been shown to be essential for glucocorticoid receptor binding in several glucocorticoid-induced genes (Morishima el u l . , 1987), although no functional significance of this sequence in the CYP2CI 1 gene has so far been reported. It is also of interest that androgens seem to act via receptor binding to the same sequence, i.e., the glucocorticoid-responsive element (Beato, 1989). Few studies if any have addressed the question whether gonadal hormones can act directly on the liver in concert with GH. In view of the finding that estrogen receptors in the liver are partially upregulated by GH (Norstedt et al., 1981), it cannot be excluded that, whereas the livers of hypophysectomized animals are nonresponsive to estrogens, they may become responsive following treatment with GH. Indeed, a direct estrogenic effect on the rat liver has been shown in the case of renin substrate formation (Nasjletti and Masson, 1972).

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34 1

Insulin is also a hormone which can have dramatic effects on the hepatic expression of P-450 genes (Thummel and Schenkman, 1989; Yamazoe et al., 1989). These effects may be indirect and could be explained by the fact that, in the absence of insulin, GH secretion from the pituitary is suppressed and GH binding to its receptor is decreased (Tannenbaum, 1981; Baster et al., 1980). In line with this, we have recently shown that insulin potentiates the induction of P-450 2C12 by GH in primary hepatocytes, most likely via an action on the GH receptor (Tollet et al., 1990). Recent studies in our own laboratory indicate that female hypophysectomized rats respond more efficiently to continuous GH treatment than hypophysectomized males and this is also true for primary hepatocytes obtained from female rats, as compared to male hepatocytes even after several days of culture (Westin et al., 1990; and unpublished data). Organization of chromatin structure has been considered to be an important determinant in the differential sensitivity of specific genes to various regulatory signals (Svaren and Chalkley, 1990). For example, sex differences in DNase I hypersensitivity or hypomethylation of cytosine residues of a gene may be important to consider in this respect. With regard to P-450 2C12, which is more efficiently induced in primary cells from female rats than from male rats, however, no sex difference in DNase I hypersensitivity or methylation has been observed (Zaphiropoulos et a / ., 1990a). The molecular mechanisms of GH action are poorly understood. GH has long been thought to mediate its growth-promoting effects via the action of insulinlike growth factor I (IGF-I) (Daughaday, 1989). However, in primary hepatocyte cultures, IGF-I does not seem to be a mediator of the GH induction of P-450 2C12 (Guzelian et a/., 1988; Tollet et a / . , 1990). The GH receptor has recently been cloned from several species including rat (Leung et al., 1987; Mathews et al., 1989). The primary structure of this receptor has not yet provided any clues as to its signaling mechanisms but it is structurally related to the cytokine receptors (Cosman e t a / ., 1990). Ligand binding to the receptor results in phosphorylation of the receptor on tyrosine residues but the receptor does not contain the characteristic ATP binding sequence of a tyrosine kinase (Carter-Su et al., 1989). sn- 1,2-Diacylglycerol formation has been shown following GH binding, indicating the possible subsequent involvement of a protein kinase C (Doglio et al., Fig. 1 (A) Northern analysis of poly(A) RNA from control (C), hypophysectomized (Hx), and hypophysectomized Sprague-Dawley rats treated with growth hormone (GH) by single daily injections (Inj) or by osmotic minipumps (Mp) and probed with 2C12 sequences. The treatment of the rats with human G H was for 3 days using a daily dose of 120 p g for both the continuously (Mp) and the discontinuously (Inj) treated animals. ( B ) Northern analysis of poly(A)+ RNA from control (C), hypophysectomized (Hx I ,2). and hypophysectomized Sprague-Dawley rats treated with GH by single daily injections (Inj I ,2) or by osmotic minipumps (Mp I .2) and probed with 2CI I sequences. The treatment of the rats with human GH was for 6 days using a daily dose of 120 p g for both the continuously (Mp 1,2) and the discontinuously (Inj 1.2) treated animals. +

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1989; Johnson et al., 1990). Most excitingly, a GH-responsive element has recently been identified in the serine protease inhibitor 2.1 gene (Yoon et al., 1990). With genomic clones of GH-responsive genes, primary cell culture systems which express these genes, and the possibilities to cotransfect cell lines with GH receptor cDNA and target genes, the prospects are bright for the identification of second messengers, cis-acting elements, and trans-acting factors involved in regulation of P-450 gene expression by GH.

VI. Regulation of Cytochrome P-450 in the Prostate, Pituitary, and Brain The prostate, pituitary, and brain will be considered together as an important group of tissues where many forms of P-450 remain to be characterized. All three tissues contain P-450, and there is evidence for both endocrine function and endocrine regulation of these enzymes. The functions of all three tissues are influenced by circulating levels of steroid hormones. In the hypothalamic preoptic area (HPOA) of the brain and the anterior pituitary gland, this function includes feedback regulation of steroid biosynthesis in the adrenals and gonads by glucocorticoids, estrogen, and androgen, while the maintenance of prostatic size and secretion is androgen dependent. A major form of P-450 in all three tissues is an enzyme whose main function is the inactivation of androgens (Warner et d.,1989a). This enzyme, Sa-androstane-3p, 17P-diol hydroxylase (3pdiol OHase), was first described in the prostate (Isaacs et al., 1979), where it was shown to be so efficient in the elimination of 5a-androstane-3f3,17P-diol (3P-diol) that this steroid could not be detected in prostate homogenates or on incubation of these homogenates with dihydrotestosterone (DHT). 3p-Diol OHase has been purified from the prostate (Sundin et al., 1987). The apparently single form of P-450 catalyzes the hydroxylation of 3p-diol at the 6p, 7p, and 6a positions. The levels of this enzyme activity in the prostate, pituitary, and brain in an 8-week-old male rat are 2000, 300, and 100 nmol triols formed hg- tissue, respectively. The enzyme is present in the brains of fetal rats at day 18 of gestation at about 30% of adult levels and increases prior to puberty to adult levels. In the prostate, activity is highest in early puberty and decreases to 10% of its maximal level by the age of 3 months. Treatment of intact adult rats with DHT has little or no effect on the level of the enzyme in the prostate, brain, or pituitary. On castration of rats, the prostate decreases in size and the enzyme becomes undetectable. Administration of DHT to castrated rats causes regrowth of the prostate and an induction of the enzyme in the prostate but does not influence the enzyme in the brain or pituitary. This pattern of androgen regulation, i.e., high levels during early puberty

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followed by a decline in activity in the mature prostate despite continued presence of androgen, has been observed for two other enzymes. These are the steroid 5a-reductase (Andersson et al., 1989b), the enzyme which catalyzes the formation of DHT from testosterone, and P-450 IVA9 (Stromstedt et al., 1990), a member of the P-450 subfamily of fatty acid hydroxylases whose function in the prostate is not known. Once the genes have been cloned, prostatic P-450s will provide useful tools to investigate the mechanisms by which androgens regulate gene expression in the prostate. In view of the postulated role of 3P-diol OHase in the elimination of androgens, the absence of sex differences and the lack of regulation by androgens of this enzyme in the pituitary and brain are unexpected. It is possible that the role of the enzyme is not limited to the elimination of DHT from tissues. An alternative role could be protection of certain organs from the estrogenic effects of 3P-diol. This steroid has been shown to bind to the estrogen receptor and have estrogenic activity at concentrations well within the concentration range in which it is produced physiologically (Thieulant et al., 1981). It is also possible that 5areduced glucocorticoids and progesterone are substrates of the enzyme and that its role in the brain is to regulate the level of these hormone metabolites, called neurosteroids, which are potent y-aminobutyric acid (GABA) receptor agonists (Majewska et al., 1986). This role could explain the wide distribution of the enzyme in the central nervous system. It may also be of significance that dehydroepiandrosterone (DHEA), which is thought to be synthesized in the brain and have a role in neuronal function (Corpechot et al., 1981), is hydroxylated by 3P-diol OHase (Warner et al., 1989b). The localization of the mRNA for this enzyme in the brain and colocalization with specific neuronal or glial markers will indicate which cell types contain the enzyme and will likely provide some insight into its function. The anterior pituitary contains approximately 2 nmol P-450 g - wet weight. of which only 10% can be accounted for by 3P-diol OHase (Warner el al., 1989b). The high level of catechol estrogens in the pituitary (Paul and Axelrod, 1977) suggests that estrogen 2- and 4-hydroxylases may be quantitatively important forms of the enzyme in this gland, but this has not yet been shown to be the case. The major hepatic forms of the enzyme which catalyze the formation of catechol estrogens are not detectable in the pituitary samples on Western or Northern blots (M. Warner, unpublished data). Most of the P-450 in the pituitary therefore remains to be characterized. In the brain there are other forms of P-450 which have not yet been characterized but which show strong evidence of endocrine regulation. During pregnancy, lactation, and on treatment of rats with DHT there is a 5- to 10-fold induction in the overall concentration of microsomal P-450 in the HPOA and in the olfactory lobes. There is no concomitant increase in the level of 3P-diol OHase, nor in the hydroxylation of estrogen or androstenedione (Warner et al., 1989b). Be-

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cause of the low level of P-450 (300-500 pmol g - ' tissue) and the small size of these brain areas, it is difficult to obtain large quantities of these enzymes for characterization. Attempts to characterize these forms of P-450 by crosshybridization of cDNAs using P-450s cloned from other tissues or with antibodies raised against hepatic P-450s have so far not been successful. During pregnancy and lactation there is also an induction of mitochondria1 P-450 in the HPOA and olfactory lobes (Warner et al., 1989b). Two mitochondria1 P-450s, cholesterol 26-hydroxylase and P-450,,,, have been detected in the HPOA with the use of the polymerase chain reaction (PCR) (Ahlgren er al., 1990a). The induction of these enzymes during pregnancy is currently under investigation. The physiological functions of these cholesterol-metabolizing enzymes in the brain and the reason for their induction during pregnancy are not known, but they could be involved in the regulation of cholesterol levels in the cells. This could be achieved by conversion of cholesterol either to excretable metabolites such as pregnenolone or to metabolites which regulate the uptake and synthesis of cholesterol. There is strong evidence for a role for P-450 in the synthesis of oxysterols, which are potent regulators at the transcriptional level of the low-density lipoprotein (LDL) receptor and enzymes involved in cholesterol biosynthesis (Kandutsch et al., 1978; Gupta et al., 1986). In the ovary, 26hydroxycholesterol has been shown to regulate steroid synthesis (Rennert et al., 1990) and, although the brain does synthesize cholesterol (Anderson et al., 1990), the regulation of this pathway and the role of oxysterols have not yet been examined. Much remains to be learned about the physiological functions and regulation of the P-450s in the prostate, pituitary, and brain. Only when the endogenous substrates and products of the reactions are identified will it be possible to investigate the influences of therapeutic agents on these reactions and perhaps understand the mechanisms by which many useful drugs cause undesirable, tissue-specific side effects.

VII. General Conclusions As is evident from this review, P-450 in many tissues is strictly regulated by a variety of endocrine control mechanisms. The major control is exerted at the transcriptional level and the regulatory signals operate through both membranebound and intracellular/soluble receptors. Sometimes one and the same P-450 gene appears to be multihormonally regulated, indicating the necessity of fine tuning of the specific enzyme activity as a well-balanced response to the current physiological demands of the organism. Such delicate and sophisticated interactive regulatory networks certainly suggest that all P-450 genes express physiologically essential proteins. This in turn opens up interesting possibilities in the

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future for development of new therapeutic agents aimed at manipulating tissue levels of those P -450s which have key roles in various physiological and pathophysiological processes.

VIII. Future Perspectives-Novel

Endocrine Systems?

When considering mechanisms for the regulation of P-450. it is extremely exciting to follow the dynamic development of the research on the so-called orphan receptors within the steroid receptor supergene family. The orphan receptors have no known ligands but share the principal three-domain structure (N-terminal domain with as yet incompletely understood function, middle DNA-binding domain, and C-terminal ligand-binding domain) with the well-characterized steroid hormone receptors and other known members of the family, e.g., the thyroid hormone and retinoic acid receptors. These receptors usually interact with specific response elements in the 5’ flank of the respective target genes. Recently, clofibrate, a well-known inducer of certain forms of P-450, was shown to act via a member of the steroid receptor supergene family, even though the physiological ligand of the receptor is still unknown (Issemann and Green, 1990). Likewise, it may be suggested that other P-450 inducers such as dioxin, phenobarbital, I6a-cyanopregnenolone, isosafrole, and butylated hydroxyanisole act via related receptor molecules. Such transcriptional regulators could well be activated, physiologically, by as yet unknown endocrine signals, perhaps in an age-specific fashion. In this regard, oxysterols, the cholesterol metabolites formed by P-450-catalyzed reactions, which regulate cholesterol metabolism and steroid synthesis at a transcriptional level, are thought to act via a soluble intracellular receptor, and the search for this protein is being actively pursued in several laboratories (Dawson et a/., 1989; Rajavashisth et al., 1989; Taylor et ul., 1989). Thus, endocrine control of P-450 may eventually turn out to be a very general phenomenon, perhaps common to most if not all P-450s.

Acknowledgments These authors are supported by grants from the Swedish Medical Council and the Swedish Work Health and Environment Fund.

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Yamamoto, K. K., Gonzalez, G . A., Biggs, W. H., 111, and Montminy, M. R. (1988). Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature (London) 334, 494-498. Yamazoe. Y., Shimada, M . , Murayama, N., and Kato, R. (1987). Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone. J. Biol. Chem. 262, 7423-7428. Yamazoe, Y., Murayama, N., Shimada, M., Yamauchi, K., and Kato, R. (1989). Cytochrome P450 in livers of diabetic rats: Regulation by growth hormone and insulin. Arch. Biochem. Biophys. 268, 567-575. Yamazoe, Y., Murayama, N., Shimada, M., and Kato, R. (1990). Thyroid hormone suppression of hepatic levels of phenobarbital-inducible P-450b and P-45Oe and other neonatal P-450s in hypophysectomized rats. Biochem. Biophys. Res. Cornmun. 160, 609-614. Yanagida, A., Sogawa, K., Yasumoto, K.-I., and Fujii-Kuriyama, Y. (1990). A novel cis-acting DNA element required for a high level of inducible expression of the rat P-45Oc gene. Mol. Cell. Bid. 10, 1470-1475. Yoon, J.-B., Berry, S. A., Seelig, S., and Towie, H. C. (1990). An inducible nuclear factor binds to a growth hormone-regulated gene. J. Biol. Chem. 265, 19947- 19954. Yoshioka, H., Morohashi, K., Sogawa, K., Miyata, T., Kawajiri, K . , Hirose, T., Inayama, S., FujiiKuriyama. Y., and Omura, T. (1987). Structural analysis and specific expression of microsomal cytochrome P-450 (M-1) mRNA in male rat livers. J. Biol. Chem. 262, 1706-171 I . Yoshioka, H., Lang. M., Wong, G., and Negishi, M. (1990). A specific cis-acting element regulates in vitro transcription of sex-dependent mouse steroid 16a-hydoxylase (C-P450 16a) gene. J. Biol. Chem. 265, 14612-14617. Zaphiropoulos, P. G., Mode, A , , Strom, A,, Moller, C., Fernandez, C . , and Gustafsson, J.-A. (1988). cDNA cloning, sequence, and regulation of a major female-specific and growth hormoneinducible rat liver cytochrome P-450 active in I5a-hydroxylation of steroid sulfates. Proc. Nail. Acad. Sci. U.S.A. 85, 4214-4217. Zaphiropoulos, P. G., Westin, S., Strom, A., Mode, A., and Gustafsson, J.-A. (1990a). Structural and regulatory analysis of a cytochrome P-450 gene (CYP2C12) expressed predominantly in female rat liver. DNA Cell Biol. 9, 49-56. Zaphiropoulos, P. G . , Strom, A., Robertson, 1. A., and Gustafsson, J.-A. (1990b). Structural and regulatory analysis of the male-specific rat liver cytochrome P-450 g: repression by continuous growth hormone administration. Mol. Endocrinol. 4, 53-58. Ziff, E. (1990). Transcription factors: a new family gathers at the CAMPresponse site. Trends Genet. 6, 69-72. Zuber, M. X., John, M. E., Okamura, T., Sirnpson, E. R., and Waterman, M. R. (1986). Bovine adrenocortical cytochrome P-45017a. Regulation of gene expression by ACTH and elucidation of primary sequence. J. Biol. Chem. 261, 2475-2482.

Subject Index

Absorption. molecular asymmetry and, 67-68, I09 Acceptors, molecular asymmetry and, 9 I ACE, see Angiotensin-converting enzymes ACEls. see Angiotensin-converting enzyme inhibitors Acetylcholine blood-brain barrier and, 145 delivery to central nervous system and, 312. 314 Acetylcholinesterase, delivery to central nervous system and, 306, 312 Achiral drugs, molecular asymmetry and. 112113, 119-120 Acquired immune deficiency syndrome (AIDS), acyclovir and, 19 Acquired immune deficiency syndrome dementia complex, calcium channel antagonists and. 273, 290-291 ACTH, cytochrome P-4.50 gene expression and, 332-334 Actinomycetes. renin inhibitors and, 2 16 Activation protein kinase and, 183. 187. 191 receptor-activated calcium entry and, 253. 256-258.260 Activator calcium, receptor-activated entry and, 252 Acute ishemic stroke, calcium channel antagonists and. 288-289 ACVMP, see Acyclovir monophosphate Acyclovir, 1-3, 22-23 antiviral action human DNA polymerase. I I - I2 potentiation. 13-19 viral DNA polymerase, 6- I3 hypersensitivity to. 21-22 metabolic activation cellular kinases. 5 cytoplasmic 5'-nucleotidase, 5-6 thymidine kinase, 3-5 resistance to, 19-2 I transport, 3 Acyclovir diphosphate, 5 . 15, 19 Acyclovir monophosphate (ACVMP). 22

antiviral action. 7-13, 19 metabolic activation, 5 Acyclovir triphosphate, 3, 23 antiviral action, 6-10. 13, 18-19 metabolic activation, 3 resistance, 21 Acylation, molecular asymmetry and. 69 Acyl glucuronides, molecular asymmetry and, 94 Adenosine. blood-brain barrier and, 146 Adenosine analogs, acyclovir and, 2 Adenosine deaminase, acyclovir and, 2 Adolescence, osteoporosis therapy and, 32, 35, 39-40 Adrenaline, molecular asymmetry and, 59-61 , 111

Adrenals, cytochrome P -450 gene expression and, 326, 329-33 I , 342 Adriamycin, protein kinase inhibitors and, 182 Adsorptive endocytosis, blood-brain barrier and, 150- I52 ADTN, delivery of drugs to central nervous system and, 30.5 Affinity chromatography, acyclovir and, 4. 12-14 Age blood-brain barrier and, 138 cytochrome P-450 gene expression and. 335,337 delivery of drugs to central nervoub system and, 300 molecular asymmetry and, 7 I osteoporosis therapy and, 29, 32-34, 38, 40-4 I Ape-related bone loss, osteoporosis therapy and. 31-33, 35 AIDS, see Acquired immune deticiency syndrome Albumin blood-brain barrier and, 150 modified, 150 molecular asymmetry and, 64, 69. 71, 102, I05 Aldosterone, renin inhibitors and, 234

355

356 Allosteric regulatory site, protein kinase inhibitors and, 169 Aluminum, blood-brain barrier and, 155- 156 Aluminum salts, blood-brain barrier and, 156 Alzheimer’s disease blood-brain barrier and, 155 calcium channel antagonists and, 289-290 delivery of drugs to central nervous system and, 300, 312-314 Amide bonds, renin inhibitors and, 219-220, 226,238 Amides, renin inhibitors and, 223, 229 Amiloride, protein kinase inhibitors and, 179 Amino acids acyclovir and, 2 I blood-brain barrier and, 144-145, 147, 153, 155-156 calcium channel antagonists and, 280 cytochrome P-450 gene expression and, 327 delivery of drugs to central nervous system and, 301. 305, 307 molecular asymmetry and, 91, 97, 116 renin inhibitors and, 209-21 1, 213-214 structure, 215-217, 220, 222-223 Amino acid sequences protein kinase inhibitors and, 185 renin inhibitors and, 215 Amino acridines, protein kinase inhibitors and, I83 7-Aminobytyric acid (GABA) cytochrome P-450 gene expression and, 343 delivery of drugs to central nervous system and, 306 Amphetamines, molecular asymmetry and, 68, I I4 Amphipathicity, protein kinase inhibitors and, 183 Analogs, delivery of drugs to central nervous system and, 305 Androgens cytochrome P-450 gene expression and, 342-343 osteoporosis therapy and, 32, 36-37, 43, 46-47 5a-Androstane-3P,l7~-diol(3P-diol). cytochrome P -450 gene expression and, 342 Sa-Androstane-3P, I7P-diol hydroxylase, cytochrome P -450 gene expression and, 342-343

Subjed Index

ANF, see Atrial natriuretic factor Angiotensin, see also Renin-angiotensinaldosterone system; Renin-angiotensin system blood-brain barrier and, 152 protein kinase inhibitors and, 188 Angiotensinase inhibitor, 21 3 Angiotensin-converting enzyme inhibitors (ACEIS), 207-208, 210, 233-234, 241 Angiotensin-converting enzymes (ACE), 208209,214-215 Angiotensin I, renin inhibitors and, 208-209, 211-216 Angiotensin I1 blood-brain barrier and, 142, 149 receptor-activated calcium entry and, 263 renin inhibitors and, 208-209, 212-213 pharmacology, 232-234, 239-240 structure, 2 14-2 15 Angiotensinogen, renin inhibitors and, 209, 21 1-215,217 Anoxia, calcium channel antagonists and, 279-280.283 Antagonists, protein kinase inhibitors and, 183-184 Antibiotic inhibitors, protein kinase and, 175-179 Antibodies cytochrome P -450 gene expression and, 333,337,344 delivery of drugs to central nervous system and, 303 molecular asymmetry and, 94-95 monoclonal, see Monoclonal antibodies protein kinase inhibitors and, 169 renin inhibitors and, 212-214 Anticancer drugs, protein kinase inhibitors and, 181 Anticonvulsants, molecular asymmetry and. 84, 87, 110, I I9 Antigens protein kinase inhibitors and, 175 renin inhibitors and, 214 Antihypertensive therapy, renin inhibitors and, 234 Anti-inflammatory drugs, molecular asymmetry and,71, 112. 114 Antiluminal membrane, blood-brain barrier and, 147 Antiresorptive therapy, osteoporosis and, 39-45,48

Subject lndex

Antitumor activity. protein kinase inhibitors and, 183 Antiviral action of acyclovir mechanism, 6-13 potentiation, 13- 19 A l l I O U , acyclovir and, 15-19, 22 Apomorphine, protein kinase inhibitors and, I79 Arbaprostil, molecular asymmetry and, I15 2-Arylpropionates, molecular asymmetry and enantioselectivity, 104 pharmacokinetics, 77-8 1 therapy, 112, 114-1 15 Aspartic acid, renin inhibitors and, 219 Aspartic proteinase specificity. renin inhibitors and, 210-21 1 Astrocytes, blood-brain barrier and. 142, 153-154, 157 Atenolol, molecular asymmetry and, 68 ATP acyclovir and, 4 blood-brain barrier and, 139, 148 protein serineithreonine kinase inhibitors and, 170-180, 183-184, 186 protein tyrosine kinase inhibitors and, 187189,191-192 ATPase. see also Na+ , K + -ATPase receptor-activated calcium entry and. 259 ATP-binding sites, protein kinase inhibitors and, 169, 174, 191 Atrial natriuretic factor (ANF), blood-brain barrier and, 148- 149 Atropine, molecular asymmetry and, 78, 93, I15 Atropisomerism, molecular asymmetry and, 74 Axial bone mass. osteoporosis therapy and, 32-33,38,46-47 Axial skeleton, osteoporosis therapy and, 30, 32.34

Baclofen blood-brain barrier and, 144 delivery to central nervous system and. 312 Barium, receptor-activated calcium entry and. 26 1-263 BCNU. delivery to central nervous system and,314-315 Benzetimide, molecular asymmetry and, 93

357 Benzodiazepines, molecular asymmetry and, 104 Benzothiazepines, calcium channel antagonists and, 279. 281 Bethanechol, delivery to central nervous system and, 312, 314 Biliary extraction, renin inhibitors and, 239 Bioavailability molecular asymmetry and, 1 17- 1 18 renin inhibitors and, 238-239 Bisphosphonates, osteoporosis therapy and, 44-45,47,49 Blood-brain barrier, 137-138, 157 calcium channel antagonists and, 279 delivery of drugs to central nervous system and, see Central nervous system, delivery of drugs to model isolated brain capillaries, 138- 140 microvessel endothelial cells, 140- 142 regulation of transport astrocytes, 153 developmental changes, 154- 155 hyperosmotic treatment, 156- 157 pathological changes, 155-156 peptides, 152-153 in vifrotransport drugs, 151 endocytosis, 151- 152 ions, 146-147 neurotransmitters, 147 nutrients, 143-146 peptides, 147-150 proteins, 147- 150 Blood-cerebrospinal fluid barrier, delivery of drugs and, 300 Blood pressure, renin inhibitors and, 229-233, 235-237,239-241 Bone acquisition. osteoporosis and, 38, 40 Bone-forming agents, osteoporosis therapy and, 45-47 Bone loss, osteoporosis therapy and, 29, 31, 37, 39.48 age-related changes, 32-33 bone-forming agents, 41, 43 Bone mass, osteoporosis therapy and, 29-3 I , 38-39.48-49 age-related changes. 32-34 antiresorptive therapy. 40, 42-45 bone-forming agents, 46-47 regulation, 34-37

358

Subjed fndex

Bone modeling, osteoporosis therapy and, 30 Bone remodeling, osteoporosis therapy and,

protein kinase inhibitors and, 168,170,180,

183,186,188 Calcium/calmodulin-dependent protein kinase 11, inhibitors and, 170-171,177, 185Bone repair, osteoporosis therapy and, 30 Bone resorption, osteoporosis therapy and, 30186 31.34,39 Calcium channel antagonists, prevention of bone-forming agents, 43-44 neurotoxicity and. 272.291-292 contributions of various types, 275-276 experimental approaches, 47 Bone strength, osteoporosis therapy and, 33efficacy, 272-273 34 human trials, 288-290 hypothesis, 275 Bone turnover, osteoporosis therapy and, 40ischemia, 283-288 41.43 Bovine serum albumin, blood-brain barrier types of neurotoxicity, 273-275 in virro models, 279-283 and, 150 voltage-dependent channels, 276-279 Brain, see also Blood-brain barrier calcium channel antagonists and, see Calcium entry, receptor-activated, 251-252, 266-267 Calcium channel antagonists, epithelial cells, 252-254 prevention of neurotoxicity and mechanisms cytochrome P-450gene expression and, 326,342-344 capacatative model, 256-261 Brain capillaries entry pathway, 261-265 inositol phosphates, 255-256 blood-brain barrier and, 144,149 isolated, 138-140,143,147-148 1,3,4,5-tetrakisphosphate,265-266 30-31,37,43,47-48

Brain capillary endothelium, blood-brain barrier and, 138,145-149,153-154,157 Brain edema blood-brain barrier and, 147,156 delivery of drugs to central nervous system

and.313-314 Brain microvessels, blood-brain barrier and,

146.152-153 Brain natriuretic peptide, blood-brain barrier and, 149 Brain tumors, delivery of drugs to central nervous system and, 300,304,314 BVdU, acyclovir and, 20-21

Calcitonin, osteoporosis therapy and, 43-44,

47 Calcitriol, osteoporosis therapy and, 41-42,47 Calcium blood-brain barrier and, 156 cytochrome P-450 gene expression and,

335-336 osteoporosis therapy and, 32,39,48 absorption, 40-41 antiresorptive therapy, 39-41,45 bone mass regulation, 35-38 experimental approaches, 47

Calcium overshoot, receptor-activated calcium entry and, 257-259 Calcium phosphate, receptor-activated calcium entry and, 251 Calcium salts, osteoporosis therapy and, 40 Calmodulin, see also Calcium/calmodulindependent protein kinase calcium channel antagonists and, 273 protein kinase inhibitors and, 168 protein serine/threonine kinase inhibitors and, 170,173,178,181,184-185 Calphostin C, protein kinase inhibitors and,

I82 CAMP, see Cyclic AMP Cancer, protein kinase inhibitors and, 169 Capacitative model for receptor-activated calcium entry, 256-263,265-267 Capillaries, see also Brain capillaries blood-brain bamer and, 155-157 Carbon, molecular asymmetry and, 59 Carboxypeptidase, renin inhibitors and, 215 Carboxy-terminal modifications, renin inhibitors and, 220-221 Cardiac effects, renin inhibitors and, 233-234 Cardiovascular activity, renin inhibitors and,

238 Cardiovascular disease, renin inhibitors and,

208,214

Subjed Index

Carrier systems. delivery of drugs to central nervous system and, 307-308, 319 Casein kinase, inhibitors and, 173, 179 Casein kinase I , inhibitors and, 171, 173 Casein kinase 11, inhibitors and, 171, 179 Casein kinase 111, inhibitors and, 179 Catalysis acyclovir and antiviral action, 6-7. 9- I 1 , 13. 18 metabolic activation, 3 cytochrome P-450 gene expression and, 327,336-337.343 Cathespin D, renin inhibitors and, 210-21 1 cDNA, cytochrome P-450 gene expression and, 336-337, 342, 344 Central nervous system blood-brain barrier and, 148. 157 calcium channel antagonists and, 275. 279, 29 1 cytochrome P-450 gene expression and, 343 disorders, 138, 155 Central nervous system, delivery of drugs to, 299-302,319-320 alteration of banier, 302-304 altering agents, 304 carrier systems. 307-308 latentiation, 304-307 circumventing barrier. 308-309 implantable polymer systems, 3 13-3 16 implantation of tissues, 316-318, 320 pumps, 309-3 13 Cerebellar cells, calcium channel antagonists and, 279 Cerebral blood flow, calcium channel antagonists and, 288 Cerebrospinal fluid (CSF) blood-brain bamer and. 146-147 delivery of drugs to central nervous system and, 300-301. 305, 309, 313 CGP 38 560A. renin inhibitors and, 232-233, 239 Chemotactic response, molecular asymmetry and, 100 Chemotherapy, delivery of drugs to central nervous system and. 303. 312, 316 Chimeric peptides. delivery of drugs to central nervous system and, 307 Chiral inversion pharmacokinetics of. 77-81 therapy and, 115

359 Chirality, see Molecular asymmetry Chloronaphthalene, protein kinase inhibitors and, 170, 173 Chloronaphthalene sulfonamides, protein kinase inhibitors and, 174 Cholesterol, cytochrome P-450 gene expression and, 344-345 Choline, blood-brain banier and, 145 Cholinergic denervation, delivery of drugs to central nervous system and, 314 Chromatography acyclovir and, 4, 12-14 molecular asymmetry and, I19 Chronic heart failure, renin inhibitors and, 233 &-acting DNA elements BTE, 328 cytochrome P-450 gene expression and, 328,33 I , 342 XRE, 328 Clearance, molecular asymmetry and enantiomer-enantiomer interactions, 98, 101 enantiomers as biochemical probes, 109 enantioselectivity, 101 pharmacokinetics, 72-8 1 polymorphic drug disposition, 82 Clones, cytochrome P-450 gene expression and, 327, 335-337, 343-344 CMV, see Cytomegalovirus Coherence therapy. osteoporosis and, 47-48 Collagen, osteoporosis therapy and, 30. 33 Collagenase, blood-brain banier and, 140 Competition acyclovir and, 13 blood-brain barrier and, 144 molecular asymmetry and, 99- 100 protein kinase inhibitors and, 192 ATP, 170-180, 183-184, 186-189 diacylglycerol. I8 1- 183 phosphatidylserine, 180-181, 183 proteinlpeptide substrate, 185- 186, 189191

renin inhibitors and, 216 Competitive inhibition, acyclovir and, 9 , 15 Cortical bone, osteoporosis therapy and, 3233, 38, 40, 46-41 Cortical neurons, calcium channel antagonists and, 279-280, 291 Coumarin, molecular asymmetry and, 71, 104 Cross-reactivity, molecular asymmetry and, 94

360 Cyclic AMP cytochrome P-450 gene expression and, 330-335.337 protein kinase inhibitors and, 168 Cyclic AMP-dependent protein kinase, inhibitors and, 182, 184-186, 189, 191, 193 competition with ATP, 170-171, 173, 175, 177, 179-180 Cyclic AMP-responsive element (CRE), cytochrome P-450 gene expression and, 331-333 Cyclic AMP-responsive element-binding protein (CREB), cytochrome P-450 gene expression and, 331, 333 Cyclic GMP, inhibitors and, 170 Cyclic GMP-dependent protein kinase, inhibitors and, 171, 173, 177, 184 Cyclic nucleotides, protein kinase inhibitors and, 168, 170, 173, 175, 178, 183-184 Cycloheximide, cytochrome P-450 gene expression and, 332 Cyclohexyl analogs, renin inhibitors and, 223 Cyclohexylmethyl group, renin inhibitors and, 217-218 Cyclohexyl SCRIP, renin inhibitors and, 222 Cyclohexyl statine inhibitors, renin inhibitors and.217 Cytochrome P-450,molecular asymmetry and, 82.88 Cytochrome P-450 gene expression, hormonal regulation of, 325-327, 344-345 biosynthesis, 329-337 growth hormone, 338-342 prostate, pituitary, and brain, 342-344 steroids, 337-338 transcription, 327-329 Cytochrome P-450, gene expression, hormonal regulation of, 333 Cytochrome P-45O1,, gene expression, hormonal regulation of, 332-334 Cytochrome P-450,,,, gene expression, hormonal regulation of, 333-334 Cytochrome P-45OC,, gene expression, hormonal regulation of, 333 Cytochrome P -450,,, gene expression, hormonal regulation of, 331-334, 337 Cytoplasm, receptor-activated calcium entry and, 251, 256, 257, 261 Cytoplasmic 5’-nucleotidase, acyclovir and, 5-6

Subject Index dATP, acyclovir and, 6-7 antiviral action, 10-11, 16, 18-19 dCTP, acyclovir and, 6-7,9-12, 18 Debrisoquin, molecular asymmetry and, 8183, 105, 110 Deletion, cytochrome P-450 gene expression and, 331 Delivery of drugs to central nervous system, see Central nervous system, delivery of drugs to Delta sleep-inducing peptide, blood-brain barrier and, 150 Dementia acquired immune deficiency syndrome dementia complex, 273,290-291 senile, 290 2-Deoxy-o-glucose (2DG). blood-brain barrier and, 143, 152, 154-155 Depression phase, osteoporosis therapy and, 47 Depressor activity, renin inhibitors and, 233234 Dexamethasone cytochrome P-450 gene expression and, 327 delivery of drugs to central nervous system and,313-314 Dextrorotatory, definition of, 58-59 dGTP, acyclovir and, 2, 21, 23 antiviral action, mechanism of, 6-7, 9, 1 1 , 13 antiviral action, potentiation of, 13, 16, 18 Diacylglycerol, protein kinase inhibitors and, 169, 180-183, 188 Diastereoisomers, molecular asymmetry and, 62-63 Diastereomers molecular asymmetry and, 62, 68 protein kinase inhibitors and, 184 Diet, osteoporosis therapy and, 31-32 Dietary calcium, osteoporosis therapy and, 32, 35-36,39,42 a-Difluoroketones, renin inhibitors and, 225 Di hydropyridines calcium channel antagonists and, 277-279, 281-282,288-289 delivery of drugs to central nervous system and, 306 Dihydrotesterone (DHT), cytochrome P -450 gene expression and, 342-343 Dihydroxy dipeptide isosteres, renin inhibitors and, 220, 222-223, 225

Subjed Index

Dihydroxyphenylalanine (DOPA), delivery of drugs to central nervous system and, 3 18 Dilazep, acyclovir and, 3 Diltiazem, calcium channel antagonists and, 279,28 I 2,4-Dinitrophenol (DNP), blood-brain barrier and, 143, 145 Dipeptide isosteres, renin inhibitors and, 220227 Dipeptides, renin inhibitors and, 216, 219, 241 Dipeptide transition-state inhibitors, renin and. 2 19-227 Diphenylalkylamines, calcium channel antagonists and, 279 Diphenylpiperazines, calcium channel antagonists and, 279-281 Dipyridamole, acyclovir and, 3 Disopyramide, molecular asymmetry and, 74, 76-77 Distribution, molecular asymmetry and, 67 Ditomers, molecular asymmetry and. 62, 104, I06 pharmacodynamics, 91 -92 therapy, 113, 1 15 Diuretics, renin inhibitors and, 232-233 10 DNA acyclovir and, 2 antiviral action, 6-7, 11-13 resistance, 22 blood-brain banier and, 156 cytochrome P-450 gene expression and, 327-330.332 molecular asymmetry and, 109-1 10 protein kinase inhibitors and, 183, 189-190 DNA polymerase, acyclovir and, 2-3, 23 antiviral action, 6-14, 19 resistance, 20-21 DNase I, cytochrome P-450 gene expression and, 341 DNA sequences. cytochrome P-450 gene expression and, 327-328, 331 Dobutamine, molecular asymmetry and, 113 Dopa, molecular asymmetry and, 67, 1 I I , I 14 L-Dopa, blood-brain barrier and, 147 Dopamine blood-brain barrier and, 147 delivery of drugs to central nervous system and, 306-307,3l3,316-3l7 Dose-response curve calcium channel antagonists and, 282, 289 molecular asymmetry and, 96

36 1 renin inhibitors and, 230, 240 Drug-free interval, osteoporosis therapy and, 47 Drug penetration, molecular asymmetry and, 64

Electronically controlled pumps, delivery of drugs to central nervous system and, 309, 31 I Elimination, molecular asymmetry and, 67 Emopamil, calcium channel antagonists and, 285-286,288 Enalkiren, renin inhibitors and, 214, 226, 230, 232,234-237 Enantiomers definition of, 58 molecular asymmetry and, 58-64, 120 biochemical probes, 108-1 10 clearance, 72-8 1 drug absorption, 67-68 enantioselectivity, 101- 105 impurity, 105-108 interactions, 97- 10I pharmacodynamics of, 88-90, 92, 94-97 pharmacokinetics, 64-67 polymorphic drug disposition, 8 1-85 protein binding, 68-72 therapy and, 11 1-1 18 tissue binding, 68-69 D-Enantiomers, molecular asymmetry and, 68, 107, 109, 115-1 I7 L-Enantiomers, molecular asymmetry and, 6768, 107, 109, I17 Enantioselectivity, 64 biochemical probes and, 1 I I , 113 drug-enantiomer interactions and, 101- 105 immunological, 94-95 pharmacodynamics of, 89-92, 95 pharmacokinetics of, 64-68, 70-72, 76 polymorphic drug disposition and, 81-85. 87 therapy and, 119 Enantiospecificity, molecular asymmetry and, 64, 79, 87, 114, 117 Endocrine regulation, cytochrome P -450 gene expression and, 325, 342-344 Endocrine reproductive status, osteoporosis therapy and, 36-37

Sobiect lndex Endocrine responses, renin inhibitors and, 232-233.239 Endocy tosi s adsorptive, 150- 152 fluid-phase, 151 receptor-mediated, 139, 148 P-Endorphin blood-brain barrier and, 150 delivery of drugs to central nervous system and, 307 Endothelial tight junctions blood-brain barrier and, 142, 154, 157 delivery of drugs to central nervous system and, 301, 303 Endothelium blood-brain barrier and, 138 model, 140- I42 regulation of transport, 152-154, 156157 in v i m transport, 148-151 delivery of drugs to central nervous system and, 308 receptor-activated calcium entry and, 257, 260 renin inhibitors and, 208 Enkephalins, blood-brain barrier and, 149 Enzymes acyclovir and, 22 antiviral action, 6, 10-1 I , 13, 15-17 metabolic activation, 4-5 potentiation, 20-21 blood-brain barrier and, 138-140, 147 calcium channel antagonists and, 273. 275, 28 1 cytochrome P-450 gene expression and, 325, 327, 334, 336, 343-344 delivery of drugs to central nervous system and, 303-304, 307 molecular asymmetry and, 58, 104, 120 enantiomer-enantiomer interactions, 97, 100

pharmacodynamics, 9 I pharmacokinetics, 65, 76 polymorphic drug disposition, 87 protein kinase inhibitors and, 168-169, 194 protein serinelthreonine kinase inhibitors and, 169-170, 173, 175, 178, 184-186 protein tyrosine kinase inhibitors and, 192I93 receptor-activated calcium entry and, 25 1

renin inhibitors and evaluation, 21 I structure, 214-216, 220, 222-223, 226 Epidermal growth factor receptors protein serinelthreonine kinase inhibitors and, 175, 177, 180, 183 protein tyrosine kinase inhibitors and, 187, 189-191 Epilepsy, delivery of drugs to central nervous system and, 299, 306 Epithelial cells, receptor-activated calcium entry and, 252-254 Erbstatin, protein kinase inhibitors and, 189191, 193 Erythrocytes, acyclovir and, 3, 5 Escherichia coli lac Z gene, acyclovir and, 22 Established osteoporosis, 39, 49 Estrogen cytochrome P-450 gene expression and, 338-339,342-343 osteoporosis therapy and, 32, 36-39, 4849 antiresorptive therapy, 42-45 experimental approaches, 47 Estrogen receptors, protein kinase inhibitors and, 181 Ethylenediaminetetraacetic acid (EDTA), renin inhibitors and, 213 Ethylene vinyl acetate (EVA) polymers. delivery of drugs to central nervous system and, 313-314 Eudismic index, molecular asymmetry and, 91,96, 106 Eutomers, molecular asymmetry and, 62 enantioselectivity, 104 pharmacodynamics, 90-91 pharmacokinetics, 68, 7 I therapy, 114-115, 117 Excitotoxins, prevention of neurotoxicity and, 273-275,283,288,290-291 Excretion molecular asymmetry and, 85, 94, 116 renal, 67, 77 Exercise, osteoporosis therapy and, 37, 48 Exocytosis, receptor-mediated, 139, 148 Experimental efficacy models, renin inhibitors and, 229-230 Extensive metabolizers, molecular asymmetry and, 106

Subjed Index Fenfluramine, molecular asymmetry and, 74, 1 I7 Fetal tissue, implantation of, 316-317 Fibroblasts, protein kinase inhibitors and, 193 Fluid-phase endocytosis, blood-brain b e e r and, 151 Flunarizine. calcium channel antagonists and, 279,281,284-286 Fluorescence receptor-activated calcium entry and, 253254,258, 264 renin inhibitors and. 212 Fluoride, osteoporosis therapy and, 45-46, 49 Flurbiprofen, molecular asymmetry and, 98100 Fracture, osteoporosis therapy and, 33, 37-39, 48-49 antiresorptive therapy, 40, 42-43, 45 bone-forming agents, 46-47 Fura-2. receptor-activated calcium entry and, 258

Gastricsin, renin inhibitors and, 210-21 I Gastrointestinal absorption, renin inhibitors and, 241 Geminal diphosphonates, osteoporosis therapy and, 44-45 Gene expression, cytochrome P-450, see Cytochrome P-450 gene expression Genetics, molecular asymmetry and, 81 Genistein, protein kinase inhibitors and, 187I89 Genital herpesvirus infection, acyclovir and, 1-2 Glaucoma, molecular asymmetry and, 95, 97 Glial cells, blood-brain barrier and, 153 Glioma cells, blood-brain barrier and, 154 Glomerular filtration molecular asymmetry and, 71, 76-77 renin inhibitors and, 234 Glucocorticoids, cytochrome P-450 gene expression and, 326, 334, 339, 342-343 Glucose blood-brain barrier and, 143-144, 152-155 transport, I52 calcium channel antagonists and, 280 delivery of drugs to central nervous system and, 301

363 Glucuronides, molecular asymmetry and, 81, 94 Glutamate, calcium channel antagonists and, 273-274,280-282.291 Glutamate receptor, calcium channel antagonists and, 273-274 Glutamic acid, molecular asymmetry and, 116 T-Glutamyl transpeptidase, blood- brain barrier and, 144-145, 153 Glycogen phosphorylase kinase, inhibitors and, 167-168 a,-Glycoprotein, molecular asymmetry and, 70,108 Gonadal hormones, cytochrome P-450 gene expression and, 338 Gonads, cytochrome P-450 gene expression and, 326, 329-331, 342 Gossypol, molecular asymmetry and, 74 gp120, calcium channel antagonists and, 281283,290-29 I Growth factors, cytochrome P-450 gene expression and, 334 Growth hormone (GH) cytochrome P-450 gene expression and, 326-327, 336,338-342 osteoporosis and, 49 Guanosine 5’monophosphate (GMP) kinase, acyclovir and, 5

H-7,protein kinase inhibitors and, 170-171, 174- 175 H-9, protein kinase inhibitors and, 171, 173 H-89, protein kinase inhibitors and, 171, 173 Hapten, molecular asymmetry and, 94 Hemodynamic responses, renin inhibitors and, 232-235,239 Heparin, receptor-activated calcium entry and, 255 Herbimycin A, protein kinase inhibitors and, 192-193 Heroin, delivery of drugs to central nervous system and, 305 Herpes simplex virus (HSV), acyclovir and, 3, 13, 15, 19 Herpes simplex virus type 1 (HSV- I ), acyclovir and, 2 antiviral action, 6-16 metabolic activation, 4

Subject Index

Herpes simplex virus type 2 (HSV-2). acyclovir and, 2 antiviral action, 11, 15-16, 18 metabolic activation, 4-5 Heterocyclic dipetide isosteres, renin inhibitors and, 225-227 Hippocampus calcium channel antagonists and, 274, 27928 1 delivery of drugs to central nervous system and, 314 Hormonal regulation of cytochrome P -450 gene expression, see Cytochrome P -450 gene expression Hormone replacement therapy, molecular asymmetry and, 107 Hormones delivery of drugs to central nervous system and, 300,312 osteoporosis therapy and, 30-31, 38, 4243,47-48 receptor-activated calcium entry and, 253, 261,266 renin inhibitors and, 207 HPPH, molecular asymmetry and, 87 Human immunodeficiency virus type 1 (HIV- I ) , calcium channel antagonists and, 281-282.290-291 Huntington’s disease, delivery of drugs to central nervous system and, 306 Hydrogen blood-brain barrier and, 147 protein kinase inhibitors and, 179 Hydrophilicity, protein kinase inhibitors and, 182 Hydrophobicity, protein kinase inhibitors and, 173, 180-181 Hydroxycinnamamides, protein kinase inhibitors and, 190-191 Hydroxyethylene dipeptide isosteres, renin inhibitors and, 220-222, 225 5-Hydroxytryptamine (5-HT), blood-brain barrier and, 147 Hypercalcemia, osteoporosis therapy and, 42 Hypercalciuria, osteoporosis therapy and, 42, 45 Hyperosmotic treatment, blood-brain barriet and, 156-157 Hypersensitivity acyclovir and, 21-22 molecular asymmetry and, 95

Hypertensin I, renin inhibitors and, 214 Hypertensin 11, renin inhibitors and, 214 Hypertension molecular asymmetry and, 96 renin inhibitors and, 208, 241 pharmacology, 232,235,239-240 structure, 214 Hyperthyroidism, osteoporosis therapy and, 46 Hypocalcemia, cytochrome P -450 gene expression and, 335-336 Hypogonadism, osteoporosis therapy and, 37, 39 Hypophosphatemia, cytochrome P -450 gene expression and, 335-336 Hypophysectomy, cytochrome P-450 gene expression and, 338-341 Hypotension, renin inhibitors and, 232-233, 235,240 Hypothalamic preoptic area (HPOA), cytochrome P-450 gene expression and, 342-344 Hypoxanthine, blood-brain barrier and, 146 Hypoxia, blood-brain barrier and, 155

Ibufenac, molecular asymmetry and, I12 Ibuprofen, molecular asymmetry and, 62 enantiomer-enantiomer interactions, 98-99 pharmacokinetics, 72-73, 76, 78-80 therapy, 113, 115, I18 Immunological renin inhibition, 2 14 Implantable polymer systems, delivery of drugs to central nervous system and, 313-318 Implantation of biological tissues, delivery of drugs to central nervous system and, 3 16318,320 Impurity, enantiomeric, 105- 108 Indole carbazole group, protein kinase inhibitors and, 175-179 Indoprofen, molecular asymmetry and, 73-74 InRammation, molecular asymmetry and, 107, 112 Infusion in animals, renin inhibitors and, 235 Inheritance, osteoporosis and, 32 Inhibitors acyclovir and, 2-4, 20-22 antiviral action, 6, 9-13, 15-18 blood-brain barrier and, 144-145, 147, 150, 153, 156

Subject Index calcium channel antagonists and, 275, 281 cytochrome P-450 gene expression and, 332,334,342 delivery of drugs to central nervous system and, 301, 306 molecular asymmetry and, 62.78, 82.93 enantiomer-enantiomer interactions, 97, 99- 100 enantioselectivity, 101- 102, 104- 105 therapy, 114 osteoporosis therapy and, 44, 47 protein kinase. 167-169, 193-194 protein serinelthreonine kinase, see Protein serinelthreonine kinase inhibitors protein tyrosine kinase, see Protein tyrosine kinase inhibitors receptor-activated calcium entry and, 263 renin, see Renin inhibitors Inosine, acyclovir and, 6 Inosine 5’-monophosphate, acyclovir and, 6 Inositol phosphates, receptor-activated calcium entry and, 255-256, 259-260, 266 lnositol 1.4.5-trisphosphate. receptor-activated calcium entry and, 255-257, 260-263, 265-267 Insulin blood-brain barrier and, 143, 147-148, I55 cytochrome P-450 gene expression and, 341 delivery of drugs to central nervous system and, 312 Insulinlike growth factors blood-brain barrier and, 148 cytochrome P-450 gene expression and, 34 1 protein kinase inhibitors and, 175, 177 Insulin receptors blood-brain barrier and, 148 protein kinase inhibitors and, 167-168, 175, 177, 190-191 Introns, cytochrome P-450 gene expression and, 327 Inversion, chiral, 77-8 I . I 15 Iodoacetamide, protein kinase inhibitors and, I93 Ionization, delivery of drugs to central nervous system and, 300-301 lonomycin, receptor-activated calcium entry and, 263 Ions, blood-brain bamer and, 146- 147 Iron, acyclovir and, 15- 16

365 Ischemia blood-brain barrier and, 155 calcium channel antagonists and, 273-274, 280,283,291 acute ischemic stroke, 288-289 animal models, 283-288 Isoflavones, protein kinase inhibitors and, I87 Isolated brain capillaries, blood-brain bamer and, 138-140, 143, 147-148 Isoprenaline, molecular asymmetry and, 100, 106, 114 Isoquinoline, protein kinase inhibitors and, 170, 173 Isoquinoline sulfonamides, protein kinase inhibitors and, 170-172, 174 Isozymes, protein kinase inhibitors and, 180, 182-183 Isradipine, calcium channel antagonists and, 285-286

K252a. protein kinase inhibitors and, 178-179 Kainate, calcium channel antagonists and, 273-274 KB-2796, calcium channel antagonists and, 285.287 Ketamine, molecular asymmetry and, 99, I17 Ketone dipetide isosteres, renin inhibitors and, 223,225 Kidney cytochrome P -450 gene expression and, 335-337 renin inhibitors and, 210-212, 229 Kinetics, acyclovir and, 3-4, 7. 20-21 KN-62, protein kinase inhibitors and, 185

Lacrimal acinar cells, receptor-activated calcium entry and, 266 Lactate, blood-brain bamer and, 154- 155 Lactation, cytochrome P-450 gene expression and, 326, 335, 343 Latentiation, delivery of drugs to central nervous system and, 304-308, 319 Lavendustin A, protein kinase inhibitors and, I89 Lead intoxication, blood-brain bamer and, I56

366 Leucine-enkephalin uptake, blood-brain barrier and, 149 Leukotrienes, delivery of drugs to central nervous system and, 304 Levodopa, delivery of drugs to central nervous system and, 307, 3 13 Levorotatory, definition of, 59 Lidoflazine, calcium channel antagonists and, 285-286 Ligands, cytochrome P-450 gene expression and, 329, 341, 345 Lipids, delivery of drugs to central nervous system and, 300, 304-306 Lipid solubility, blood-brain barrier and, 151 Liposomes, delivery of drugs to central nervous system and, 304, 307 Lithium, molecular asymmetry and, 105 Liver cytochrome P-450 gene expression and, 326,337-342 renin inhibitors and, 210 Lorazepam acetate, molecular asymmetry and, 104-105 Ltype calcium channel, prevention of neurotoxicity and, 276-278, 282, 291 Lymphomas, delivery of drugs to central nervous system and, 303

Magnesium, acyclovir and, 4, 13 Manganese, receptor-activated calcium entry and, 258-260 Mean arterial blood pressure (MABP), renin inhibitors and, 231-232 Meclofenamate, molecular asymmetry and, 102- I03 Memory, delivery of drugs to central nervous system and, 312, 314 Menopause, osteoporosis therapy and, 38-39, 48 antiresorptive therapy, 42, 44-45 bone mass, 33, 36-37 Mephenytoin, molecular asymmetry and, 110 Mephenytoin phenotype, molecular asymmetry and, 83-87 Messenger RNA (mRNA) cytochrome P-450 gene expression and, 331-337.339 protein kinase inhibitors and, 179, 184

Subject Index

Metabolism blood-brain barrier and, 138 molecular asymmetry and, 67, 99, I 13 Methacholine, receptor-activated calcium entry and, 259-260 Methotrexate delivery of drugs to central nervous system and, 303, 305, 312 molecular asymmetry and, 67.77, 11 I , 114 D-Methotrexate, molecular asymmetry and, 67-68, 109 Methylation, cytochrome P-450 gene expression and, 329 N-Methyl-D-aspartate (NMDA), calcium channel antagonists and, 273, 275, 280281,283 N-Methyl-D-aspartate (NMDA) receptors, calcium channel antagonists and, 274, 279,282 3-0-Methyl-D-glucose (3MG), blood-brain barrier and, 143, 152, 156 Methyldopa, blood-brain barrier and, 144 N-Methylpyridinium-2-carbaldoximechloride (2-PAM), delivery of drugs to central nervous system and, 306 Metoprolol, molecular asymmetry and, 76-77, 82-83. 106 Metrazol, delivery of drugs to central nervous system and, 302 Microsomes, molecular asymmetry and, 99 Microvessel endothelium, blood-brain barrier and, 138 model, 140- 142 regulation of transport, 152, 156-157 in virro transport, 143, 145, 148-151 Microvessels blood-brain bamer and, 156-157 brain, 152-153 Mineral density, osteoporosis therapy and, 3335, 39, 42, 46 Mineralization, osteoporosis therapy and, 3031,44 Mitochondria, cytochrome P-450 gene expression and, 326, 335-337, 344 ML-89, protein kinase inhibitors and, 171, 173 MLCK, see Myosin light-chain kinase (MEW Molecular asymmetry, 58, 120 enantiomer-enantiomer interactions, 97- 101 enantiomeric impurity, 105-108 enantioselectivity, 101- 105

Subject lndex

nomenclature, 58-64 pharmacodynamics, 88-89 acceptors, 91-92 asymmetric senses, 89-90 discrimination, 90-91 enantioselectivity, 94-95 receptors, 91-94 p receptors, 95-97 timolol, 95-96 pharmacokinetics, 64-66 pharmacokinetics of enantiomers, 67 clearance, 72-81 drug absorption, 67-68 protein binding, 68-72 tissue binding, 68-69 polymorphic drug disposition mephenytoin phenotype, 83-88 sparteine-debrisoquin phenotype, 81-83 therapy, 1 10- 1 12 achiral drugs, 112-1 13 bioavailability, I 17- I18 drug monitoring, I19 enantiomeric drugs, 1 13- 1 17 nonracemic drugs, 1 12 racemate, I17 racemic drugs, 113 Monoamine oxidase. blood-brain barrier and, 147 Monoclonal antibodies blood-brain barrier and, 148 renin inhibitors and, 214 Morphine, delivery of drugs to central nervous system and, 305, 312 Morphology. protein kinase inhibitors and, 192-193 mRNA, see Messenger RNA Multiple-dose clinical studies, renin inhibitors and, 235 Mutation acyclovir and, 4, 16, 19-22 cytochrome P-450 gene expression and, 330-332 Myosin, protein kinase inhibitors and, 170 Myosin light-chain kinase (MLCK). inhibitors and, 170-171, 173, 177, 185-186

Na+, K + -ATPase, blood-brain barrier and, 146-147, 153

367 Naphthalene, protein kinase inhibitors and, 170, 173, 191 Naphthalene sulfonamides, protein kinase inhibitors and, 170, 177 NBMPR, acyclovir and, 3 Nephrotic syndrome, molecular asymmetry and, I16 Nervous tissue, protein kinase inhibitors and, 170 Neurosteroids, cytochrome P-450 gene expression and, 343 Neurotoxicity, prevention of, calcium channel antagonists and, see Calcium channel antagonists Neurotransmitters blood-brain barrier and, 147 delivery of drugs to central nervous system and, 312, 316-318, 320 receptor-activated calcium entry and, 253. 260-26 1,266 renin inhibitors and, 233 Neutrophils, protein kinase inhibitors and, 174, 191 Nicardipine, prevention of neurotoxicity and, 284-287 Nifedipine, prevention of neurotoxicity and, 280-28 1 Nilvadipine, prevention of neurotoxicity and, 285 Nimodipine, prevention of neurotoxicity and, 279,281-282,284-286,288-290 Nitrendipine, prevention of neurotoxicity and, 286 Nonracemic drugs, molecular asymmetry and. 112 Noradrenaline, molecular asymmetry and, 61, 68,96-97 Norepinephrine, blood-brain barrier and, 147 Norstatine, renin inhibitors and, 213 Nucleoside 5'-monophosphate, acyclovir and, 5 Nucleosides acyclovir and, 3-4, 7, 13, 19 blood-brain barrier and, 145-146 protein kinase inhibitors and, 179 Nucleotides acyclovir and, 10-11, 19, 21-22 blood-brain barrier and, 145-146 protein kinase inhibitors and, 179 receptor-activated calcium entry and, 255 Nutrients, blood-brain barrier and, 143-146

368

Subjed Index

Oncogenes, protein kinase inhibitors and, 192 Optical activity, molecular asymmetry and, 59-60 Optical isomers, molecular asymmetry and, 58 Optical purity, molecular asymmetry and, 105-107 Oral efficacy, renin inhibitors and, 238-240 Orphan receptors, cytochrome P-450 gene expression and, 345 Osteoblasts, osteoporosis therapy and, 36, 41, 45 Osteocalcin, osteoporosis therapy and, 48 Osteoclasts, osteoporosis therapy and, 30, 36, 43,47 Osteomalacia, osteoporosis therapy and, 4 I , 44 Osteoporosis, cytochrome P-450 gene expression and, 335 Osteoporosis therapy, 29-30, 37-39.48-49 AFDR therapy, 47 age-related changes in bone mass, 32-34 antiresorptive therapy, 39-45 bone-forming agents, 45-47 bone mass regulation, 34, 37 calcium, 35-36 endocrine reproductive status, 36-37 physical activity, 34-35 experimental approaches, 47-48 nonpharmacological considerations, 48 prevention of osteoporosis, 39 skeletal organization, 30-31 Ouabain, blood-brain barrier and, 144- 146, 155

Oxidation, molecular asymmetry and, 81-83, 87, 101, 104 Oxygen blood-brain barrier and, 155 protein kinase inhibitors and, 184 renin inhibitors and, 225

central nervous system and, 300, 307, 313,316-318 Parotid, receptor-activated calcium entry and, 26 I Parotid acinar cells, receptor-activated calcium entry and, 256-258 Pathological changes, blood-brain barrier and, 155-157 Penicillamine, molecular asymmetry and, 115I I7 Pepsin, renin inhibitors and, 210-21 1 Pepstatin, renin inhibitors and, 214, 216-217 Peptide-binding site, protein kinase inhibitors and, 169 Peptides blood-brain barrier and, 147- 150 regulation, 152-153 delivery of drugs to central nervous system and. 301,304-305, 307-308 receptor-activated calcium entry and, 253 renin inhibitors and, 213-214, 238, 241 Peptide substrates, protein kinase inhibitors and, 185-186, 189-190 Peptide transition-state inhibitors, renin and, 2 16-219 Peripheral skeleton, osteoporosis therapy and, 30 Peristaltic pump, delivery of drugs to central nervous system and, 3 I I Permeability blood-brain barrier and, 151-154, 156-157 delivery of drugs to central nervous system and, 302, 305 protein kinase inhibitors and, 174, 186 receptor-activated calcium entry and, 254255, 257-258.260, 263 pH, renin inhibitors and, 21 1-212, 219. Pharmacodynamics, molecular asymmetry and, 58-59,63,66,71, 88-89, 120 acceptors, 91-92 discrimination, 90-91 enantiomer-enantiomer interactions, 97, 100-101

Paget’s disease, osteoporosis therapy and, 44 Parathyroid hormone (FTH) cytochrome P-450 gene expression and, 336-337 osteoporosis therapy and, 40-41, 46-48 protein kinase inhibitors and, 184 Parkinson’s disease, delivery of drugs to

enantiomeric impurity, 105 enantioselectivity, 94-95, 101 pharmacokinetics, 66, 71 receptors, 91 -97 senses, 89-90 therapy, I 13, I15 timolol, 95-96

Subjed Index

Pharmacokinetics of enantiomers, 67 clearance, 72-81 drug absorption, 67-68 enantioselectivity, 103 impurity, 105, 107 interactions, 97, 101 protein binding, 68-72 tissue binding, 68-69 molecular asymmetry and, 58, 63, 120 concepts, 64-66 pharmacodynamics, 94 therapy, 115, 117 renin inhibitors and, 210, 239-240 Phenotype acyclovir and, 20-21 cytochrome P-450 gene expression and, 330 molecular asymmetry and, 81-88, 110 protein kinase inhibitors and, 192- 193 Phenylalkylamines, calcium channel antagonists and, 279-281, 288 Phenylbutazone, molecular asymmetry and, 78, 101-103 Phenylethylhydantoin, molecular asymmetry and, 87 Phenytoin, molecular asymmetry and, 87 Phorbol esters blood-brain barrier and, 143 protein kinase inhibitors and, 169, 175, 1; 78, 182, 188 Phosphate transfer, protein kinase inhibitors and, 178, 191-192 Phosphatidylinositol kinase, inhibitors and, I89 Phosphatidylserine, protein kinase inhibitors and, 180-181, 183 Phosphodiesterase, protein kinase inhibitors and, 183-184 Phospholipase C. receptor-activated calcium entry and, 256, 266 Phospholipids blood-brain bamer and, 145 protein kinase inhibitors and, 18 1 Phosphorus cytochrome P-450 gene expression and, 335-336 osteoporosis therapy and, 47 protein kinase inhibitors and, 184 Phosphorus, renin inhibitors and, 226-229

369 Phosphorylase kinase, inhibitors and, 179 Phosphorylation acyclovir and, 2-3, 22 antiviral action, 12, 16, 19 metabolic activation, 3-6 resistance, 20-21 blood-brain barrier and, 143 cytochrome P-450 gene expression and, 331,341 protein kinase inhibitors and, see Protein kinase inhibitors; Protein serinelthreonine kinase inhibitors; Protein tyrosine kinase inhibitors receptor-activated calcium entry and, 25 I , 255,265 Physical activity, osteoporosis therapy and, 32, 34-35,37,39 Pindolol, molecular asymmetry and, 76-77 Pinocytosis, blood-brain barrier and, 140, 151-152, 156 Pituitary, cytochrome P-450gene expression and, 326,341-344 Plasma cytochrome P-450gene expression and, 335-336 molecular asymmetry and, 82. 85, 102, 106-107, 1 I9 renin inhibitors and, 210, 229 pharmacology, 230, 232-235, 238-240 Plasma membrane blood-brain barrier and, 146 receptor-activated calcium entry and, 252 epithelial cells, 253 mechanisms, 255-257. 259, 261-263, 266 Plasma proteins, molecular asymmetry and, 69-71, 77, 81, 98, 120 Plasma renin activity (PRA), inhibitors and, 212-214, 231-235.239 Platelet-activating factor molecular asymmetry and, I 13 protein kinase inhibitors and, 188 Platelet-derived growth factor, protein kinase inhibitors and, 187 Platelets, protein kinase inhibitors and, 170, 174-175, 188 Polyanhydride polymers, delivery of drugs to central nervous system and, 314-315 Polymerase chain reaction, cytochrome P-450 gene expression and, 344

370 Polymerases, acyclovir and, 11-13. 22 Polymer systems, implantable, delivery of drugs to central nervous system and, 313318 Polymorphic drug disposition, molecular asymmetry and, 81-88 Polypeptides, renin inhibitors and, 215 Poor metabolizer phenotype, molecular asymmetry and, 81-82, 87, 97 Portacaval anastomosis (PCA), blood-brain barrier and, 156 Potassium, see also Na+, K+ -ATPase blood-brain barrier and, 146-147, 155 receptor-activated calcium entry and, 253, 257 Potency, renin inhibitors and, 212 Potentiation of antiviral action, acyclovir and, 3, 13-19 PRA, see Plasma renin activity Practolol, molecular asymmetry and, 95 Pregnancy, cytochrome P-450 gene expression and, 326, 336, 343-344 Prevention of neurotoxicity, calcium channel antagonists and, see Calcium channel antagonists Primary osteoporosis, 37-38 Prodrugs delivery of drugs to central nervous system and, 305-308,319 protein kinase inhibitors and, 191 Prolactin, cytochrome P-450 gene expression and, 336 Propranolol blood-brain barrier and, 151 enantiomers and biochemical probes, 109-1 10 interactions, 99- 101 molecular asymmetry and pharmacodynamics, 94 pharmacokinetics, 68-73 therapy, 11I , 113, 119 Prorenin, inhibitors and, 210 Prostaglandins blood-brain barrier and, 152 molecular asymmetry and, 114 Prostate, cytochrome P-450 gene expression and, 326, 341-344 Protein blood-brain barrier and, 147-150, 153-155 cytochrome P-450 gene expression and, 325,344

Subject lndex

biosynthesis, 330, 332-337 transcription, 327-328 delivery of drugs to central nervous system and, 301,303 altering agents, 305, 307-308 circumventing barrier, 309, 3 12-3 13, 3 16 molecular asymmetry and, 58, 104, 120 pharmacodynamics, 9 1,94 pharmacokinetics, 64,81 renin inhibitors and, 212 Proteinase specificity. renin inhibitors and, 210 Protein binding enantiomers and biochemical probes, 101- 102 interactions, 98- 100 molecular asymmetry and, 68-72 Protein kinase A , cytochrome P-450 gene expression and, 330-33 1 Protein kinase C blood-brain barrier and, 143 cytochrome P-450 gene expression and, 342 inhibitors of, 169-170, 180-183, 185-186, 193

competition with ATP, 171, 173-175, 177, 179-180 receptor-activated calcium entry and, 253 Protein kinase inhibitors, 167-169, 193-194, see also Protein serine/threonine kinase inhibitors; Protein tyrosine kinase inhibitors Proteinlpeptide substrates, protein kinase inhibitors and, 185-186, 189-190 Protein products, protein kinase inhibitors and, I92 Protein serine/threonine kinase inhibitors, 169-170, 187, 189-190, 193 calmodulin-dependent protein kinases, 184185

competition with ATP, 170-180 cyclic nucleotides, 183- 184 protein kinase C, 180-183 protein/peptide substrate, 185- 186 Protein tyrosine kinase inhibitors, 177, 179180, 183, 186-187 competition with ATP, 187-189 modes of action, 192-193 proteinlpeptide substrate, 189-191 transition state analogs, 191-192 Prothrombin, molecular asymmetry and, 103, 119 PTH, see Parathyroid hormone

Subject Index Pumps, delivery of drugs to central nervous system and, 309-313, 319 PY 108-068, prevention of neurotoxicity and, 287,289

Quercetin, protein kinase inhibitors and, I87 Quinidine, molecular asymmetry and, 62, 74 Quinine, molecular asymmetry and, 62-63 Quisqualate, prevention of neurotoxicity and, 273,280

Racemates enantiomers and biochemical probes, 110 enantioselectivity, 103 interactions, 98, 100-101 molecular asymmetry and pharmacokinetics, 74.76-78 therapy, 113-117, 119 Racernic drugs definition of, 58 molecular asymmetry and, 59, 119-120 enantiomer-enantiomer interactions, 99, 101 enantiomers as biochemical probes, I10 enantioselectivity. 102, 104 pharmacodyndmics, 92.94-95 pharmacokinetics, 80-8 1 polymorphic drug disposition, 82-83 therapy, 111-113, 115, 117, 119 Racemization, molecular asymmetry and, 78, 111,114-115 Radioimmunoassay, renin inhibitors and, 2 12, 235 Receptive bodies, optically active, 59 Receptor-activated calcium entry, see Calcium entry, receptor-activated Receptor-mediated endocytosis, blood-brain banier and, 139, 148 Receptor-mediated exocytosis, blood-brain barrier and, 139, 148 Receptors blood-brain banier and, 148- 149 calcium channel antagonists and, 273 cytochrome P-450 gene expression and, 339, 341-343, 345

37 1 biosynthesis, 329, 331, 334 enantiomers and, 97, 100 molecular asymmetry and, 60-61, 65 biochemical probes, 108 pharmacodynamics, 91-92 polymorphic drug disposition, 89-90 protein kinase inhibitors and, 174, 178 renin inhibitors and, 209 P-Receptors, molecular asymmetry and, 6869.95-97 Rectus molecule, definition of, 61 Red blood cells, blood-brain banier and, 143 Redox chemical systems, delivery of drugs to central nervous system and, 306 Remodeling space, osteoporosis therapy and, 43 Renal clearance, molecular asymmetry and, 76-77,94, 109 Renal dysfunction, molecular asymmetry and, 81 Renal effects, renin inhibitors and, 234 Renal excretion, molecular asymmetry and, 67,77 Renal mitochondria, cytochrome P-450 gene expression and, 326,335-337 Renin, cytochrome P-450 gene expression and, 339 Renin-angiotensin-aldosterone system (RAAS), 207-208,241 pharmacology, 229,233,238 properties, 208-209 structure, 214 Renin-angiotensin system, inhibitors and, 212 Renin inhibitors, 207-208, 241 evaluation, 210-213 pharmacology acute effects, 230-234 chronic administration, 234-235 experimental efficacy models, 229-230 hypotensive effect, 235-238 oral efficacy, 238-240 side effects, 208 plasma renin activity assays, 213 properties, 208-210 proteinase specificity, 210 species specificity, 213-214 structure design, 214-215 dipeptide transition-state inhibitors, 2 19227 immunology, 2 14

372 Renin inhibitors ( c o n / . ) nonpeptide inhibitors, 227-229 peptide transition-state inhibitors, 216219 substrates, 215-216 Renin-inhibitory peptide (RIP), 2 16 Replication, acyclovir and, 2, 16, 28 Reproduction, osteoporosis therapy and, 3637, 39,48 Resistance, acyclovir and, 19-21 Retinal ganglion cell neurons, calcium channel antagonists and, 280-282 Rheumatoid arthritis, molecular asymmetry and,70, 1 1 1 , 117 Ribonucleotide reductase, acyclovir and, 2-3, 23 antiviral action, 13-17, 19 hypersensitivity, 2 1-22 Ricin, blood-brain barrier and, 141 RK-286C, protein kinase inhibitors and, 179 RNA polymerase 11, cytochrome P-450 gene expression and, 327, 329, 331

Sarcoplasmic reticulum, receptor-activated calcium entry and, 252, 256 Secondary osteoporosis, 37-38 Second messengers cytochrome P-450 gene expression and, 33 1,342 protein kinase inhibitors and, 169, 186, 188 receptor-activated calcium entry and, 256 Selective inhibition, acyclovir and, 2-3 Sequence homology protein kinase inhibitors and, 177 renin inhibitors and, 217 Sequences cytochrome P-450 gene expression and, 327-328,330-333,339 crsl, 332-333 CTE, 328 SDI, 328 protein kinase inhibitors and, 185, 191 renin inhibitors and, 213 Signal transduction cytochrome P -450 gene expression and, 326.33 I , 334-335 protein kinase inhibitors and, 184

Subject Index Sinister molecule, definition of, 61 Site-specific inhibitors, protein kinase and, 169 Skeletal organization, osteoporosis therapy and, 29-3 I Sodium, see ulso Na+, K - ATPase blood-brain barrier and, 144, 146-147, 155 calcium channel antagonists and, 273, 282 protein kinase inhibitors and, I79 renin inhibitors and, 235 Sodium azide, blood-brain barrier and, 145 Solenoid pump, delivery of drugs to central nervous system and, 3 I I Somatostatin, blood-brain barrier and, 149 Sparteine, molecular asymmetry and, 105 Sparteine-debrisoquin phenotype, molecular asymmetry and, 81-83 Spatial memory, delivery of drugs to central nervous system and, 314 Species specificity, renin inhibitors and, 213214 Specificity. renin inhibitors and, 210, 213214,220 Sphingosine, protein kinase inhibitors and, 1x2-I83 Statine, renin inhibitors and species specificity, 213-214 structure, 216-21 7,219-220, 222-223 Statine-containing renin-inhibitory peptides (SCRIP), 217,222 Statine dipeptide isosteres, renin inhibitors and, 222,224-225 Staurosporine, protein kinase inhibitors and, 175, 177-179 Stereoisomers, molecular asymmetry and, 62, 74 Stereoselectivity, molecular asymmetry and, 64,89 Stereospecificity blood-brain bamer and, 143 molecular asymmetry and, 64,104, 107 protein kinase inhibitors and, 184 Steroidogenesis, cytochrome P-450 gene expression and, 328-331, 334-335 Steroids blood-brain barrier and, 147 cytochrome P-450 gene expression and, 326,337-338,342-343,345 biosynthesis, 329, 331, 333 Strepfomyces, protein kinase inhibitors and, 189, 192 +

373 Striatuni, delivery of drugs to central nervous system and, 316-317 Stroke, acute ishemic, calcium channel antagonists and. 288-289 Structure-activity relationships molecular asymmetry and, 60. 62. I0X protein kinase inhibitors and. 179 renin inhibitors and, 212, 229 Substrate analog inhibitors, renin and, 216 Substrate enantioselectivity, 66 Sulfur, protein kinase inhibitors and, 184 Sulindac, molecular asymmetry and, 107 Suramin, protein kinase inhibitors and, 183 Susceptibility, protein kinase inhibitors and, 178

Tamoxifen, protein kinase inhibitors and. 181 Tartrates, molecular asymmetry and, 59 T cells, protein kinase inhibitors and, 175, 187, 193 Teratogenicity, molecular asymmetry and, I I I , 1 I6 Testosterone, osteoporosis therapy and, 36. 46 Tetradecapeptides, renin inhibitors and, 2 I5 Tetrafluorotyrosine, protein kinase inhibitors and, 191 Thalidomide, molecular asymmetry and, I I I , I I6 Thapsigargin, receptor-activated calcium entry and, 259-261, 265 Therapeutic drug monitoring, molecular asymmetry and, 119 Therapeutics blood-brain barrier and, 157 for cancer treatment, 169 Thiazide diuretics, osteoporosis therapy and. 45 Thrombin, protein kinase inhibitors and. 174 Thymidine acyclovir and, 3, 15, 19-20 blood-brain barrier and, 146 Thymidine kinase, acyclovir and. 2-3, 22 antiviral action, 16, 19-21 metabolic activation, 3-5 Thyroid hormone, molecular asymmetry and, 107 Thyroxine

molecular asymmetry and. 107- 108. 1 14- I 15 protein kinase inhibitors and, I85 L-Thyroxine, molecular asymmetry and, 107 Tiaprofenic acid, molecular asymmetry and. 72-73 Tight junctions, endothelial. see Endothelial tight junctions Timolol, molecular asymmetry and, 68-69, 89.95-97, I14 Tissue binding, molecular asymmetry and, 6869, 91,98-99 Tissue specificity, cytochrome P-450 gene expression and, 326. 344 Tofisopam, molecular asymmetry and, 72 Total body clearance, molecular asymmetry and, 72-76 Toxicity cytochrome P-450 gene expression and, 325-326 delivery of drugs to central nervous system and, 303, 305, 308 molecular asymmetry and, 117 protein kinase inhibitors and, 189 TPA, cytochrome P-450 gene expression and, 337 Trabecular bone, osteoporosis therapy and, 30, 32-34.46-48 Transcription cytochrome P-450 gene expression and, 338,344-345 biosynthesis, 330-333 regulation, 327-329 protein kinase inhibitors and, 179 Transcription factors, cytochrome P -450 gene expression and, 327, 329, 33 I , 333 Transcytosis, blood-brain barrier and, 148 Transfemn, blood-brain bamer and, 142. 148 Transforming growth factor+, osteoporosis therapy and, 47 Transition-state analogs, protein kinase inhibitors and, 191-192 Transition-state inhibitors dipeptide, 219-227 peptide, 216-219 Transplantation, delivery of drugs to central nervws system and, 316-319 Triiodothyronine, protein kinase inhibitors and, I85 Trypsin, renin inhibitors and, 215

374

Subiecf Index

n m o r promoters cytochrome P-450 gene expression and, 337

protein kinase inhibitors and, 169, 178 receptor-activated calcium entry and, 259 'hmors delivery of drugs to central nervous system and, 312,315 protein kinase inhibitors and, 181-182, 189

Type I osteoporosis, 38 Type 11 osteoporosis, 38 Tyrosine hydroxylases, delivery of drugs to central nervous system and, 318 Tyrosine kinase, cytochrome P-450 gene expression and, 341

Vapor-pressure-powered pump, delivery of drugs to central nervous system and, 309312

Verapamil, prevention of neurotoxicity and, 279-281.284

Vero cells, acyclovir and, 2, 4-6, 13, 16, 19 Virus, delivery of drugs to central nervous system and, 300, 303 Vitamin D, osteoporosis therapy and, 36, 39, 41-42

Vitamin D, la-hydroxylase, cytochrome P-450 gene expression and, 335-337 Voltage activated calcium signaling, 252 Voltage-dependent calcium channels, prevention of neurotoxicity and, 272-283, 288,291

W-7, protein kinase inhibitors and, 170 Warfarin, molecular asymmetry and, 71, 94 enantioselectivity, 101- 105 therapy, 119 Wilson's disease, molecular asymmetry and, 116-117

Varicella zoster virus (VZV), acyclovir and, 2, 4-5, 15-16

Vasoactive peptide, blood-brain barrier and, 152-153

Vasopressin, blood-brain barrier and, 149150

Xenobiotics, cytochrome P-450 gene expression and, 326, 337