No title

ADVANCES I N

PHARMACOLOGY VOLUME 35

J. Thomas August

Ferid Murad

Baltimore, Maryland

Lake Bluff, Illinois

M. W. Anders

Joseph T. Coyle

Rochester, New York

Belmont, Massachusetts

ADVISORY BOARD

R. Wayne Alexander

Floyd E. Bloom

Boston, Massachusetts

La Jolla, California

Thomas F. Burke

Leroy Liu

Houston, Texas

Piscataway, New Jersey

Anthony R. Means

G. Alan Robison

Durham, North Carolina

Houston, Texas

John A. Thomas

Thomas C. Westfall

Houston, Texas

St. Louis, Missouri

ADVANCES IN

PHARMACOLOGY VOLUME 35

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 Molecular Geriatrics Corporation Lake Bluff, Illinois

Joseph T. Coyle Harvard Medical School McLean Hospital Belmont. Massachusetts

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1996 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. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX International Standard Serial Number: 1054-3589 International Standard Book Number: 0-12-032936-0 PRINTED IN THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 0 1 B B 9 8 7 6 5

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Contents

Contributors xiii

Interactions between Drugs and Nutrients C. Tschanz, W . Wayne Stargel, and J. A. Thomas

I. Introduction 1 11. Factors Affecting Gastrointestinal Absorption 3 111. Bioavailability and Metabolism 8 IV. Excretion and Elimination 8 V. Special Interactions/Conditions 10 A. Antimicrobials 10 B. Drug-Ethanol Interactions 11 C. Drug-Vitamins 14 D. Drug-Minerals 15 E. Genetic Differences 17 F. Geriatrics 20 G. Hypoglycemics 22 H. Parenteral Nutrition 23 VI. Concluding Remarks 24 References 24

Induction of Cyclo-Oxygenase and Nitric Oxide Synthase in Inflammation Ian Appleton, Annette Tomlinson, and Derek A. Willoughby

I. General Introduction 27

V

vi

Contents

11. Cyclo-Oxygenase 28 A. Isoforms of Cyclo-Oxygenase 29 III. Regulation of COX-1 and COX-2 30 IV. Prostanoids 31 A. Prostanoids and Inflammation 33 B. Prostanoids and Chronic Inflammation 37 V. Prostaglandins and Pain 38 A. Nonsteroidal Anti-Inflammatory Drugs and Inflammatory Pain 38 VI. COX-1 and COX-2 in Inflammation 40 VII. Pharmacological Inhibition of COX-1 and COX-2 41 VIII. Conclusion 44 IX. Nitric Oxide Synthase 45 A. Induction and Inhibition of Nitric Oxide Synthase 46 B. Cellular Production and Activity of NO at Inflammatory Sites 47 C. Arginine Metabolism at Inflammatory Sites 48 D. Nitric Oxide Production by Human Macrophages 50 E. Cell Types in Which Nitric Oxide Synthase Is Induced 50 F. Cytotoxicity and Tissue Damage 50 X. Nitric Oxide in Mechanisms of Acute Inflammation 51 A. Complement-Mediated and Cell-Mediated Inflammatory Models 52 B. Neurogenic Inflammation 53 XI. Nitric Oxide in Mechanisms of Chronic Inflammation 53 A. Nitric Oxide in Angiogenesis 54 XII. Nitric Oxide in Inflammatory Disease 55 A. Rheumatoid Arthritis 55 B. Multiple Sclerosis 56 C. Graft-Versus-Host Reaction 56 D. Renal Inflammation 57 E. Gastrointestinal Inflammation 57 F. Other Inflammatory Disease States 58 [. Involvement of Nitric Oxide in Inflammatory Pain 5 XIV. Conclusion 59 XV. Interactions between the Nitric Oxide Synthase and CycloOxygenase Pathways 59 References 61

Current and Future Therapeutic Approaches to Hyperlipidemia John A. Farmer and Antonio M. Gotto, Jr.

I. Introduction

79

Contents

vii

II. Agents That Predominantly Lower Cholesterol 80 A. Bile-Acid Sequestrants 81 B. 3-Hydroxy-3-MethylglutarylCoenzyme A Reductase Inhibitors 87 C. Probucol 94 D. Estrogen-Replacement Therapy 96 111. Agents That Predominantly Lower Triglyceride 98 A. Nicotinic Acid 99 B. Fibric-Acid Derivatives 101 C. Fish Oil 104 IV. Combination-Drug Therapy 105 V. Future Developments 105 VI. Conclusion 107 References 107 In Vivo Pharmacological Effects of Ciclosporin and Some Analogues Jean F. Borel, G ~ Q Baurnann, Ian Chapman, Peter Donatsch, Alfred Fahr, Edgar A. Mueller, and Jean-MarieVigouret

I. Introduction and Summary 115 11. Molecular Mechanism of Immunosuppression 124 111. Suppressive Effects on Cell-Mediated Immunity 128 A. T Cell-Mediated Cytotoxicity 128 B. T-cell Functions for Help, Memory, and Delayed-Type Hypersensitivity (DTH) 133 C. Cell-Mediated Suppressor Function 137 D. Induction of Antigen-Specific Hypo- or Unresponsiveness 143 IV. Other Biological Effects Associated with the Immunosuppressive Activity 154 A. Chronic Inflammation 154 B. Prolactin Antagonism 154 C. Possible Interactions with the Central Nervous System 156 D. Major Side Effects 158 V. Biological Effects Possibly Correlated with the Immunosuppressive Activity 165 A. Development of Suppressor Cells 165 B. Interference in the Regulation of Tolerance to Self and Nonself 165 C. TherapeutiGEffects in Psoriasis 172 D. Therapeutic Effects in Asthma? 175 E. Effects on Hair Follicles 179

viii

Contents

VI. Biological Effects Appearing Independently of Immunosuppressive Activity 180 A. Effects on Nonlymphoid Cells 180 B. Effects on Various Cellular Functions 187 C. Antibiotic Effects 189 VII. Chronic Allograft Rejection 194 A. Clinical Situation 194 B. Experimental Approaches 195 C. Factors Involved in Chronic Allograft Rejection 199 VIII. Impact of Galenic Formulation on Pharmacokinetics 204 A. Clinical Pharmacokinetics of CS (SANDIMMUN) 204 B. New Galenical Formulation of CS (SANDIMMUN NEORAL) 207 References 208

Mono-ADP-ribosylation: A Reversible Posttranslational Modification of Proteins Ian J. Okazaki and Joel Moss

I. Introduction 247 11. Mono-ADP-ribosyltransferases 250 A. Avian ADP-ribosyltransferases 250 B. Mammalian ADP-ribosyltransferases 256 C. Inhibitors of Mono-ADP-ribosyltransferase 26 1 111. Conserved Regions among ADP-ribosyltransferases 262 A. Region I 263 B. Region11 265 C. Region In 266 IV.ADP-ribosylarginine Hydrolases 268 A. Turkey ADP-ribosylarginine Hydrolase 269 B. Mammalian ADP-ribosylarginine Hydrolases 270 V. Summary 271 References 272

Activation of Programmed (Apoptotic) Cell Death for the Treatment of Prostate Cancer Samuel R. Denmeade and John T. lsaacs

I. Overview of the Problem 281 11. Androgen Sensitivity of Prostate Cancer 282

Contents

ix

111. Cell Kinetics during Progression of Prostate Cancer 283

IV. Proliferation-Independent Therapeutic Approach for AndrogenIndependent Proliferate Cancer Cells 285 V. Summary of the Temporal Sequences Involved in the Programmed Death of Normal Prostatic Glandular Cells Following Androgen Ablation 287 VI. Prostate Gene Expression during Programmed Cell Death Pathway Induced by Androgen Ablation 290 VII. Role of Cell Proliferation in the Prostatic Death Process Induced by Castration 291 VIII. Androgen Ablation Induced Programmed Cell Death Does not Require Recruitment into a Perturbed Cell Cycle 293 IX. p53 Expression Is not Required for Androgen Ablation-Induced Programmed Death of Go Prostatic Glandular Cells 294 X. Redefining the Prostate “Cell Cycle” 295 XI. Therapeutic Implication of Programmed Cell Death for Prostatic Cancer 295 XII. Ability of Thapsigargin (TG) to Activate Programmed Cell Death 297 XIII. Thapsigargin as Therapy for Prostate Cancer 300 References 302

Reversal of Atherosclerosis with Therapy: Update of Coronary Angiographic Trials Howard N. Hodis

I. Overview 307 11. Coronary Angiographic Trials Utilizing Pharmacological Intervention 308 A. The NHLBI Type I1 Coronary Intervention Study 308 B. The Cholesterol-LoweringAtherosclerosis Study (CLAS) 308 C. The Familial Atherosclerosis Treatment Study (FATS) 309 D. The University of California, San Francisco, Specialized Center of Research Intervention Trial (UCSF SCOR) 309 E. The Monitored Atherosclerosis Regression Study (MARS) 310 F. The Canadian Coronary Atherosclerosis Intervention Trial (CCAIT) 311 G. Multicentre Anti-Atheroma Study (MAAS) 314 H. The Stanford Coronary Risk Intervention Project (SCRIP) 314 I. The St. Thomas’ Atherosclerosis Regression Study (STARS) 315

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Contents

111. Coronary Angiographic Trials Utilizing Nonpharmacological Intervention 3 15 A. The Leiden Intervention Trial 3 15 B. The Lifestyle Heart Trial 316 C. Heidelberg Exercise-Diet Study 3 16 D. The Program on the Surgical Control of the Hyperlipidemias (POSCH) 317 IV. Summary of the Coronary Angiographic Trials 3 17 V. Conclusions from Coronary Angiographic Trials 317 References 3 18

Unnatural Nucleotide Sequences in Biopharmaceutics Lawrence A. Loeb

I. Introduction 321 11. Site-SpecificMutagenesis and Rational Drug Design 322 111. Molecular Evolution and Its Consequences 323 IV. Random Molecular and Chemical Libraries 324 A. Random Genetic Selection for Biologically Active Proteins 324 B. Phage Display Libraries for Binding Proteins 324 C. Peptide Libraries for Modeling Peptide Hormones and Drugs 326 D. Nucleic Acid Libraries 326 E. Combinatorial Chemical Libraries 328 V. Random Sequence Selection 328 A. General Protocol 328 B. Choice of a Plasmid Vector and Host 329 C. “Dummy” or Nonfunctional Inserts 330 D. Oligonucleotide Inserts Containing Random Nucleotide Sequences 330 E. Randomization of Oligonucleotides 33 1 F. Combinatorial Consideration 332 VI. Applications of Random Sequence Selection 333 VII. Regulatory DNA Sequences and Binding Proteins 333 VIII. Production of Mutant Enzymes 334 A. P-Lactamase 334 B. Related Studies 335 IX. Gene Therapy for Human Cancer 336 A. Herpes Thymidine Kinase 336 B. Protection of Bone Marrow 341 X. Status, Summary, and Future Prospects 343 References 344

Contents

Pharmacology of the Neurotransmitter Release Enhancer Linopirdine (DuP 996), and Insights into I t s Mechanism of Action Simon P. Aiken, Robert Zaaek, and Barry S. Brown

I. Introduction 349 A. The Unmet Medical Need of Alzheimer’s Disease 349 B. The Cholinergic Hypothesis of Alzheimer’s Disease 350 C. Rationale behind the Use of Linopirdine 351 11. Pharmacology of Linopirdine 353 A. Enhancement of Evoked Neurotransmitter Release by Linopirdine 353 B. Behavioral Effects of Linopirdine 358 C. Other Effects of Linopirdine 361 111. Mechanistic Studies on Linopirdine 364 A. Neurochemical Studies 364 B. Electrophysiological Studies 371 References 380 Index 385 Contents of Previous Volumes

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Contributors

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

Simon P. Aiken (349) Department of Pharmacology, Zeneca Pharmaceuticals, Wilmington, Delaware 19850 Ian Appleton (27) Department of Experimental Pathology, London EClM 6BQ, United Kingdom Gotz Baumann (115)Preclinical Research Transplantation, Sandoz Pharma AG, CH-4002 Basel, Switzerland Jean F. Borel (115) Preclinical Research Transplantation, Sandoz Pharma AG, CH-4002 Basel, Switzerland Barry S. Brown (349)Department of Preclinical Pharmacology, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880 Ian Chapman (115) Preclinical Research Transplantation, Sandoz Pharma AG, CH-4002 Basel, Switzerland Samuel R. Denmeade (281) Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 Peter Donutsch (115) Drug Safety, Toxicology, Sandoz Pharma AG, CH4002 Basel, Switzerland Alfred Fahr (115)Technical Research Development, Drug Delivery Systems, Sandoz Pharma AG, CH-4002 Basel, Switzerland John A. Fanner (79) Baylor College of Medicine, Houston, Texas 77030 Antonio M . Gotto, Jr. (79) Deptartment of Medicine, Baylor College of Medicine, Houston, Texas 77030 Howard N. Hodis (307) Division of Cardiology, University of Southern California School of Medicine, Los Angeles, California 90033 xiii

xiv

Contributors

John T. Isaacs (281) Department of Oncology and Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2123 1 Lawence A. Loeb (321)Department of Pathology, Joseph Gottstein Memorial Laboratory, University of Washington School of Medicine, Seattle, Washington 98195 Joel Moss (247)Pulmonary-CriticalCare Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Edgar A. Mueller (115)Drug Safety, Clinical Pharmacology, Sandoz Pharma AG, CH-4002 Basel, Switzerland Ian J . Okazaki (247) Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 W . Wayne Stargel (1)Monsanto Company, Deerfield, Illinois 60015 J . A. Thomas (1)University of Texas Health Science Center, San Antonio, Texas 78284 Annette Tomfinson (27) Department of Experimental Pathology, London EClM 6BQ, United Kingdom C. Tschanz (1)Monsanto Company, Deerfield, Illinois 60015 Jean-Marie Vigouret (115) Preclinical Research, Central Nervous System, Sandoz Pharma AG, CH-4002 Basel, Switzerland Derek A. Willoughby (27) Department of Experimental Pathology, London EClM 6BQ, United Kingdom Robert Zuczek (349) Department of Central Nervous System Diseases Research, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880

C. Tschanz" W. Wayne Stargel* J. A. Thomas+*'

* Monsanto Corporation, Deerfield, Illinois 600 I 5 t University of Texas Health Science Center, San Antonio, Texas 78284

Interactions between Drugs and Nutrients

1. Introduction It is well recognized that food ingestation concomitant with drug administration can profoundly affect the rate and the degree of drug and nutrient absorption (cf., Welling, 1984). Studies by Welling (Welling, 1980; Welling and Tse, 1982) revealed that 51 of 55 and 100 of 130 drugs tested exhibited abnormal absorption patterns when taken with food. Certain clinical conditions, where prolonged medication is prescribed, can accentuate such potential interactions (Stiefeld et al., 1991). Increasing attention is being devoted to patient counseling programs potentially involving drug-food interactions (Wix et a1.,1992; Thomas and Tschanz, 1994). Both the pharmacist and the dietitian should assist in identifying food-drug interactions and should be involved in patient education and counseling programs, particularly at the

' To whom correspondenceshould be addressed. Advances in Phamcology, Volume 35 Copyright Q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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time of hospital discharge. Unfortunately, the majority of teaching hospitals involved in a nationwide survey do not have formal drug-food interaction counseling programs (Wix et al., 1992). Whether the introduction of genetically engineered foods will impact the incidence of drug-food interaction is unknown. At this time, most attention to recombinant DNA-derived food has focused on it safety (Kessler et al., 1992). There is, however, no reason to believe that genetically engineered foods differ significantly from natural foodstuffs with regard to drug-nutrient interactions. Perhaps the impact of plant genetic engineering on foods and nutrition is too embryonic to accurately forecast (Comai, 1993). Plant genetic engineering can improve agronomic and quality traits such as nutritional value, yet virtually nothing is known about potential nutrient-drug interactions. Increasing attention has focused on nutrientdrug interaction due perhaps to drugs becoming more and more potent and having greater specificity. Potent drugs with extended durations of action have also led to an increased incidence of nutrient-drug interactions. With an aging population using an increasing number of prescription drugs, there is a greater likelihood of affecting the nutritional status of the elderly patient (Munro et al., 1987; Chen et al., 1985; Smith, 1990). The elderly, along with conditions such as pregnancy, breastfeeding, and malnourishment, can be predisposed to food-drug interactions. Among other factors, the disposition of drugs (e.g., half-life) can be affected by malnutrition (Mehta et al., 1982). Drugs and nutrients share a host of characteristics including physicochemical properties which may affect certain biochemical actions and other dose-related toxicities. Frequently, the mechanism of action of a drug may involve a nutrient(s)in a manner comparable to a nonnutrient component(s). The anatomical or cellular sites of nutrient-drug interactions can occur in the gastrointestinal tract, in the bloodstream, or at the drug’s receptor(s). Further, some drugs may modify body composition as evidenced by cationic amphophilic drugs affecting phospholipid storage (Kodavanti and Mehendale, 1990). The clinical relevance of nutrient-drug interactions is not entirely understood. Mainly, interactions depend on the specific drug and may increase, delay, or reduce a particular pharmacologic effect. Not all patients experience the same degree of risk for nutrient-drug interactions (Skaar, 1991). There are many contributing factors involved in nutrient-drug interactions, but the elderly are at particular risk (Roe, 1993). Pathophysiologic changes associated with aging, endocrine changes, alcoholism, and restricted diets all increase risk (Lee, 1991). Several factors are included in assessing the potential for nutrient-drug interactions (Skaar, 1991) (Table I). Specialnutritional-drug interactions are important in diabetes mellitus (Roe, 1988), cardiovascular disease (Roe, 1988), and even in certain genetic disorders (Montgomery et al.,1991; Kitler, 1994).

Interactions between Drugs and Nutrients

3

TABLE I Risk Factors for DrugNutrient Interactions’

Socioeconomic status Eating habits Nutrient loss due to food Frocessingkookkg Restrictive diets Anorexiateatingdisorders Alcoholism and/or drug addiction Chronic wasting diseases Multiple medications Renal and/or hepatic dysfunction Protein deficiency

Modified from Skaar, 1991.

II. Factors Affecting Gastrointestinal Absorption The most clinically significant nutrient-drug interactions involve the absorption process. Very few drugs are absorbed to any significant degree in the stomach with the exception of ethanol, which can be readily absorbed. Drugs that are acidic or basic are usually absorbed in the small intestine. It is recognized that gastric function exerts a major effect on both the rate and the degree of drug absorption. Alterations in gastric motility can affect the residence time of the food andlor drug in the gastrointestinal (GI)tract. Both the composition of the diet and the timing of meals can influence drug absorption. Delays in gastric emptying time caused by fatty foodstuff can likewise affect a drug’s absorption. Foods can enhance drug absorption (Table 11), can delay drug absorption (Table 111), or can decrease drug absorption (Table IV)(Randle, 1987; Smith and Bidlack, 1984). There are several mechanisms whereby foodstuffs and drugs can interact, leading to altered pharmacological response (Tables 2-4). Very often, the mechanisms involve the physiological alteration of drug blood levels caused by food increasing or decreasing a drug’s rate of absorption. Physiological interactions between drugs and nutrients involve those factors by which a drug affects processes related to eating, sensory appreciation of food, swallowing, digestion, gastric emptying, nutrient absorption, nutrient metabolism, or renal excretion of nutrients (cf., Roe, 1939). Thus, physiological interactions can aiso include reactions in which the absorption metabolism or elimination of a drug is changed by food ingestion. The mechanisms of food-drug interactions are not well characterized. These interactions involve both direct and indirect factors (Table V). Although the exact number of drugs influencing gastrointestinal absorption is not known, some estimates reveal that about 100 to 150 separate agents can exert such effects. Actually, this number probably represents a very small fraction of total marketed products. Understandably, the oral administration

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TABLE II Drugs Whose Absorption Is Enhanced by Food or Nutrients" Drug

Mechanism

Remarks

Carbamazepine

Increased bile production; enhanced dissolution and absorption Food enhances enterohepatic recycling of drug; increased dissolution secondary to gastric acid secretion Increased bile flow;delayed gastric emptying permits dissolution and absorption Unknown Drug is lipid soluble; enhanced absorption

Take with food

Diazepam

Dicumarol

Erythromycin Griseofulvin

Hydralazine

Hydrochlorothiazide La betalol Lithium citrate Metoprolol Nitrofurantoin

Phenytoin

Propoxyphene Propranolol Spironolactone

a

Food reduces first-pass extraction and metabolism, blocks enzymatic transformation in GI tract Delayed gastric emptying enhances absorption from small bowel Food may reduce first-pass extraction and metabolism Purgative action decreases absorption Food may reduce first-pass extraction and metabolism Delayed gastric emptying permits dissolution and increased absorption Delayed gastric emptying and increased bile production improves dissolution and absorption Delayed gastric emptying improves dissolution and absorption Food may reduce first-pass extraction and metabolism Delayed gastric emptying permits dissolution and absorption; bile may solubilize

None

Drug taken with meal

Take with food Take with high-fat foods, or suspend in corn oil unless contraindicated Take with food

Take with food Take with food Take on full stomach Take with food Take with food

Always take the same time in relation to meals

Take with food Take with food Take with food

Modified from Randle, 1987.

of a drug is convenient, and associating drug doses with daily routines such as mealtimes often improves patient compliance. However, this association can result in an increased incidence of nutrient-drug interactions. Certain foods can decrease, delay, or increase the absorption of drugs, hence altering their bioavailability, their solubility in gastric fluid, and their gastric emptying time (Trovato et al., 1991). Delayed drug absorption does not necessarily imply that less total drug is actually absorbed, but that peak

Interactions between Drugs and Nutrients

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TABLE 111 Selected Drugs Whose Absorption can be Delayed by Food or Nutrients Drug

Mechanisms

Acetaminophen High pectin foods act as absorbent and protectant Reduction in stomach fluid volume Ampicillin Amoxicillin Reduction in stomach fluid volume Aspirin Direct interference; change in gastric pH Aten o1oI Mechanism unknown, possibly physical barrier Cephalosporins Mechanism unknown Cimetidine Mechanism unknown Digoxin High-fiber, high-pectin foods bind drug Furosemide Glipizide

Mechanism unknown Unknown

Metronidazole Piroxicam Quinidine

Mechanism unknown Mechanism unknown Possibly protein binding

Sulfonamides

Mechanism unknown, may be physical barrier Mechanism unknown

Valproic acid

Remarks Take on empty stomach if not contraindicated Take with water Take with water Taking on empty stomach is not advisable Take on empty stomach if tolerated None May not be clinically significant Take drug same time with relation to food; avoid taking with high fiber foods May not be clinically significant Affects blood glucose; more potent when taken 1/2hour before meals None None May take with food to prevent GI upset Taking with meals may prolong gastric emptying Delayed absorption may give uniform blood levels

Modified from Randle, 1987;Smith and Bidlock, 1984;Garabedian-Ruffalo et al., 1988.

blood levels of the drug may require a longer period of time to be achieved. Drugs that bind or complex to nutrients are often unavailable for absorption or at least their absorption is delayed. Food can affect the bioavailability of drugs by directly binding to the drug by components in the foodstuff, or by changing luminal pH, gastric emptying, intestinal transit, mucosal absorption, and splanchnic-hepatic blood flow (Anderson and Kappas, 1987; Anderson, 1988). Food-induced changes in the bioavailability of some drugs may partially depend on hepatic biotransformation as evidenced by absorbed nutrients competing with drugs for first-pass metabolism in the intestine or in the liver. Some drugs can undergo metabolic transformation by enteric organisms and, since nutrients might also affect these microorganisms, they can influence the drug’s metabolism. The metabolism of drugs can occur by two basic processes, which are called Phase I and Phase I1 reactions. Phase I reactions include oxidation, hydroxylation, reduction, or hydrolysis resulting in changes in a functional

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TABLE IV Selected Drugs Whose Absorption can be Decreased by Food or Nutrients“ Drug

Mechanism

Remarks

Ampicillin Atenolol

Take with water Take on empty stomach if tolerated Take before meals None

Lincomycin

Reduction in stomach fluid volume Mechanism unknown, possibly physical barrier Mechanism unknown Drug undergoes first-pass metabolism in gut; delayed gastric emptying affects bioavailability Mechanism unknown; also impaired by water Food raises gastric pH preventing dissolution and absorption; also delayed gastric emptying Drug competes with amino acids for absorption and transport Mechanism unknown

Methyldopa

Competitive absorptions

Nafcillin

Mechanisms unknown; may be alteration of gastric fluid on pH May form chelates with calcium or iron

Captopril Chlorpromazine

Erythromycin Stearate Isoniazid Levodopa

Penicillamine

Penicillin G Penicillin VK

Delayed gastric emptying, gastric acid degradation; impaired dissolution More rapid dissolution in gastric fluids

Propantheline

Mechanism unknown

Rifampin

Mechanism unknown; conflicting reports

Tetracyclines

Binds with calcium ions or iron salts forming isoluble chelates

a

None Take on empty stomach if tolerated Avoid taking drug with high protein foods Take on empty stomach; food impairs absorption Avoid taking with high protein foods Take on empty stomach Avoid taking with dairy products or iron rich foods or supplements Take on empty stomach Take on empty stomach with full glass of water Evaluate “take with meals” directions Absorption limited with dose less than 150 mg; unaffected at dose greater than 700 mg Take 1 h before, 2 h after meals; do not take with milk

Modified from Randle, 1987; Smith and Bidlock, 1984; Garabededian-Ruffalo et al., 1988.

group on the drug molecule (TablesVI and VII). The mixed function oxidase system (MFOS) is an inducible enzyme system that catalyzes the oxidation of a wide variety of drugs. The MFOS is found primarily in the endoplasmic reticulum of the liver and other tissues. Phase I1 reactions include conjugation to glucuronate or glutathione and acetylation or sulfonation to functional groups on the drug molecule. Modification of functional groups frequently

Interactions between Drugs and Nutrients

TABLE V

Drug-Food/Drug-Fluid

7

interactions Affecting Absorptive Processes"

Indirect Mechanisms Drug-induced alterations in GI motility (e.g., anticholinergics) Drug-induced malabsorption syndromes (e.g., Neomycin) Direct mechanisms Drug-induced p H alterations in G-I tract (e.g., antacids) Drug-induced changes in bioavailability (e.g., absorption to drug-kaolidpectin) Drug-induced retardation of absorption (e.g., charcoal) Drug-bindingchelation (e.g., anionic exchange resins-Cholestyramine; metal ions-iron, calcium) Modified from Welling, 1984.

TABLE VI

Nutrients in Phase I (Oxidation) Reactions'

Nutrients

Component of reaction requiring nutrient

Nicotinic acid Riboflavin (vitamin B2) Glycine Pantothenic acid Iron Copper Protein Ascorbic acid (vitamin C)* Calcium Zinc Magnesium

NADPH FMN and FAD in NADPH-cytochrome c reductase Heme (Cytochrome P-450) CoA (ALA synthesis) Heme Ferrochelatase in heme synthesis Apo-enzymes ? Maintenance of membranes Maintenance of membranes Maintenance of membranes

a

Modified from Hoyumpa and Schenker, 1982. Some species

TABLE VII

Factors Affecting Drug Dispositiono

Diet Nutritional state Genetic traits Age Sex Pregnancy a

Renal and CVS function Pharmacologic variables (dose, route, duration, etc.) Stress Diseases states (e.g., liver, kidney) Other medication

Modified from Hoyumpa and Schenker, 1982.

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renders the drug more water soluble (or polar) and thus more readily excreted by the kidney. Conjugation enzymes are present in the endoplasmic reticulum or the cytoplasm. Several oxidized products of the MFOS are substrates for conjugating enzymes. The metabolism of drugs by Phase I and Phase I1 reactions is catalyzed by various enzymes, and the formation of metabolites necessitates that other substances be provided by the body through nutrition. Several nutrients and micronutrients (e.g., vitamins) exert significant roles in Phase I oxidation reactions (Tables VI and VII) (Hoyumpa and Schenker, 1982). Phase I1 reactions involving conjugation depend on the body to provide carbohydrates, amino acids, fats, and proteins. Acute starvation may depress MFOS. The state of nutrition thus has a major influence on drug metabolism. 111. Bioavailability and Metabolism

Bioavailability describes that portion of the drug’s dosage that actually reaches the systemic circulation metabolically unchanged (Winstanley and Orme, 1989). Food can alter many of the factors that affect bioavailability, which may modify a drug’s pharmacokinetics. Food can also alter the pharmacodynamics of drugs or can influence drug bioavailability due to physicochemical or chemical interactions between a specific nutrient or other food component(s) and the drug molecule in the GI tract. Additionally, gastrointestinal processes can affect a drug’s bioavailability. There are many other factors that can affect drug disposition Table VII) (Hoyumpa and Schenker, 1982).

IV. Excretion and Elimination Drugs are excreted from the body either unchanged or as metabolites. The organs of excretion (e.g., kidney, skin, liver, and lungs) eliminate polar compounds (i.e., water soluble) more efficiently than drugs that are lipid soluble. Ordinarily, lipid soluble drugs are poorly excreted unless they have undergone some degree of biotransformation to render them more water soluble and hence more suitable for elimination. The kidney plays a major role in the excretion of drugs and their metabolites. The renal excretion of drugs involves three processes: glomerular filtration, active tubular secretion, and passive tubular absorption. Drugs excreted in the feces are primarily unabsorbed orally ingested drugs (or their metabolites) excreted into the bile and are not reabsorbed from the GI tract. The organic acid and base renal transport mechanisms play an important role in the elimination of nonfilterable molecular species. Many drugs undergo such elimination processes via these organic acid and organic base

Interactions between Drugs and Nutrients

9

systems (Table VIII) (Bennett and Porter, 1993). The mechanism of action some drugs may depend on these transport systems, whereas other drugs involve proximal tubular transport systems as a major route of elimination from the body. Drugs transported by the organic ion system may produce nephrotoxicity either directly or indirectly. Rapidly metabolized drugs or those that undergo conjugation are generally more readily eliminated by the kidney. The degree of protein binding to a drug (i.e., bound vs free or unbound) can influence its rate of metabolism. Drugs that modify electrolytes can also affect the excretion of a drug (Table IX) (Bennett and Porter, 1993).Loop of Henle and thiazide diuretics increase urinary excretion of sodium, potassium, and magnesium. Loop diuretics increase the urinary excretion of calcium; thiazides may diminish its elimination. Cardiac glycosides can also facilitate potassium excretion. Conversely, anti-inflammatory steroids and certain antihypertensive agents can lead to sodium retention. There are several major clinical syndromes in nephrology produced by drugs and by chemicals which can ultimately impact the nutrient-drug interaction (Table X ) (cf., Bennett and Porter, 1993). The degree of nephrotoxicity depends on the dose, duration of treatmendexposure, and several other factors known to affect pharmacologic activity (e.g., age, sex, hepatic function, etc.). It is noteworthy that aminoglycoside antibiotics commonly lead to proximal tubular injury in 10 to 15% of therapeutic regimens. Similar nephrotoxicity is seen following amphotericin B and cis-platinum treatment. Although hepatotoxicity or liver damage may vary depending on the type, severity, and duration of injury, there are only a few mechanisms that alter a drug’s elimination (Hoyumpa and Schenker, 1982). Drug elimination may be modified due to decreased enzyme activity resulting from hepatic parenchymal cell disease, altered hepatic blood flow, hypoalbuminemia, or a combination of these factors or conditions. Hypoalbuminemia can affect TABLE Vlll Some Drugs Eliminated by Organic Transport Systems.

Organic acid system

Organic base system

Phenylbutazone Salicylate Cephalothin Sulfonarnides Chlorothiazide Furosernide Penicillin Methotrexate Probenecid

Isoproterenol Quinidine Morphine Procaine Tolazoline Macanylamine Piperidine

* Modified from Bennett and Porter, 1993.

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C.Tschanz et a\. TABLE IX Drug-Induced Electrolyte Disturbances" Sodium Hyponatremia-drugs that impair water excretion Hypernatremia-saline Anti-inflammatory steroids Potassium Cardiac glycosides Anti-inflammatory steroids Hypokalemia Diuretics Antibiotics Tocolytic agents Hyperkalemia Potassium supplements Potassium-sparingdiuretics Selected antihypertensive drugs Calcium Hypocalcemia Aminoglycoside antibiotics Hypercalcemia Thiazide diuretics Vitamin D supplements Phosphorus Hypophosphatemia Parenteral nutrition Hyperphosphatemia Cytotoxic drugs

Modified from Bennett and Porter, 1993.

the protein binding of a drug in the serum and change its pharmcodynamics. In acute liver disease, some drugs are poorly eliminated. In cirrhosis, reduced functional hepatic cell mass may lead to diminished enzyme complement.

V. Special InteractionslConditions A. Antimicrobials The relationship between circulating levels of antibiotics and their therapeutic efficacy is particularly well studied relative to other classes of pharmacologic agents (Welling and Tse, 1982). Further, the importance of food on the absorption of antimicrobials is well documented. The effects of food and fluid volumes on the absorption of ingested antimicrobials have clinical relevancy as they are related to the drug's efficacy (Toothaker and Welling, 1980; Royer et al., 1984). Different classes of antibiotics may be affected

Interactions between Drugs and Nutrients

TABLE X

II

Drug-Induced or Chemical-Induced Nephropathies"

Syndrome

Drughhemical

Acute renal failure (Direct tubular injury)

Aminoglycosides, radiocontrast agents, chemotherapeutic drugs, amphotericin B, cephaloridine, heavy metals Nonsteroidal anti-inflammatory drugs (NSAIDs), angiotensin-converting enzyme (ACE) inhibitors, cyclosporine, diuretics Penicillins, sulfonamides, phenytoin, diuretics, allopurinol, NSAIDs, many miscellaneous drugs High-dose methotrexate, acyclovir, methysergide Analgesic-associated nephropathy, lead nephropathy, cyclosporine, nitrosoureas, intravenous drug abuse Gold, penicillamine, captopril, NSAIDs, heroin Beta blockers, NSAIDs, ACE inhibitors, potassium-sparing diuretics, cyclosporine NSAIDs, chlorpropamide, thiazide diuretics Lithium, demeclocycline, clofibrate

Prerenal azotemia due to impaired perfusion Acute interstitial nephritis Acute obstructive uropathy Chronic renal failure

Nephrotic syndrome Hyperkalemia Hyponatremia Nephrogenic diabetes insipidus ~

Modified from Bennett and Porter, 1993.

differently by the presence of food. Some antibiotics have their absorption reduced by foods (e.g., certain penicillins and tetracyclines), some may be delayed (e.g., sulfonamides), some may be unaffected (e.g., ampicillin, amoxycillin), and some antibiotics may actually have their absorption increased by the presence of food (e.g., griseofulvin, nitrofurantoin) (Table XI) (cf., Welling and Tse, 1982).Different formulations of erythromycin are affected differently by the presence of food (viz., delayed, reduced, increased, or unaffected). The effect of food on the bioavailability of ingested erythromycin is influenced by the chemical derivative, the dosage formulation, and the timing of the meal relative to the administration of the antibiotic. Ironically, there may be circumstances in which the food-antibiotic interaction may reduce the absorption of the antimicrobial yet the presence of the food may lessen GI associated side-effects attributed to the drug. However, these special circumstances need not compromise the therapeutic efficacy of the antibiotic.

B. Drug-Ethanol Interactions The ingestion of ethyl alcohol with other drugs can cause clinically significant interactions. Not surprisingly, these interactions are more frequent in alcoholics than in persons who consume only small amounts of ethanol. Lieber (1994) has recently reviewed the mechanisms involving ethanol-drug-nutrition interactions.

I2

C.Tschanz et at. TABLE XI

Effect of Food on the Absorption of Antibioticsa

Reduced Amoxycillinb Tetracycline(s) Isoniazid

Penicillin G Penicillin V(K)b Ampicillin

Delayed Sulfonamide(s) Metronidazole

Cephalexin Cephradine

Unaffected Penicillin V(acid)b Ampicillinb

Amoxycillinb Clarithromycin

Increased Griseofulvin

Nitrofurantoin

Variable (i.e., formulations reduced, increased, delayed, or unaffected) Erythromycin(s) Representative antibiotics (see Welling and Tse, 1982; Chu et al., 1992). * Literature studies not in complete agreement.

a

Chronic alcoholism is a major cause of liver disease leading to abnormal drug metabolism. The use of therapeutic agents in the alcoholic is complicated by underlying hepatic disease and by acute and chronic ethanol-drug interactions. Drug metabolism is affected by both acute and chronic use of ethanol. Chronic use results in enzyme induction, which tends to increase metabolism and lead to a greater dose in order to achieve the desired therapeutic effect. Depending on the class of drugs (e.g., sedatives), many alcoholics exhibit a tolerance to or lack of effect of the drug. The acute use of alcohol may simply overwhelm metabolic enzymes and tends to diminish the normal hepatic metabolism of drugs. Many different classes of drugs can be affected by the use of alcohol (Table XII) (Hoyumpa and Schenker, 1982; Lieber, 1994).The acute ingestion of ethanol and agents with sedative action leads to greater psychomotor impairment than that produced by each agent separately. Several mechanisms may explain these interactions, including a combined CNS depressant action, altered drug metabolism by ethanol, or acute impairment of the degradation process of the sedative(s). Lieber (1994) has described at least twelve sites of ethanol-drug interaction (Table XIII). The major portion of ethanol is catalyzed by alcohol dehydrogenase (ADH) in the liver, yet ADH is only marginally involved in alcohol-drug interactions. Chronic alcohoVdrug interactions lead to enhanced hepatic drug metabolism sometimes referred to as metabolic drug tolerance. In addition to

Interactions between Drugs and Nutrients

13

TABLE XI1 Effect of Ethanol on Degradation of Drugs' Acute administration

Drugs Sedatives and tranquilizers Chlordiazepoxide Diazepam Lorazepam Clorazepate Oxazepam Meprobamate Pentobarbital Chlorpromazine Chloral hydrate Miscellaneous drugs Tolbutamide Phenytoin Warfarin Antipyrine Chlormethiazole Acetaminophen Rifamycin

Chronic ingestion

Decreased Decreased Decreased Decreased No effect(?) Decreased Decreased Decreased Decreased

Alcohol withdrawal Decreased Decreased

Increased Increased

Decreased Decreased Decreased Decreased Decreased Decreased

Increased Increased Increased Increased Decreased Increased Increased

Modified from Hoyumpa and Schenker, 1982; Lieber, 1994.

ethanol tolerance, alcoholics also display tolerance to various other drugs. Such tolerance may be partially attributed to CNS adaption, but also to metabolic adaption. The induction of the microsomal ethanol oxidizing system (MEOS)following chronic ethanol consumption affects various other drug-metabolizing systems in hepatic microsomes, leading to a generalized acceleration of drug metabolism. Transethnic differences exist with regard to the metabolism of ethanol and hence there are genetic differences in the disposition or metabolism of

TABLE Xlll

Sites of Ethanol-Drug Interactionsa ~~

Gastric ADHb Absorption from stomach Plasma protein binding Hepatic blood flow Hepatic cell uptake Hepatic cell metabolism a

Modified from Lieber, 1994. ADH, Alcohol dehydrogenase.

~

Congeners Nutrition Cirrhosis Genetic factors Conjugation Peripheral sites of action

14

C. Tschanz et al.

ethanol (Kitler, 1994). Some Orientals (e.g., Chinese and Japanese) and Native Americans exhibit a higher rate of alcohol metabolism compared to Caucasians. Jewish men and women have a lower incidence of severe alcoholrelated problems purportedly due to a heightened sensitivity to relatively low doses of alcohol. Hence, transethnic differences may affect drugethanol interactions. The ingestion of large amounts of ethanol over a brief interval (i.e., binges) or small quantities to the individual who seldom drinks can result in an additive or synergistic effect in the presence of other CNS depressants. One of the best known interactions of drugs with ethanol is the reaction with disulfiram. Disulfiram inhibits acetaldehyde dehydrogenase resulting in the accumulation of acetaldehyde and hence causing nausea and vomiting within minutes of alcohol ingestion. C. Drug-Vitamins

Many drugs can change the body's requirements for vitamins (Munro et al., 1987; Christakis and Christakis, 1983; Katz and Dejean, 1985).These vitamin-drug interactions can occur with either water-soluble or fat-soluble vitamins (Table XIV). Some antibiotics can modify enteric organisms and TABLE XIV

Some Drug-Vitamin Interactions"

Vitamin

D w

Interaction

Vitamin BI2

K++supplements Colchicine Oral contraceptives Hydralazine Isoniazid Penicillamine Salicylates Tetracyclines Mineral oil

Gastric pH slows vitamin Blz absorption Vitamin Blz absorption impaired Increased requirement for vitamin B g

Cholestyramine

Retards absorption of vitamins A, D, and K

Cathartics (irritants) Neomycin Ethanol Tetracycline Glutethimide Dicumarol Digoxin Anticonvulsants Dicumarol

Retards absorption of vitamin D Retards absorption Hepatotoxicity causing hypervitaminosis A Intracranial hypertension Produces vitamin D deficiency Enhances anticoagulant action Hypercalcemia and arrhythmias Induces enzymatic inactivation Inhibits hypoprothrombin action

Vitamin B6 (pyridoxine)

Vitamin C Fat-soluble vitamins (A, D, E, and K) Vitamin A, D, and K Vitamin D Vitamin A

Vitamin D Vitamin E Vitamin K

Decreased uptake of vitamin C Depletion of vitamin C Retards absorption of fat-soluble vitamins

Modified from Christakis and Christakis, 1983; Smith and Bidlock, 1984.

lnteactions between Drugs and Nutrients

I5

thus affect the absorption of fat-soluble vitamins. The principal mechanism of interaction usually involves impaired absorption of the vitamin by a particular drug. However, some drugs can induce enzyme systems that can accelerate the metabolism of the particular vitamin (e.g., ethanol). In addition, ethanol consumption can depress hepatic levels of Vitamin A. Ethanol interacts with the clearance of beta-carotene. Glutathione, which acts as one of the scavenging mechanisms for toxic free radicals, can be reduced by acute ethanol administration. Physiologically, glutathione can spare and potentiate Vitamin E, but ethanol can interfere with such metabolic events (cf., Lieber, 1994). A number of mechanisms of drug-folate interaction have been reported (Table XV) (Roe, 1974).Folate may be required as a cofactor in the hydroxylation of different drugs and chemicals. There is variation among the classes of drugs capable of affecting folate metabolism. The mechanism of action also varies considerably, ranging from impaired absorption, to competitive binding to serum proteins, to enzyme inhibition. Folate intake shows wide variation, particularly in the elderly (Munro et al., 1987). Folate absorption is strongly pH dependent. Gastric atrophy and atrophic gastritis with achlorhydria and hypochlorhydria lead to malabsorption of folate and Vitamin Biz. Ethanol can change the kinetics of folate metabolism and increase its excretion; chronic alcoholism can cause megaloblastic anemia. Folate antagonism decreases the availability of substrates required for nucleic acid biosynthesis. The inhibition of nucleic acid metabolism, and subsequently nucleic acid synthesis, has been suggested as a mechanism responsible for developmental toxicity caused by drugs such as methotrexate and aminopterin (Farrar and Blumer, 1991). D. Drug-Minerals

There are three types of drug-mineral interactions: (1) malabsorption of the mineral and/or drug, (2) mineral depletion and retention, and TABLE XV

Drugs Affecting Folate Activity"

Agentddmgs

Mechanism of action

Barbiturates Primidone Oral contraceptives Ethinyl estradiol Cycloserine Aspirin Methotrexate

Malabsorption Folate metabolism Malabsorption of polyglutamate Unknown Secondary to vitamin Bs antagonism Competitive serum protein binding Inhibits dihydrofolate reductase

Modified from Roe, 1974; see also Thomas and Markovac, 1994.

a

16

C. Tschanz et al.

(3) drug-mineral interactions induced by simultaneous antacid ingestion (Murray and Healy, 1991). There are six fnajor minerals and approximately a dozen minor or trace elements that are generally considered essential for physiologic process. Due to their relative abundance in foods, minerals such as sodium, potassium, magnesium, calcium, and phosphorous, are involved in drug interactions (Hazell, 1985). The minor elements include arsenic, cobalt, chromium, copper, fluoride, iron, iodide, manganese, molybdenum, nickel, selenium, silicon, tin, vanadium, and zinc. While these trace elements have important physiological functions, most are not important contributors to drugmineral interactions. Many drug-mineral interactions have little clinical importance. Malnourished individuals, the elderly, and patients with chronic diseases are more frequently viewed as having these interactions (Hussar, 1988; Smith and Bidlack, 1984; Hansten and Horn, 1989). Drugs cause malabsorption of minerals by two mechanisms: (1) by primary drug-induced malabsorption whereby the drug directly prevents the absorption of one or more minerals, and (2)by influencing a drug’s absorption, disposition and metabolism. Primary malabsorption involves direct binding with the nutrient through chemical complexing (e.g., chelation) and through direct adverse action by the drug on the mucosa of the small intestine preventing the mineral from being absorbed (Table XVI). Drugs can also secondarily prevent mineral absorption. For example, drug-induced effects on Vitamin D might secondarily cause calcium malabsorption (Table XVI). While drugs may cause mineral depletion through primary and secondary malabsorption processes, depletion can also be caused by pharmacologic agents (e.g., diuretic) acting on renal function. Undoubtedly the largest group of agents producing interaction on the GI tract are gastric antacids. Antacids may alter a drug’s dissolution by modifying gastric pH and even by chelation (Hussar, 1988; Cardinale, 1988; Royer et al., 1984). Antacids are thus capable of interfering with a drug’s absorption. Furthermore, aluminum, which is a constituent of many antacid preparations, can cause a relaxing action on gastric-smooth muscle causing a delay in gastric emptying time. Antacids exert two actions to affect changes in gastric pH and to chelate with minerals to prevent their absorption. Alkinity, or increasing gastric pH, will lead to diminished absorption of calcium, iron, magnesium, and zinc (D’Arcy and McElnay, 1985). Several important classes of drugs have their pharmacologic actions modified by minerals (Smith and Bidlack, 1984). Calcium, iron, magnesium, and zinc can interfere with the GI absorption of tetracyclines. Iron can reduce the absorption of penicillamine (Table XVI). It is evident that a very diverse group of pharmacologic agents can have their actions modified by minerals.

Interactions between Drugs and Nutrients

17

E. Genetic Differences Differences in response to drug action among various ethnic and racial subpopulations have long been recognized, and have been accentuated by specific pharmacologic agents causing exaggerated biological reactions. Altered drug metabolism has been studied extensively among different populations (cf., Kitler, 1994). Among the many conditions to which the human organism must adapt is the nutritional environment (Childs, 1988). Genetic variation can impact certain nutritional states. Ethnic differences can be related to individual variation in response to drugs (Ghoneim et al., 1981).Such variations are most frequently controlled genetically, although there are other environmental factors. There are marked interethnic differences with some groups exhibiting the ability to metabolize drugs rapidly whereas other groups metabolize drugs slowly. There are also racial differences in body fat composition, lean tissue density, and adipose tissue metabolism (Ama et al., 1986). Indeed, these factors can affect the overall pharmacokinetics of a drug(s). There are numerous examples of classes of pharmacologic agents that reveal transethnic differences (Table XVII). Genetic differences are evident in absorption, distribution, and elimination processes. Other pharmacologic or biologic endpoints are marked by genetic differences including pain threshold, immune responsiveness, and metabolic parameters. Such ethnic and racial differences in response to pharmacologic agents have the potential to involve drug-nutrient interactions. Lactose intolerance can be genetically related to lactase phenotypes as evidenced by differences among various subpopulations throughout the world (cf., Montgomery et ul., 1991 ). Lactase-phlorizin hydrolase, which hydrolyzes lactose, has an important role in the nutrition of the neonate. There are transethnic differences in lactase activity and in lactase mRNA. For example, Orientals frequently reveal low levels of lactase activity and lactase mRNA concentrations (Montgomery et al., 1991). Lactose intolerance is a common form of carbohydrate malabsorption affecting all ages. Lactose, a disaccharide of glucose and galactose, is present in human milk, in cow’s milk, and in standard infant formulas and many dairy products. Lactose is also an additive in foodstuffs including baked goods, cereals, and soft drinks (Montes and Perman, 1991). Normally, lactase hydrolyzes lactose in the small intestine to glucose and galactose. Thus a reduction or absence of lactase results in lactose being malabsorbed. The unabsorbed lactose residing in the distal small intestine and colon exerts a significant osmotic pressure resulting in fluid and electrolyte secretion into the lumen. This condition causes a watery, acidic fecal output. The most common etiology of lactose intolerance in adults is primary lactase deficiency. Low lactase levels are due to either intestinal injury or

TABLE XVI Selected Drug-Mineral Interaction and Possible Mechanisms" Mechanisrn(s)

Interaction

D w

Mineral($

WOH)p Ampicillin Anabolic steroid Aspirin Atenolol Atropine Bisacodyl Chlordiazepoxide Cholestyramine Colchicine Corticoids Dicumarol Digoxin Diphosphates Estrogens Ethanol

Po4 MgOH Na Fe, K AUW3 WOHL K, Na Antacids Fe Ca,Fe,K,Na Ca MgOH Antacids Ca Na Mg,K,Zn

l Malabsorption

2 Malabsorption

Mineral depletion

Mineral retention

+ +

+

+

+ + +

+

+ +

Antacidinduced

+

+ +

+ +

+

Furosemide Guanethidine Hydralazine Hydrochlorothiazide Indomethacin Lithium Methotrexate Methy1dopa Methyldopa Metropolol Mineral oil Naproxen Neomycin Nitrofurantoin Oral contraceptives Penicillamine Phenobarbital Penytoin Primadone Quinidine Tetracyclines Triamterene

Cl,Mg,K,Na,Zn,Ca Na Na Ca,Mg,K,Na,Zn Fe Cu,Na Ca Fe Na Ca,PO,,K Antacids Ca,Fe,K,Na Mg Antacids Cu,Fe,Po4,Na,Zn c a m CaNg Ca WOHh Ca,Fe,Mg,Zn K

‘Modified from Murray and Healy, 1991.

+ +

+ +

+

+ + +

+

+

+

+

+ + +

+

+ + + + +

+ +

20

C. Tschanz et al.

TABLE XVll

Transethnic Responsiveness to Selected Pharrnacologic Agents"

DruglAgent

General Ethnic Biologic Responseb

Ethanol Analgesic Benzodiazepines Insulin Diuretics (thiazides) Propranolol Phenytoin (DPH) Lithium Dermatologic preparations Haloperidol Immunosuppressive agents rt-PA

Exaggerated in Orientals Higher pain threshold in Asians Lower dosages required in Asians Hyperinsulinemia in Native Americans More effective in Blacks than Caucasians Renal clearance in Chinese twice that of Caucasians Eliminated faster in Eskimos Lower doses required in Japanese populations Reduced absorption in Blacks Asians exhibit more extrapyramidal symptoms Reduced allograph survival in Blacks Enhanced thrombolytic activity in Blacks

Modified from Kitler, 1994. Individual pharmacologic responses may vary.

more commonly through alterations in the genetic expression of lactase. Lactose intolerance increases with advancing age. However, congenital lactase deficiency, evident at birth, is very rare and is inherited as an autosomal recessive gene (Savilathi et al., 1983). Many drugs can induce lactose intolerance (Roe, 1985). Drug-induced lactose intolerance is induced by certain drugs that cause malabsorption. Unlike mineral oil, which can produced malabsorption at luminal sites, drugs such as methotrexate, neomycin, and colchicine interact at mucosal sites. Such agents, acting at mucosal sites, can produce lactose intolerance. Some of the drugs that induce lactose intolerance are also cytotoxic (e.g., neomycin, colchicine, and methotrexate), which may be a contributing factor to malabsorption. F. Geriatrics

The elderly are more at risk for adverse and clinically important outcomes of drug-nutrient interactions (Roe, 1984). Such increased risks are due to multiple drug usage, age-related modifications in drug disposition, geriatric pathologies which might impair drug clearance, and simply because subgroups of the elderly may suffer from nutritional inadequacies. The GI tract of the elderly is often more vulnerable to drug-nutrient interactions. The elderly frequently take more drugs due to various biological deteriorations. Oftentimes, the elderly consume over-the-counter (OTC)drug products such as laxatives, vitamidminerals, and antacids. It has been estimated that by the year 2000 about 50% of all chronic care drugs will be OTC products (Cardinal, 1988).Perhaps the most common form of drug-nutrient

Interactions between Drugs and Nutrients

21

interaction in the elderly is a mineral deficiency caused by the frequent use of diuretics leading to potassium and magnesium loss (Roe, 1984; Larmy, 1982). Drug-nutrient interactions in the elderly have been classified as physicochemical,' physiological,2 and pathophysi~logical~ (Roe, 1993). Physicochemical interactions would be represented by chelation or chelation complexes as well as by modifications in the stability of the nutrient. Physiological interactions would include drug-induced changes in appetite, digestion, gastric emptying, biotransformation, and renal clearance. Pathophysiological interactions can occur when a drug impairs nutrient absorption or when its toxicity produces an inhibition of metabolic processes or events. Mooradian (1988) discussed nutritional modulation in the elderly suggesting that alterations in the micronutrient and micronutrient constituents of the diet can affect gene expression. Nutritional problems in the elderly are not only related to multiple drug use, but also to the consumption of specialized diets for one or more chronic illnesses. There are several major mechanisms involved in drug-nutrient interactions in the elderly (Table XVIII) (Mooradian, 1988). Many drugs possess side-effects (e.g., nausea, vomiting, and diarrhea) that can secondarily affect drug-nutrient responses. Drugs that are cytotoxic can damage different cell populations including mucosal cells in the GI tract. Enteric microflora can be suppressed by a variety of antibiotics leading to altered digestive processes. Based on the incidence of certain diseases in the elderly (e.g., hypertension, cardiac failure, renal insufficiency),there are particular concerns about selected classes of pharmacologic agents and to what extent they affect nutrition. Some of the more common interactions seen in concomitant disease states are shown in Table XIX (Roe, 1993). Drug-induced adverse outcomes can complicate therapies by affecting the nutritional status of the geriatric patient. Digoxin, while an important therapeutic agent for congestive heart disease, has inherent anorexic properties such as nausea and vomiting. Loop diuretics not only facilitate the loss of sodium, but also TABLE XVlll

Mechanism of Drug-Nutrient Interaction in

the Elderly'

Appetite suppression (anorexic) Appetite stimulation Diminished nutrient absorption; toxicity to mucosal cells Facilitated renal elimination Decreased nutrient use Antagonisdcompetitive (e.g., Coumarin and Vitamin K) Inhibition or facilitation with metabolism or transport system(s) Hormonal effects of nutrients Indirect effect due to components of drug formulation a

Modified from Mooradian, 1988.

22

C.Tschanz et al.

TABLE XIX Common Drug Therapies in the Elderly in Relation to Risk and Adverse Effects" Drug therapy

Risk factor(s)

Adverse outcome(s)

Digoxin-cardiac failure

High dose

Loss of appetite Low food intake Cachexia Thiamine deficiency

& Furosemide-hypertension NSAIDsb-arthritis

a

Renal function

High prescription dose Ethanol intake High dose Frequent use

Iron deficiency Gastritis leading to anorexia

Modified from Roe, 1993. NSAIDs-Nonsteroidal anti-inflammatorydrugs.

the loss of potassium, magnesium, calcium, and thiamin. Thus, osteoporosis can be exacerbated in the elderly postmenopausal patient undergoing therapy with loop diuretics. It would be evident that the elderly represent a high-risk population with respect to drug-nutrient interactions. The aging process can profoundly affect the pharmacokinetics of a drug. G. Hypoglycemics In diabetics as well as nondiabetics, it is well known that injections of insulin can provoke a sensation of hunger. However, insulin-induced hypoglycemia can also be associated with nausea and a sensation of weakness, rather than the desire for food (Roe, 1979). Because diabetic patients with renal and/or hepatic disease are often more vulnerable to hypoglycemia, certain oral antidiabetic agents must be used with clinical discretion. The coadministration of sulfonylurea drugs and thiazide diuretics can exacerbate the diabetic condition. A decreased alcohol tolerance may also be manifest in patients ingesting sulfonylurea agents. Several other drugs may enhance the hypoglycemic actions of the sulfonylurea drugs including propranolol, salicylates, phenylbutazone, chloramphenicol, probenecid, and the sulfonamides (cf., Thomas and Thomas, 1994). Tolbutamide and chlorpropamide purportedly increase appetite in some diabetics. These oral hypoglycemic agents may enhance appetite by stimulating the release of pancreatic insulin. Certain antibiotics can affect hypoglycemia (Stiefeld et al., 1991). Cotrimoxazole and fuconazole can both interact with oral hypoglycemic agents leading to a further reduction in blood glucose. Rifampin can antagonize the action(s) of oral hypoglycemics. While there is continuing interest in developing new hypoglycemic agents, recent focus has been devoted to

Interactions between Drugs and Nutrients

23

compounds that act directly on the GI tract (cf., H u h , 1994).This anatomical site of action may affect absorption processes leading to potential drugnutrient interactions. The absorption of both simple and complex carbohydrates from the intestine is mediated by a family of enzymes called alphaglucosidases. The alpha-glucosidases hydrolyze oligo- or polysaccharides to monosaccharides. The inhibition of alpha-glucosidases leads to a delay in the absorption of carbohydrates. A number of alpha-glucosidase inhibitory drugs are under development as new hypoglycemic agents including acarbose and miglitol. Acarbose is a reversible competitive inhibitor of glucoamylase and sucrase (Saperstein et al., 1990). Miglitol, a compound that appears to mimic glucose, also inhibits alpha-glucoamylase and sucrase (cf., Hulin, 1994).These compounds can reduce postprandial hyperglycemia and diminish insulin secretion, but other potential interactions have not been revealed or otherwise studied.

H. Parenteral Nutrition Total parenteral nutrition (TPN) can affect the metabolism of drugs (Anderson, 1988). Experimental evidence suggests that TPN can reduce hepatic clearance of barbiturates. Antipyrine pharmacokinetcs can be altered by intravenous nutritional regimens leading to increased renal clearance and a shortened plasma half-life. Accordingly, antipyri1,e metabolism can be increased by nutritional repletion (Anderson, 1988). Ethanol interferes with a host of nutritional factors including the type and amount of dietary fat, protein, and amino acids. These interactions provide the rationale for the parenteral administration of complete amino acid mixtures to patients with severe alcoholic liver disease (cf., Lieber, 1994). Although dietary deficiencies (i.e., reduced food intake) may play a role in alcoholic liver injury, supplementation with S-adenosyl-L-methionine (SAM) and polyunsaturated lecithin may significantly offset some of the toxic manifestations of ethanol. Short-chain peptides are being considered as new candidates in parenteral nutrition (Furst et al. 1990).Their potential use is based on the assumption that specially concocted amino acid solutions will enhance the therapeutic benefits to patients receiving parenteral nutrition. Dipeptide-based parenteral solutions exhibit low osmolarity thus enabling them to fulfill nitrogen requirements of patients with severe fluid restriction. Further, synthetic peptides are rapidly eliminated and substantial amounts of these solutes do not accumulate in biological fluids. L-alanyl-L-glutamine has undergone clinical evaluation and other dipeptides are certain to be tested for their potential efficacy. At this time, it is difficult to predict any clinical significant drug-dipeptide interaction, but there would appear little likelihood of such events.

24

C. Tschanz et al.

Concluding Remarks Nutritional status plays a significant role in a drug’s pharmacologic response. Certain disease states and other special subpopulations affect nutrient status and a drug’s therapeutic efficacy. Certain classes of drugs such as antimicrobials can have their absorption modified by the presence of food in the GI tract. Although a drug’s pharmacokinetic profile can usually be predicted, it can be modified by nutrients and by certain pathophysiologic conditions, including aging. References Ama, P. F. M., Poehlman, E. T., Simoneau, J. A., Boulay, M. R., Theriault, G., Tromblay, A., and Bouchard, C. (1986). Fat distribution and adipose tissue metabolism in non-obese male black African and Caucasian subjects. Int. J. Obes. 10, 503. Anderson, K. E. (1988). Influences of diet and nutrition on clinical pharmacokinetics. Clin. Pharmacokinet. 14, 325. Anderson, K. E., and Kappas, A. (1987). How diet affects drug metabolism. Hosp. Ther. April, 93. Bennett, W. M., and Porter, G. A. (1993).Overview of clinical nephrotoxicity. In “Toxicology of the Kidney” 2nd ed, (J. B. Hook and R. S. Goldstein, eds.) Target Organ Toxicity Series, p. 61, Raven Press, New York. Cardinale, V. (1988).Stemming the tide of polymedicine. Drug Topics 132, 36. Chen, L. H., Liu, S., Cook Newell, M. E., and Barnes, K. (1985). Survey of drug use by the elderly and possible impact of drugs on nutritional status. Drug-Nutr. Interact. 3, 73. Childs, B. (1988). Genetic variation and nutrition. Am. J. Clin. Nutr. 48, 1500. Christakis, P. and Christakis, P. (1983).Part 11: Drug interactions-nutrients, vitamins, foods. Pharma. Times Nov, 68. Chu, S., Park, Y., Locke, C., Wilson, D. S., and Cavanaugh, J. C. (1992).Drug-food interaction potential of clarithromycin, a new macrolide antimicrobial. J. Clin. Pharmacol. 32, 32. Comai, L. (1993). Impact of plant genetic engineering on foods and nutrition. Annu. Rev. Nuh. 13, 191. D’Arcy, P. F., and McElnay, J. C. (1985). Drug interactions in the gut involving metal ions. Rev. Drug Metabol. Drug Interact. 5, 83. Farrar, H. C., and Blumer, J. L. (1991).Fetal effects of maternal drug exposure. Annu. Rev. Pharmacol. Toxicol. 31, 525. Furst, P. Albers, S., and Stehle, P. (1990). Dipeptides in clinical nutrition. Proc. Nutr. SOC. 49, 343. Garabedian-Ruffalo, S . M., Syrja-Farber, M., Lanius, P. M., and Plucinski, A. (1988).Monitoring of drug-drug and drug-food interactions. Am. /. Hosp. Pharm. 45, 1530. Ghoneim, N. M., Kortilla, K., Chiang, C. H., et al. (1981).Diazepam effects and kinetics in Caucasians and Orientals. Clin. Pharmacol. Ther. 29, 749. Hansten, P. D., and Horn, J. R. (1989).“Drug Interactions” 6th ed., Lea & Febiger, Philadelphia, PA. Hazell, T. (1985).Minerals in foods: dietary sources, chemical forms, interactions, bioavailability. World Rev. Nuh. Diet. 46, 14. Hoyumpa, A. M., and Schenker, S. (1982). Major drug interactions: Effect of liver disease, alcohol, and malnutrition. Annu. Rev. Med. 33, 113. H u h , B. (1994). New hypoglycaemic agents. Prog. Med. Chem. 31, 1.

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Hussar, D. A. (1988). Drug interactions in the older patient. Geriatrics 43, 20. Katz, N. L., and Dejean, A. (1985).Interrelationships between drugs and nutrients. Pharmlndex Dec, 9. Kessler, D. A., Taylor, M. R., Maryanski, J. H., Flamm, E. L., and Kahl, L. S. (1992). The safety of foods developed by biotechnology. Science 256, 1747. Kitler, M. E. (1994). Clinical trials and transethnic pharmacology. Drug Saf. 11, 378. Kodavanti, U. P., and Mehendale, H. M. (1990).Cationic amphiphilic drugs and phospholipid storage disorder. Pharmacol. Rev. 42, 327. Larmy, P. P. (1982).Effects of diet and nutrition on drug therapy.]. Am. Geriat. SOL.30,599. Lee, C. R., McKenzie, C. A., and Mantooth, R. (1991). Food and drug interactions. U.S. Pharmacist May, 44. Lieber, C. S. (1994).Mechanisms of ethanol-drug-nutrition interactions. Clin. Toxicol. 32,631. Mehta, S., Nain, C. K., Sharma, B., and Mathur, V. S. (1982). Disposition of four drugs in malnourished children. Drug-Nutr. Interact. 1, 205. Montgomery, R. K., Buller, H. A., Rings, E. H. H. M., and Grand, R. J. (1991). Lactose intolerance and the genetic regulation of intestinal lactase-phlorizin hydrolase. FASEB J. 5, 2824. Montes, R. G., and Perman, J. A. (1991). Lactose intolerance. Postgrad. Med. 89, 175. Mooradian, A. D. (1988).Nutrition modulation of life span and gene expression. Ann. Intern. Med. 109, 890. Munro, H. N., Suter, P. M., and Russell, R. M. (1987).Nutritional requirements of the elderly. Annu. Rev. Nutr. 7, 23. Murray, J. J., and Healy, M. D. (1991). Drug-mineral interactions: A new responsibility for the hospital dietitian. 1.Am. Diet. Assoc. 91, 66. Randle, N. W. (1987).Food or nutrient effects on drug absorption: A review. Hosp. Pharm. 22, 694. Roe, D. A. (1974). Effects of drugs on nutrition. Life Sci. 15, 1219. Roe, D. A. (1979).Interactions between drugs and nutrients. Med. Clin. North Am. 63, 985. Roe, D. A. (1984).Therapeutic significance of drug-nutrient interactions in the elderly. Pharmacol. Rev. 36, 109s. Roe, D. A. (1985).Prediction of the cause, effects, and prevention of drug-nutrient interactions using attributes and attribute values. Drug-Nutr. Interact. 3, 187. Roe, D. A. (1988).Drug and nutrient interactions in the elderly diabetic. Drug-Nutr. Interact. 5 , 195. Roe, D. A. (1988). Drug and nutrient interactions in elderly cardiac patients. Drug-Nutr. Interact. 5, 205. Roe, D. A. (1993).Drug and food interactions as they affect the nutrition of older individuals. Aging Clin. Exp. Res. 5, 51. Royer, R. J., Debry, G., Ulmer, M., and Bannwarth, B. (1984). Food and drug interactions. World Rev. Nutr. Diet. 43, 117. Saperstein, R., Chapin, E. W., Brady, E. J., and Slater, E. E. (1990).Effects of an a2-adrenoceptor antagonist on glucose tolerance in the genetically obese mouse. Metabolism 39,445. Savilathi, E., Launiala, K., and Kuitunen, P. (1983). Congenital lactase deficiency: A clinical study on 16 patients. Arch. Dis. Child. 58, 246. Skaar, D. J. (1991). Drug-nutrient interactions: Implications for pharmaceutical care. Purtn. Pbarmaceut. Care Oct, 11. Smith, C. H. (1990).Drug-food/food-drug interactions. In “Geriatric Nutrition: A Comprehensive Review” Raven Press, NY. Smith, C. H., and Bidlack, W. R. (1984).Dietary concerns associated with the use of medications. 1. Am. Diet. Assoc. 84, 901. Stiefeld, S. M., Graziani, A. L., MacGregor, R. R., and Esterhai, J. L. (1991). Toxicities of antimicrobial agents used to treat osteomyelitis. Orthop. Clin. North Am. 22, 439.

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Thomas, J. A., and Tschanz, C. (1994).Nutrient-Drug interactions, In “Nutritional Toxicology” (E. N. Kotsonis, M. Mackey, and J. J. Hjelle eds.), Target Organ Toxicity Series, p. 139, Raven Press, NY. Thomas, J. A., and Thomas, M. J. (1994).Insulin, glucagon, somatostatin, and orally effective hypoglycemic drugs In “Modern Pharmacology” 4th ed., Little, Brown & Co., Boston. Thomas, J. A., and Markovac, J. (1994). Aspects of neural tube defects: a minireview. Toxic Subst. J. 13, 303. Toothaker, R. D., and Welling, P. G. (1980).The effect of food on drug bioavailability. Annu. Rev. Pharmacol. Toxicol. 20, 173. Trovato, A., Nuhlicek, D. N., and Midtling, J. E. (1991). Drug-nutrient interactions. Am. Fam. Physician 44, 1651. Welling, P. G. (1984). Interactions affecting drug absorption. Clin. Pharmacokinet. 9, 404. Welling, P. G., and Tse, F. L. S. (1982).The influence of food on the absorption of antimicrobial agents. J. Antimicrob. Chemother. 9, 7. Winstanley, P. A., and Orme, ML’E. (1989). The effects of food on drug bioavailability. Br. J. Clin. Pharmacol. 28, 621. Wix, A. R., Doering, P. L., and Hatton, R. C. (1992). Drug-food interaction counseling programs in teaching hospitals. Am. J. Hosp. Pharm. 49, 855.

Ian Appleton' Annette Tomlinson Derek A. Willoughby Department of Experimental Pathology Charterhouse Square London, EC I M 684 United Kingdom

Induction of Cyclo=Oxygenase and Nitric Oxide Synthase in Inflammation

1. General Introduction Inflammation is a complex series of overlapping cellular and plasma derived events which occur in response to injury or infection. The classic signs of inflammation as defined by Celsus (30 BC-38AD) and later added to by Virchow (1865)are heat, redness, swelling, pain, and loss of function. In fact, inflammation can be considered as not just one but a series of processes each characterized by different cellular populations, extracellular matrix components, and mediators. The initial inflammatory event leads to the sequential release of mediators starting with histamine, S-hydroxytryptamine (5-HT) and bradykinin (BK).Subsequently, the response is maintained by the prostaglandins (PGs)and a plethora of cytokines and growth factors. This sequential release occurs in a variety of inflammatory responses but

' To whom correspondence should be addressed. Advances in Pharmacology, Volume 35

Copyright 8 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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varies in time scale. For a review on cytokines and vasoactive mediators in inflammation and wound repair see Appleton, (1994). Inhibition of prostanoid formation by use of nonsteroidal anti-inflammatory drugs (NSAIDs) ameliorates the classical signs of inflammation, indicating their pivotal role in the inflammatory response. Due to the cytoprotective effects of the PGs in the stomach and kidney, inhibition of their biosynthesis with NSAIDs results in gastric and renal side effects. Recently, a second isoform of cyclo-oxygenase (COX), the enzyme which liberates the prostanoids, has been identified. This discovery has given new impetus as to the role of COX isoforms in inflammation and has fueled the search for selective inhibitors free from side effects. A body of evidence is also accumulating to indicate a role for nitric oxide as a mediator of inflammation. Like COX, the enzyme nitric oxide synthase (NOS), which liberates NO, exists in different isoforms. The use of inhibitors of these isoforms has uncovered a role for NO in all stages of inflammation. This chapter focuses on the role of prostanoids and NO at each stage of the inflammatory response with particular emphasis given to the cellular source and the factors which may modulate the activity of their respective enzymes. II. Cyclo-Oxygenase

One of the first events in an inflammatory response is the liberation of arachidonic acid from membrane bound glycerophospholipids by the enzyme phospholipase. Several forms of phospholipase have been identified which act on different substrates. It is not our intention to elaborate further on these enzymes, rather the reader is referred to Bonventre (1992). Once liberated, arachidonic acid is converted to the biologically active PGs and thromboxanes (Txs), collectively termed prostanoids, by the enzyme COX, also known as prostaglandin H synthase or prostaglandin endoperoxidase synthase. COX has two functions: (1) a cyclo-oxygenase activity that catalyzes PGG, formation, and (2) peroxidase activity that reduces the 15hydroperoxyl group of PGG, to PGH2 (Miyamoto et al., 1976; Pagels et al., 1983); for detailed description of the biochemistry of COX see Smith et al. (1991).These two reactions occur on the same enzyme but the active sites differ. Inhibition of COX, using NSAIDs, has no effect on peroxidase activity (Mizuno et al., 1982; Van der Ouderaa et al., 1980). Arachidonic acid is also metabolized by other enzymes including the lipoxygenases leading to generation of the hydroxyeicosatetranoic acids (HETEs) and leukotrienes (LTs; see Sigal, 1991; Ford-Hutchinson, 1990), and by the epoxygenase pathway, resulting in the formation of dihydroxy acids and epoxy eicosatrienoic acids (see Fig. 1). For extensive review of PG metabolism see Granstrom and Kumlin (1987).The generation of specific

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eytOkiIlW

LPS h0IM

C l

5 WETE

4

FIGURE I The major pathways of arachidonic acid metabolism.

arachidonic acid products is controlled by numerous complex mechanisms. The rest of this chapter concentrates on those mechanisms which may be in operation during inflammation.

A. lsoforms of Cyclo-Oxygenase Sir John Vane demonstrated in 1971 that the mechanism of action of the aspirin-like NSAIDs is via inhibition of COX (Vane, 1971). It soon became clear, however, that different NSAIDs had varying degrees of efficacy which were dependent on the tissue source of the enzyme (Flower and Vane, 1974). This led to the suggestion that different intracellular pools of COX may exist. However, it is only within the last few years that Xie et al. (1991) and Kujubu etal. (1991),working independently, identified a second isoform of cox. I. CycIo-Oxygenose I Cyclo-oxygenase 1 (COX-1) is a constitutive isoform found in virtually all cell types and is highly conserved throughout species. It is a homodimeric enzyme with a subunit molecular weight of 72 kDa and a central heme group. The gene for COX-1 contains multiple transcription start sites (Wang etal., 1993). The genomic DNA for COX-1 has been identified in numerous sources including human, bovine, and mouse tissues (Funk et al., 1991;

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DeWitt and Smith, 1988; DeWitt et al., 1990). It encodes a 600 amino acid protein with four glycosylation sites. Additionally an epidermal growth factor (EGF)-like domain lies next to the signal peptide with the site for aspirin acetylation (Ser-530)lying close to the carboxyl terminus. The COX1 mRNA is found constitutively in numerous tissues and is approximately 2.8 kb. Recently the X-ray crystallography of the structure of COX-1 protein has been determined (Picot et al., 1994). 2. Cyclo-Oxygenase 2

The COX-2 gene is approximately 60% homologous to COX-1 in human, mouse, and avian species. The major differences lie in the presence of a TATA box and regulatory sites for glucocorticoids and cytokines in COX-2 (Xu et al., 1995). It encodes for a 604 (for mouse) or 603 (for chick) amino acid protein. COX-2 protein has a unique 18 amino acid insert near the carboxyl terminal. Similar to COX-1, the COX-2 protein has an EGF-like domain, a serine site for aspirin acetylation, and five possible glycosylation sites. The COX-2 enzyme is able to utilize a larger number of substrates than COX-1 because it can metabolize C18 and/or C20 carboxylic acids, whereas COX-1 has greater specificity for 20 :4 fatty acids (Smith et al., 1994). The COX-2 mRNA is approximately 4 kb. COX-2 mRNA is found in the testes, brain, and lung but is usually at very low levels in most tissues (Simmons et al., 1991). For details of distribution of COX mRNA in human tissues see O’Neill and Ford-Hutchinson (1993). See Section 111 for agents which regulate mRNA and protein levels of COX. In addition to differences in structure, substrate specificity, and regulation, it has recently been demonstrated that differences occur in the intracelMar localization of COX-1 and COX-2. The activity of COX-1 is mainly localized to the endoplasmic reticulum, although some activity is observed around the nucleus. By contrast, the activity of COX-2 is predominantly around the nucleus with trace amounts in the cytoplasm (Smith et al., 1994). The discovery of different intracellular localizations in conjunction with factors which affect regulation and different substrate specificities suggests that these two enzymes can act independently. This raises the question whether COX-1 and COX-2 are modulated independently during an inflammatory response?

111. Regulation of COX-I and COX-2 The generation of prostanoids can be controlled at two levels: phospholipase and COX. However, because COX is inactivated during catalysis (Egan et al., 1976; Kent et al., 1983) control of the turnover of COX and COX activity is obviously particularly important in prostanoid biosynthesis. Cyclo-oxygenase-1 is present in virtually all cells at a constant level, being particularly high in platelets (Funk et al., 1991) and endothelial cells

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(DeWitt et al., 1983). Stimulation with an appropriate agonist results in a moderate increase in COX-1 levels. Cyclo-oxygenase-2is undetectable in most tissues, but is rapidly induced in response to mitogenic stimuli and other agents. A list of factors which effect prostanoid production and COX-2 expression in a variety of cell types are shown in Table I but this list is by no means exhaustive. The rapid induction of COX-2 following stimulation indicates that it is part of the immediate early gene family (Herschman, 1991; Ryseck et al., 1992). There are important distinctions in the regulation of COX-1 and COX2. The presence of a glucocorticoid regulatory region on the COX-2 gene (Xu, 1994)accounts for COX-2 induction being inhibited by glucocorticoids such as dexamethasone (Masferrer et al., 1992; DeWitt and Meade, 1993; Evett etal., 1993),whereas COX-1 is unaffected (DeWittand Meade, 1993). Studies in vitro have clearly illustrated the rapid and transient expression of COX-2. However, although rapidly expressed, COX-2 in vivo can continue to be synthesized for several days or even weeks given the appropriate or persistence of a stimulus. For example, vascular injury results in the persistence of COX-2 synthesis several days after the initial insult (Rimarachin et al., 1994). In addition to activation by cytokines and growth factors, COX activity can also be modulated by products of arachidonic acid metabolism. The hydroperoxide 15-HPETEcan stimulate COX at low concentrations and can inhibit COX at high concentrations (Warso and Lands, 1983). Arachidonate itself can increase prostanoid synthesis. This may be an important factor in initiating cell to cell induced prostanoid synthesis. For example, T lymphocyte-derived arachidonate can induce macrophage ( M 4 ) TX synthesis (Goldyne and Stobo, 1983). Likewise, platelet-derived PGH2can be used for the synthesis of PGI2 by endothelial cells (Marcus et al., 1980). The signal transduction pathway leading to activation of COX is not clearly established. Lipopolysaccharide (LPS) induction of COX-2 protein and COX activity in 5774.2 M+s can be inhibited by using tyrosine kinase antagonists such as erbstatin and genistein (Akarasereemont et al., 1994). Furthermore, the LPS induction of COX-2 in 5774.2 M+s may be indirectly related to the production of TNFa and PDGF (receptors which have intrinsic tyrosine kinase activity) because the use of neutralizing antibodies to these two cytokines inhibits COX-2 induction (Akarasereemont, personal communication). The tyrosine kinase pathway is also involved in endothelin-1 (ET-1) induction of COX-2 in rat mesangial cells (Kester et al., 1994).

IV. Prostanoids The PGs are polyunsaturated Cz0fatty acids with a characteristic cyclopentane ring structure. The term was first used to describe the presence of these substances in semen and the belief that they came from the prostate.

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TABLE I Factors Regulating the Activity and Expression of COX in Different Cell Types. COX-2 Expression Refers to Protein or mRNA.

Stimuli

Cell type

cox-2 PG expression Reference

IL-1

3T3 fibroblasts synoviocytes

t t

mesangial cells chondrocytes HUVECs

t t t

human alveolar macrophages

f

LPS

PMA

human monocytes u937 human PMNs m c s

t t t t t t t

PAF

dermal fibroblasts u937 HUVECS BAEC human PMNs mouse glial astrocytes rat alveolar macrophages

GM-CSF G-CSF bFGF TGFP PDGF

human PMNs human PMNs mouse glial astrocytes MC3T3-El 3T3 fibroblasts

t t

1L-2 TNF

EGF

bovine smooth muscle cells 3T3 fibroblasts ET-1 rat mesengial cells IL-lo human monocytes chorionic rat preovulatory granulosa gonadotropin cells dexamethasone dermal fibroblasts 3T3 fibroblasts human monocytes u937 rat mesangial cells neurones heparin rat mesangial cells stretch/relaxation rat mesangial cells

f

t t f

t

1 1 1 1

t t t t t t

t t t t

t t

.1

t

1 1 J. 1

t

Burch et al. (1988) O’Neill et al. (1987) Gilman et al. (1988) Nakazato et al. (1991) Chang et al. (1986) Rossi et al. (1985) Habib et al. (1993) Monick et al. (1987) Lee et al. (1992) O’Sullivan et al. (1992) Hempel et al. (1994) Hempel et al. (1994) Bienkowski et al. (1989) Herrmann et al. (1990) Wu et al. (1988) Habib et al. (1993) Raz et al. (1989) Koehler et al. (1990) Frasier-Scott et al. (1988) Frasier-Scott et al. (1988) Herrmann et al. (1990) O’Banion et al. (1994) Thivierge and RolaPleszczynski (1994) Herrmann et al. (1990) Herrmann et al. ( 1990) O’Banion et al. (1994) Sumitani et al. (1989) Habenicht et al. (1985) Herschman et al. (1994) Bailey et al. (1985) Herschamnn et al. (1994) Kester et al. (1994) M e m et al. (1994) Sirios and Richards (1992) Raz et al. (1989) DeWitt and Meade (1993) Raz et al. (1990) Koehler et al. (1990) Kester et al. (1994) Yamagata et al. (1993) Kester et al. (1994) Akai et al. (1994)

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None of the prostanoids (or LTs) are stored within cells but rather are synthesized from fatty acid precursors on stimulation. Once formed, all are rapidly metabolized with biological half-lives of approximately 1 min. There are numerous prostanoids and derivatives (see Fig. 1). However, after the generation of PGGz,generally only one arachidonic acid metabolite is formed in abundance and this effect is cell specific. For example, TXA2 is the major arachidonic acid metabolite of platelets (Hamberg et al., 1975). Endothelial cells from large vessels produce PGIz (Weksler et al., 1977); in contrast, human foreskin capillary endothelial cells produce mainly PGFz, (Charo et al., 1984). PMNs produce TXA2 but they primarily synthesize the 5lipoxygenase product LTB4 (Borgeat and Samuelsson 1979). The major COX product of mast cells is PGDz (Lewiset al., 1982) and PGEzis produced by fibroblasts and M+s (DeWitt, 1991).

A. Prostanoids and Inflammation Elevated levels of COX derivatives have been found in numerous human inflammatory conditions including rheumatoid arthritis (RA), osteoarthritis, psoriasis, allergic eczema, gout, and ulcerative colitis (Higgs et al., 1984). However, with the recent finding of a second isoform of COX, much of the literature concerning prostanoids in inflammation must be reevaluated in terms of which isoform is responsible for their elaboration. In a number of animal models of inflammation, PGEzis the major metabolite of arachidonic acid (Willis, 1969a; Velo et al., 1973; Glatt et al., 1974; Herman and Moncada, 1975; Bonta and Parnham, 1977).However, other COX products are detectable. TXAz and PGIz have been identified in granulomas induced by carrageenin (Chang et al., 1976). Normal synovial fluid does not contain prostanoids (Herman and Moncada, 1975), whereas tissue cultures of synovium from patients with RA are able to produce PGE2, 6-keto-PGF1, and TXBz (Robinson et al., 1975; Salmon et al., 1983). 1. Prostaglandin E z

The enzyme independent conversion of PGH results in the rapid production of PGE (Hamberg and Samuelsson, 1967). PGE can also be formed by the action of PGE synthase (Ogino et al., 1977). PGEZ is the major arachidonic acid metabolite formed in inflammation. a. Pro-Inflammatory E f f e a ~ ofProstaglandin €2 The production of PGE2at sites of injury is proinflammatory due to its ability to cause vasodilatation (Vane, 1976), which augments edema formation caused by agents which increase vascular permeability such as BK and histamine (Williams and Peck, 1977). PGE2 also potentiates pain (Davies et al., 1984; see Section V and Fig. 2).

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Ian Appleton et al.

Trauma

r

spinal c o z

FIGURE 2 Diagram to show the involvementof NO and prostanoidsin acute inflammation.

One characteristic of acute inflammation is erythema. PGE2 and PG12 are potent vasodilators whereas PGFh is a vasoconstrictor. The production of PGEz and PGIZ during acute inflammation is sufficient to account for erythema as demonstrated by the injection of PGEz into human skin (Juhlin and Michaelson, 1969). Acute inflammation is also characterized by fever. The pyrexia induced by IL-1 is mediated via the production of PGE2,which is one of the most potent pyretic substances yet identified (Bernheim et al., 1980). Thus, many of the classical signs associated with acute inflammation can be accounted for by PGEZ production. Therefore a proinflammatory role for PGE2is indicated and inhibition by NSAIDs results in reduction of pain, swelling, and fever. The role of PGEZ in the chronic inflammatory autoimmune disease RA can also be considered to be proinflammatory due to its destructive effects on bone. The addition of PGEzto bone cultures stimulates osteoclast activity and bone resorption (Klein and Raisz, 1970; Tashjian et al., 1972). PGE2 also inhibits bone collagen synthesis (Hefley et al., 1986) and increases collagenase production in osteoblasts (Partridge et al., 1987). In addition to antagonizing osteoblast proliferation induced by mononuclear cells (Gowen et al., 1985), PGE2also contributes to the juxta-articular bone erosions observed in RA.

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b. lmmunonodulatory and Anti-Inflammatory Effec~ of Prostaglandin E2 Prostaglandin E2 has potent effects on the immune system. For example, it causes differentiation of immature thymocytes and B lymphocytes to mature cells (Parker, 1986). However, in general, the actions of PGE2 are immunosuppressive. The evidence that PGs of the E series downregulate the immune response is derived mainly from in vitro studies. PGE2is a potent inhibitor of lymphocyte mitogenesis (Goodwin et al., 1978), migration (Van Epps, 1981), and cell-mediated cytotoxicity (Schultz et al., 1979; Meerpohl and Bausknecht, 1986). Inhibition of cytokines including IL-1 (Kunkel et al., 1986a) and TNF (Kunkel et al., 1986b) by PGE2is observed in M+s. PGE2also inhibits the production of IgM in B cells (Phipps et al., 1990) and the production of T cell-derived lymphokines such as IL-2 and IFNT (Betz and Fox, 1991; see Fig. 3). Recently, it has been demonstrated that two groups of T helper cells (Th cells) can be identified on the basis of their cytokine elaboration following challenge with mitogen or antigen. Thl cells mainly produce the proinflammatory cytokines IL-2 and IFNT, whereas TH2 cells produce the antiinflammatory cytokines 1L-4 and IL-10. The ability of PGE2 to inhibit Thl cytokines adds further to its immunosuppressive and anti-inflammatory actions in chronic inflammation by pushing toward a Th2 cell response (Betz and Fox, 1991). Other anti-inflammatory effects of PGE2include inhibition of the oxidative burst, LTB4 production, and lysozyme release in PMNs (Weissman et al., 1971).

FIGURE 3 Diagram to show the involvement of NO and prostanoids in chronic inflammation.

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Ian Appleton et al.

The in vivo actions of PGE2 on the immune system were first demonstrated by Webb and Osheroff (1976). Following challenge with antigen, raised levels of PGE2 were found in the spleen which were inhibited by treatment with indomethacin. For an extensive review of the effects of PGE, on the immune system see Phipps et al., (1991). The potent immunosuppressiveeffects of PGE2have led to its implication in certain immunopathologies. It is well established that tumor cells (Sykes and Maddox, 1972) and monocytes from patients with Hodgkin’s disease produce elevated levels of PGE2 (Bockman, 1980). The immunosuppressive effects are reduced by indomethacin in both cases (Hial et al., 1976; Bockman et al., 1987). Recently, it has been demonstrated that human colorectal adenomas and adenocarcinomas contain high levels of COX-2 (Eberhart et al., 1994). If this isoform is responsible for the production of PGE2 with immunosuppressive actions, then selective inhibition of COX-2 may have greater therapeutic implications for tumor therapy. 2. Prostacyclin Prostacyclin was originally identified on the basis of its ability to inhibit platelet aggregation (Moncada et al., 1976).It is a bicyclic prostanoid which rapidly breaks down to the stable 6-keto-PGF1,. It is formed by the enzyme PG12 synthase. In humans the mRNA levels for PG12synthase are increased by the proinflammatory cytokines IL-1 and IL-6 (Miyata et al., 1994). Therefore, during an inflammatory response the levels of PGI2 will be controlled at the level of phospholipase, COX, and PG12 synthase. Endothelial cells are the major producers of PGIp (Weksler et al., 1977; MacIntyre et al., 1978), although vascular smooth muscle cells and fibroblasts also have this capability (Baenziger et al., 1979). PGIz release is stimulated by numerous chemical and physical agents including arachidonic acid, substance P, thrombin, BK, and pressure (see Gryglewski et al., 1988; Quadt et al., 1982). PG12 is a weak inhibitor of platelet adhesion. The inhibitory actions of PG12 on platelet aggregation and adhesion may be considered anti-inflammatory. The inhibitory effects of PG12 on phospholipase A2 and phospholipase C act to decrease substrate availability for COX, adding further to its antiinflammatory effects (Siess, 1989). PGI, also has effects similar to PGEp in that it inhibits cytokine release in several cell types including M+s and endothelial cells (Willis et al., 1986; Willis and Smith, 1989). However, an overproduction of PGIz may contribute to endotoxic shock (Naworth et al., 1984).

3. Thromboxane A2

Thromboxane A2 (TXA2)is generated by the action of the ferrihemoprotein enzyme thromboxane-A synthase on PGHz. During catalysis the enzyme undergoes inactivation (Jones and Fitzpatrick, 1991). The major source of

Induction of Cyclo-oxygenase and Nitric Oxide Synthase in Inflammation

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TXA2is the platelet (Hamberg et al., 1975), but it can also be generated by M+s (Morleyet al., 1979). It is rapidly broken down (half-lifeapproximately 30 sec) to the inactive but stable metabolite TXB2. The gene for TXA2 synthase has recently been mapped and in humans is approximately 100 kb (Shen et al., 1995). Unlike the gene for COX-2, the TXAZ synthase gene cannot be induced by agents such as LPS (Tanabe et al., 1995). Therefore, the control of TXA2generation is at the level of phospholipase and/or COX. The principal actions of TXA2in inflammation are its effects on inducing platelet aggregation and vasconstriction (Hamberg et al., 1975). Although the platelet is the major depot of circulating TXAz it is unlikely that this is the source of inflammatory TXA2. In experiments where platelets were depleted, no change was observed in the levels of TXB2,whereas in neutropenic animals, TXB2 levels in the exudate fell but were unchanged in the serum. Thus the platelet is the source of circulating TXA2 involved in blood clotting and the PMN is the cellular source in inflammation. PMNs from human and guinea-pig sources predominantly make TXA2 (Morley et al., 1979). 4. Prostaglandin Fh

In the rat carrageenin-induced pleurisy model the levels of the potent vasoconstrictor PGFh peak before those of PGEZ. This finding led to the hypothesis that PGF2, may inhibit the edema formation induced by PGEZ. Indeed the administration of PGF2, will directly inhibit the edema induced by PGE2 (Crunkhorn and Willis, 1971). PGFz, also inhibits edema formation in a number of models of inflammation and antagonizes the increased vascular permeability induced by BK, 5-HT, and histamine (Willoughby, 1968). 5. Prostaglandin D2

PGDz is formed through a nonoxidative conversion of PGH2. A role for PGD2in inflammation has not been clearly established. However, some of its properties suggest an involvement in the inflammatory response. PGDZ inhibits platelet aggregation (Whittleet al., 1978)and can cause bronchoconstriction (Wasserman et al., 1977).The main cellular source of PGDz during inflammation is the mast cell (Lewis et al., 1982). B. Prostanoids and Chronic Inflammation

The role of prostanoids in acute inflammation is well established. Their role in chronic inflammation is more elusive due to their proinflammatory, anti-inflammatory, and immunosuppressive actions (see Sections W.A. 1.a and IV.A.1.b). The growth of RA inflammatory tissue or pannus is dependent on new blood vessel formation “angiogenesis” to supply nutrients to the newly forming tissue. PGEz in vitro is proangiogenic (Form and Auerbach, 1983).

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Ian Appleton et al.

In the chick chorioallantoic membrane (CAM) model, the angiogenic response induced by basic fibroblast growth factor (bFGF) is mediated by PGEl (Spisni et al., 1992; see Fig. 3). Furthermore, neovascularization of solid tumors is due partly to PGE (Peterson, 1986; Ziche et al., 1982). Thus the action of PGE, may be considered proinflammatory due to its effects on angiogenesis, a component of many chronic inflammatory conditions. Fibroblasts, M+s, and lymphocytes are also involved in chronic inflammation. The effects of PGEz on these cells dictates anti-inflammatory activity (see Section IV.A.1.b). In a model of T-cell driven chronic granulomatous inflammation, we have shown high levels of COX activity (with PGE, being the major metabolite) at Day 14, a time when the inflammation has started to resolve. We have interpreted this result to indicate that in chronic inflammation PGE, is exerting both an anti-inflammatory and immunosuppressive effect (see Appleton et al., 1995). Many, if not all, of the chronic inflammatory conditions are T-cell driven by an exogenous or endogenous antigen; therefore, the immunosuppressive actions of the PGs, in particular PGE,, must be considered anti-inflammatory (see Section IV.A.1.b). Thus, in acute inflammation PGE, is undeniably proinflammatory whereas in chronic inflammation a number of anti-inflammatory effects may be in operation. This is particularly relevant to inflammatory diseases such as RA which is characterized by episodes of acute flare up superimposed on a chronic inflammatory state.

V. Prostaglandins and Pain The PGs alone do not produce pain, but rather sensitize afferent nociceptors to the effects of other pain producing substances such as BK and histamine (Ferreira, 1972; see Fig. 2). The major PGs involved in hyperalgesia are PGE, and PGI,. The hyperalgesic actions of these two mediators are different and point to different pathophysiological roles. The injection of PGE, results in a delayed onset but long lasting hyperalgesic state (Ferreira, 1972; Moncada et al., 1975). In contrast, the effects of PGI, are observed more rapidly and quickly decline. Additionally, PGIzis a more potent hyperalgesic than PGE, (Ferreira et al., 1978). Differences in the potency and duration of these two prostanoids implicate them in different types of painful conditions. Thus, PGIz may be involved in certain types of headache (which are ameliorated rapidly by COX inhibitors), whereas PGE, may be involved in back pain and sunburn. A. Nonsteroidal Anti-Inflammatory Drugs and Inflammatory Pain

It is generally accepted that inhibition of peripherally formed PGs is the basis for the analgesic effects of NSAIDs (Willis, 1969b; Juhlin and

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Michaelson, 1969; Ferreira, 1972). However, in models of pain, inhibition of locally formed PGs alone does not account for the analgesic properties of many of these compounds (McCormack and Brune, 1991; Weissman, 1992; Brune et al., 1993). This is best exemplified by dipyrone which at clinically effective doses has little effect on PG biosynthesis but is a potent analgesic. Salicylic acid at analgesic concentrations also exhibits no effect on COX activity (Brune et al., 1991). Similarly, aspirin at analgesic and antipyretic doses has no anti-inflammatory activity (Abramson and Weissman, 1980; Weissman, 1991). An effect of NSAIDs on centrally formed PGs can therefore not be discounted (for review see Urquhart, 1993) and actions separate from direct inhibition of COX must also be considered. For example, ketoprofen can significantly reduce the amount of the neurotransmitter substance P in the hypothalamus and spinal cord (Dubourdieu and Dray, 1989). For comparison of anti-inflammatory and analgesic effects of NSAIDs see McCormack and Brune, (1991) and for the mechanisms of action of NSAIDs on pain see McCormack (1994). Little information exists concerning the isoform of COX responsible for the production of the PGs involved in pain. However, it has recently been demonstrated that the selective COX-2 inhibitors, SC-58125 (1-[(4pyrazole) and methyl sulfonyl) phenyl]-3-triflouromethyl-5-(4-fluorophenyl) L745,337 (5-methanesulfonamido-6-(2,4-difluorothiophenyl)-l-indanone) have analgesic effects on thermal injury and the carrageenan-induced rat paw hyperalgesia assay respectively, suggesting a role for COX-2 in inflammatory pain (Seibert et al., 1994; Chan et al., 1994). Anomalies between anti-inflammatory, analgesic, and antipyretic effects of NSAIDs may be explained by recent studies on aspirin. Aspirin and sodium salicylatecan inhibit NF-KB (Kopp and Gosh, 1994),a transcription factor involved in the activation of cytokines including IL-1, IL-6, IL-8, TNFa, and IFNO (Grilli et al., 1993). Furthermore the use of large doses of aspirin will acetylate Ser 516 on ovine COX-2 resulting in the metabolism of arachidonic acid to 15-HETE, an effect not observed with COX-1 (Holtzman et al., 1991). 15-HETE has potent anti-inflammatory properties including inhibition of LTB4 formation, synovial cell proliferation, (Herlin et al., 1990) and carrageenin-induced arthritis (Fogh et al., 1989). Thus the antiinflammatory effects of aspirin in addition to inhibition of COX may also include production of the anti-inflammatory agent 15-HETE by COX-2 and inhibition of proinflammatory cytokines. Paracetamol has potent analgesic and antipyretic effects but is only a mild anti-inflammatory agent. However, it is more active at inhibiting PG production in the CNS (Flower and Vane, 1972). This discrepancy associated with the analgesic vs anti-inflammatory action of NSAIDs (in particular paracetamol) inevitably raises the question: Is there a third isoform of COX present centrally?

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VI. COX-I and COX-2 in Inflammation

The rapid induction of COX-2 following appropriate stimulation may imply a role for this isoform in acute inflammation as prostanoid levels are rapidly increased following insult. In a complement-dependent model of acute inflammation, the rat carrageenin-induced pleurisy, we have demonstrated that COX activity peaked at 2 to 6 h after injection of the carrageenin but by 24 h was significantly reduced. Western blot analysis showed that this activity correlated with COX-2 protein levels with COX-1 protein remaining constant throughout the time course (Tomlinson et al., 1994). PMNs were the major source of COX-2 immunoreactivity. A proportion of M+s and mesothelial cells (the cell type lining the pleural cavity) were also immunolabeled for COX-2. These studies have been extended by Katori et al. (1995)who demonstrated that dexamethasone suppressesthe induction of COX-2 and PG release in the rat carrageenin-induced pleurisy model. In two immune-driven models of acute inflammation, the Arthus reaction and pertussis pleurisy (cell-mediated immunity), we have shown that COX-2 protein increased with COX-1 protein remaining constant (Moore et al., 1995). Therefore, in several models of acute inflammation COX-2 is the predominant isoform and its activity can be blocked by glucocorticoids. In the acute stages of the murine air pouch model of croton oil-induced chronic granulomatous inflammation COX activity progressively rose during the first 24 h accompanied by an increase in COX-2 protein. In the chronic phase of the inflammatory response COX activity was two to three times greater than in the acute stage. This profile of activity was mirrored by COX-2 protein levels with COX-1 protein being unchanged throughout the time course (Vane et al., 1994). A number of COX metabolites have been measured in this model and PGE2 was always proportionally greater at all time points measured > 6-keto PGFI, > TXBz > PGFh. Furthermore, the major source of COX-2 protein was the M+. At later time points COX2 labeled fibroblasts and endothelial cells were also observed (Appleton et al., 1994). The profile of a number of cytokines involved in inflammation has been documented in this model of chronic inflammation (Appleton et al., 1993). On the basis of this work it is suggested that transforming growth factor P (TGFP), which is known to induce COX (see Table I), may be the endogenous cytokine responsible for the control of COX-2 activity in chronic inflammation. It is well documented that RA synovial fluids contain elevated levels of PGE2. Recently, Crofford et al. (1994)have demonstrated that under basal conditions RA synovial explants express COX-1 and COX-2 protein. Following stimulation with IL-lp or PMA, COX-2 protein and mRNA was markedly increased while COX-1 levels showed only slight elevation. Furthermore, this induction of COX-2 mRNA was blocked by dexamethasone with COX-1 mRNA levels unchanged. In addition, the same cellular ele-

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ments within the RA synovium, as we have shown in a model of chronic inflammation (Appleton et al., 1994), express COX-2 namely the macrophage, endothelial cell, and fibroblast. VII. Pharmacological Inhibition of COX- I and COX-2 It is well established that COX is the target for the therapeutic effects of NSAIDs (Vane, 1971). It is equally well documented that NSAIDs have varying degrees of efficacy dependent on the tissue source of COX (Flower and Vane, 1974). These findings may now be explained in light of the existence of two isoforms of COX. Thus the effect of an NSAID will depend on the relative amounts of COX-1 and COX-2 present in a tissue and also on its relative inhibitory capacity for COX-1 and/or COX-2. Table I1 gives a list of currently used NSAIDs and a number of experimental compounds and their inhibitory effects on COX-1 and COX-2 in different assay systems. Although differences in the ECSO values occur (depending on the assay system used), it is generally accepted that most NSAIDs are equipotent inhibitors of COX-1 and COX-2 while aspirin and salicylate have greater efficacy for COX-1. COX-1 is present constitutively in the stomach and kidney. In models of gastric ulcers, PGEI, PGE2, and PG12have been shown to protect against gastric erosions induced by a number of agents (Ferguson et al., 1973; Lee et al., 1973; Whittle, 1976). The removal of these cytoprotective effects may hence account for the gastric and renal side effects associated with the use of aspirin and indeed other NSAIDs. Cyclo-oxygenase-2 is the predominant isoform of COX in a number of models of inflammation (Vane et al., 1994; Appleton et al., 1994; Tomlinson et al., 1994; Moore et al., 1995; Katori et al., 1995). At present there are no specific COX-2 antagonists in clinical usage. However, several experimental compounds are available. NS-398 (N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide])selectively blocks COX-2 in vitro (Futaki et al., 1994) and in vivo in the rat carrageenin-air pouch model of inflammation (Futaki et al., 1993a; Masferrer et al., 1994) while sparing COX-1 and thus gastric side effects (Futaki et al., 1993b; Masferrer et al., 1994). Similar gastric sparing and anti-inflammatory effects are reported with the selective COX-2 inhibitors L-745,337 and SC-58125 on rat carrageenin-induced paw edema (Chan et al., 1994; Seibert et al., 1994). Strategies other than selectively antagonizing COX-2 may prove equally as effective for inhibiting the generation of prostanoids in inflammation. The use of a carrier molecule able to localize to the site of inflammation may increase the effect of an NSAID which has limited inhibitory effects on COX-2. In the murine model of chronic granulomatous inflammation, diclofenac in combination with the carrier molecule hyaluronic acid leads to a greater suppression in granuloma dry weight in comparison to diclofenac

TABLE II The Relative Inhibitory Capacity of Currently Available and Experimental NSAlDs on COX- I and COX-2 in Various Systems. Assays for

Drugs

cox-1

cox-2

Diclofenac Diclofenac Meloxicam Indomethacin CGP-28238 Tolfenamic acid CGP-28238 CGP-28238 SC-5812 BF389 Flurbiprofen Piroxicam CGP-28238 Tenoxicam

Human platelets Gp MOs GP MOs Human platelets Human platelets BAEC BSV TF'A-HEL cells Platelets BAEC BAEC GP MOs BSV GP MOs

IL-1 rat mesangial cells LPS GP MOs LPS GP MOs IL-1 rat mesangial cells IL-1 rat mesangial cells LPS 5774.2 MOs IL-1 murine calvarial cultures LPS human monocytes IL-1 fibroblasts LPS 5774.2 M8s LPS 5774.2 MOs LPS GP MOs IL-1 bone resorption LPS GP MOs

IGo (CLM)

cox-2 0.0012 0.0019 0.0019 0.0069 0.0147 0.019 0.02 0.038 0.07 0.09 0.1 0.175 0.3 0.322

Ratios

cox-1 0.0179 0.00085 0.0058 0.0015 72.3 0.001 >1000 >10 100 0.45 0.08 0.0053

>loo0 0.0201

cox-2/cox-1 0.067 2.23 0.33 4.5 0.0002 16.6 0.00002 0.0038 0.0007 0.2 1.25 33 0.0003 16

Piroxicam Indomethacin Indomethacin Diclofenac Diclofenac Indomethacin Indomethacin Flurbiprofen Flurbiprofen NS-398 Flurbiprofen BW755C Naproxen Indomethacin Ibuprofen Carprofen Flurbiprofen Sulindac

BAEC RSV hCOX-1 BAEC RSV Broken BAEC BAEC BAEC Murine rCOX-1 RSV GPMOs BAEC BAEC GP MOs Murine rCOX-1 BAEC Purified COX-1 Murine rCOX-1

,. From Battistini et al. (1994, Table 11, pp. 506-507).

LPS 5774.2 MOs Sheep placenta hCOX-2 LPS 5774.2 MOs Sheep placental cotyledons LPS broken 5774.2 MOs LPS 57742.2 MOs LPS 5774.2 MOs Murine rCOX-2 Sheep placenta LSP GP MOs Murine rCOX-2 LPS 5774.2 MOs LPS GP MOs Murine rCOX-2 LPS 5774.2 MOs Purified COX-2 Murine rCOX-2

0.9 0.97 1 1.1

0.0015 0.74 0.02 1.57

1.12 1.68 2.05 2.75 3.8 4.76 5.58 5.64 6.81 7.7 10.96 12.3 12.5

0.028 0.028 0.41 0.48

-

-

>loo

0.015 3.02 9.55 0.0636 11.45 10.96 1.2 0.4

600 1.31 50 0.7 4 40.1 60.1 5 5.7 0.038 317.3 1.85 0.6 107.1 0.67 1 10.25 31.25

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or hyaluronic acid alone (Seed 1994 personal communication) and could therefore be used as an adjunct for anti-inflammatory therapy. A recent development for reducing the gastrointestinal effects of NSAIDs is the linkage with a N O donor. NO helps maintain gastric blood flow and inhibits leukocyte adherence (Kubes et al., 1991) and would therefore counteract the effects of the NSAID alone. Because NO can inhibit platelet aggregation, these compounds may also have antithrombotic activity. Examples include diclofenac nitroxybutylester and ketoprofen nitroxybutylester. In models of acute and chronic inflammation it has been shown that these agents will suppress inflammation while producing less gastric side effects (Reuter et al., 1994; Cuzzolin et al., 1994). It remains to be seen whether selective inhibitors of COX-2 or NSAIDs-NO compounds will prove clinically more effective than existing NSAIDs for the treatment of chronic inflammatory diseases. Selective inhibition of COX-1 may be more beneficial in the treatment of cardiovascular diseases. Platelets cannot synthesize new COX, i.e., no COX-2. The major prostanoid produced by platelets is the prothrombic TXA2,whereas endothelial cells produce the antithrombogenic PG12. Therefore the use of low doses of aspirin, which irreversibly inhibits COX, may inhibit TXA2 formation without significantly affecting PG12, as endothelial cells are able to constitutively synthesize COX. Other selective COX-1 compounds include FR122047 which is 100 times more potent than aspirin at inhibiting platelet aggregation (Dohi et al., 1993).It seems unlikely, however, that selective COX-1 inhibitors will replace the use of aspirin, as it is readily available and inexpensive.

VIII. Conclusion Theoretically, if COX-2 is primarily responsible for the elaboration of prostanoids in inflammation, then its selective inhibition may lead to less gastric and renal side effects which are associated with traditional NSAID therapy. However, the effects of selectively antagonizing COX-2 in chronic inflammation may further inhibit the immunomodulatory and antiinflammatory properties of PGE2 and PG12. Furthermore, although COX-2 is an inducible enzyme, it is constitutively expressed in certain tissues including rat seminal vesicles, human pregnant myometrium (Zuo et al., 1994), and rat fallopian tube epithelial cells (Bryant et al., 1993). Selective COX2 inhibition in these tissues thus may have untold side effects on reproductive function. Indeed-COX-2 knock out mice are infertile (Herschmann et al., 1994). In addition, COX-2 inhibition may have effects on disease progression. Based on the work of De Brito et al. (1987) and Desa et al. (1988), it was demonstrated that treatment with NSAIDs accelerated cartilage degra-

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dation both in vitro and in vivo. This effect can be reversed however by the administration of the PGEl analogue misoprostol (Dingle, 1991, 1993). In conclusion, the use of specific COX-2 inhibitors for the treatment of inflammatory disease at present remains to be proved. However, novel compounds are being used experimentally and these questions for human pathology will undoubtedly be rapidly addressed.

IX. Nitric Oxide Synthase Important experiments performed in 1987 by Palmer, Ferrige, and Moncada revealed and confirmed previous suggestions (seeFurchgott, 1988) that the biological activity of the elusive molecule, termed endothelium-derived relaxing factor (EDRF),involved in vascular relaxation, could be accounted for by nitric oxide (NO). They observed that NO, synthesized by endothelial cells (ECs),acted as an intercellular effector molecule, causing vasodilatation by activation of guanylate cyclase and the elevation of cGMP levels in vascular smooth muscle cells (for review, see Moncada et al., 1989). This discovery opened the gates to thousands of investigations which implicated NO, not only in the physiological regulation of the vasculature, but also in neurotransmission, reproduction, host defense mechanisms and in several pathophysiological events (for review, see Moncada et al., 1991). In 1992, NO was named “molecule of the year” (Koshland, 1992) and all aspects of its biology and chemistry have been extensively reviewed (Ignarro, 1991; Nathan and Hibbs, 1991; Snyder and Bredt, 1992; McCall and Vallance, 1992; Nathan, 1992; Dinerman et al., 1993; Gorbunov and Esposito; 1993; Nussler and Billiar, 1993; Lowenstein et al., 1994; Nathan and Xie, 1994 a,b; Anggard 1994; Schmidt and Walter, 1994; Marletta, 1994; Stamler, 1994; Laskin et al., 1994). Endogenous NO3 production by mammals (in excess over dietary intake) was long thought to be a product of intestinal microorganisms and was recognized only recently to be partially independent of this source (Green et al., 1981).Urinary excretion of NO3increases in fever and can be experimentally induced in rats by injection of bacterial toxins. Cytotoxicity of NO2 to microorganisms was well established when Stuehr and Marletta (1985, 1987) showed that the pathway of mammalian biosynthesis of NO2 and NO3 was expressed on immunostimulation of M+s by exogenous bacterial LPS and endogenous INFT, but was absent from quiescent cells. L-arginine was discovered as the precursor molecule for NO2 and NO3 production (Iyengar et al., 1987; Hibbs et a1.,1987) with the additional production of citrulline (Iyengar et al., 1987). L-arginine also appeared to be essential for M+ inhibition of tumor proliferation (Hibbs et al., 1987); inhibition being blocked by an L-arginine substituted analogue. Therefore, the recognition of N O as an intercellular effector molecule suggested that it may be similarly

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operative in M+-mediated cellular cytotoxicity. Subsequently, NO was confirmed as an intermediate in M+ conversion of L-arginine to NO2 (Marletta et al., 1988); as a molecular effector of M+-induced cytotoxicity (Hibbs et al., 1988); secreted as a metabolite of arginine from activated murine M+s (Stuehr et al., 1989) and responsible for cytostasis and respiratory inhibition in tumor target cells (Steuhr and Nathan, 1989; for review, see Nathan and Hibbs, 1991). Nitric oxide is now recognized as an effector molecule of M+ cytotoxicity activated by specific cytokines and participating in the elimination and growth inhibition of tumor cells and pathogens including bacteria, parasites, fungi, and viruses. However, it is important to acknowledge that this cytotoxic ability may also have deleterious consequences for nearby normal cells, resulting in cellular death and tissue trauma.

A. Induction and Inhibition of Nitric Oxide Synthase From early experiments it was apparent that M+ production of NO was quantitatively and qualitatively different from that of the EC. NO production from ECs is synthesized on demand at low levels and released for short periods in response to receptor activation or mechanical stimulation (Bredt and Snyder, 1990). Mediators capable of inducing this activity include BK, acetylcholine, ADP, Substance P, and shear stress (see Moncada et al., 1991). In contrast, M+s are capable of sustained release of high levels of N O initiated by inflammatory cytokines and bacterial products. The enzyme catalyzing the conversion of L-arginine and molecular oxygen to NO and L-citrulline is nitric oxide synthase (NOS) and differential N O production is attributable to the isoform of NOS present in different cells. Eight cDNA sequences from three NOS genes in four species have been reported to date (see Nathan and Xie, 1994a). Two isoforms of a constitutively expressed NOS have been isolated and cloned from neurons and ECs. Neuronal NOS (nNOS or NOS I, a recently suggested nomenclature; Nathan and Xie, 1994b) is found in a population of central and peripheral nonadrenergic, noncholinergic (NANC)neurons and is also present in skeletal muscle, pancreatic islets, endometrium, and respiratory and gastrointestinal (GI)epithelia. ECs contain eNOS (NOS 111).M+s synthesize an inducible isoform iNOS or NOS 11. The isoform synthesized is dependent on cell type and cell activation. All three isoforms have sequence homology. Across species, homology averages 90% between equivalent isoforms. NOS 1 and I11 depend on Calf and calmodulin for catalytic activity, while NOS I1 is Caz+-independent and has calmodulin tightly bound to the enzyme. All isoforms require the cofactors flavin adenine nucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide phosphate, and tetrahydrobiopterin (for reviews see Forstermann et al., 1991; Marletta, 1994; Lowenstein et d., 1994).

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A number of proteins and growth factors inhibit NO production in a variety of cell types. These include EGF, TGFP, macrophage stimulating protein, IL-4, IL-8, IL-10, bFGF, PDGF, and osteopontin (see Laskin et al., 1994). Glucocorticoids in inflammation as well as selectively inhibiting COX-2 expression (see Section 111)can also inhibit N O production (Radomski et al., 1990). NOS I1 is regulated posttranscriptionally consistent with the absence of a steroid response element in the promoter region. Colleaguesin our institute have shown recently that endogenous lipocortin mediates the inhibition by dexamethasone of the expression of NOS I1 (Wu et al., 1995). Pretreatment with a neutralizing antiserum to lipocortin 1 abolished the inhibitory effect of dexamethasone on NOS I1 synthesis and nitrite accumulation in LPS stimulated 5774.2 M+s. Similarly, lipocortin 1 neutralizing antibodies abolished the dexamethasone inhibition of NOS I1 induction elicited in LPS treated rats. NOS is inhibited endogenously and pharmacologically, both in vitro and in vivo, by analogues of L-Arginine (Rees et al., 1990, McCall et al., 1991). NG,NG-Dimethylarginine is an endogenous inhibitor in man and accumulates in patients with chronic renal failure (Vallance et al., 1992). Arginine analogues have proved immensely valuable in dissecting mechanisms involving NO; however, they are capable of interacting with other iron containing enzymes, including COX and their effects may not be solely attributable to inhibition of NOS (Peterson et al., 1992).

B. Cellular Production and Activity of NO at Inflammatory Sites Polymorphonuclear neutrophils and M+s are the predominant inflammatory cells at the sites of acute and chronic inflammation respectively. Human PMNs synthesize NO (Wright et al., 1989; McCall et al., 1989) at levels sufficient to function in vitro as a vasodilator (Schmidt et al., 1989) and inhibit thrombin-induced platelet aggregation (Salvemini et al., 1989). Whether the levels of NO produced by PMNs have a role in nonspecific host defense has been questioned. It is suggested that PMN-derived NO produced in the early stages of an experimental wound model [s.c. implanted polyvinyl alcohol (PVA) sponges in the rat], mediates vasodilatation, inhibitiodreversal of platelet aggregation, and also antimicrobial activity (Albina eta]., 1990). In addition, cytoplasts prepared from human PMNs are purported to kill staphylococci (Malawista et al., 1992). Recent data on nitrite levels generated by rat, mouse and human PMNs (Padgett and Pruett, 1995) suggest that all three species are similar in their ability to produce reactive nitrogen intermediates; however, at considerably lower levels than those produced by rodent M+s. Such levels are sufficient to act as a vasodilator or neurotransmitter, but

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not to be effective as an antimicrobial or antitumor agent. Therefore, the precise role of PMN-derived N O in host defense remains equivocal. Numerous experiments, particularly in rodents and using cell lines, have demonstrated that M+s produce high levels of N O in response to bacterial products and proinflammatory cytokines including INFT, TNFa, and IL-1P (see Moncada et al., 1991; Nathan, 1992). M+s secrete the cytokines IL-1 and TNFa at sites of inflammation in both a paracrine and autocrine fashion to induce NO. M+ activation induced by cytokines is inhibited by TGFP (Tsunawaki et al., 1988), which results in the inhibition of 1NF.r-induced NO synthesis (Ding et al., 1990). TGFP acts by destabilizing the mRNA for NOS and decreasing translation (Vodovotz et al., 1993).We have demonstrated in a model of chronic granulomatous inflammation that the decline in NOS activity coincides with an increase in TGFP immunoreactivity (Vane et al., 1994). Recently Ianaro et al. (1995) have shown that TGFP delivered orally in an attenuated bacterial construct reduces inflammation induced by injection of carrageenin into the rat paw. The same experiments also showed that the ability of the draining lymph node cells to produce IL-2 and INFT was reduced and that of IL-10 enhanced. T helper (Th) lymphocytes are categorized by the profile of cytokines they produce. Thl cells produce IL2 and 1NF.r; Th2cells produce IL-4, IL-5, and IL-10 (Mosmann and Coffman, 1989). N O production by murine M+s induced by INFT is inhibited by Th2 cytokines IL-4 (Liew et al., 1991) and IL-10 (Cunha et al., 1992). IL-4 also inhibits intracellular parasite killing in murine and human M+s (Liew et al., 1989; Lehn et al., 1989). The switch between Thl and Th2cells occurs in infection and inflammation; therefore, the balance between these two cell types and the cytokines they produce may affect the outcome of disease processes. Thl cells are important in cell-mediated immunity (Cher and Mosmann, 1987; Fong and Mosmann, 1989) and Thz are important in immediate hypersensitivity reactions; therefore, N O may play a significant role in the former. Rat peritoneal M+s activated by LPS and incubated at 40"C, produce elevated levels of nitrite more rapidly than cells at 37°C (Bernard et al., 1994). Elevated temperatures in fever and at sites of inflammation may therefore contribute to host defense mechanisms via accelerated M+ NO synthesis. Thus, NO mediation and modulation at inflammatory sites will depend on the type of cell present, its state of activation, and the cytokine milieu. C. Arginine Metabolism at Inflammatory Sites

Metabolism of arginine in inflammatory cells can occur by more than one pathway: by NOS to N O and citrulline or by arginase to ornithine and urea. Ornithine can be converted into proline, which is required for collagen synthesis, and into the polyamines, putrescine, spermidine, and spermine

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which are essential for cell growth and differentiation. The products of both pathways may affect NOS activity. In M+s, high levels of NO feedback to inhibit NOS activity by binding to the heme moiety (Griscavageet al., 1993). Spermine also inhibits NOS activity in 5774.2 M+s (Szabo et al., 1994). Several factors have been implicated in activating the NOS or arginase pathway. One probable factor is the presence of bacterial antigen. In a recent investigation, LPS from nontoxic or detoxified sources specifically triggered the arginase pathway in murine bone marrow-derived M+s (Corraliza et al., 1995). NOS was induced solely by toxic LPS. The same study also showed that the arginase pathway was triggered by IL-10 and that arginase levels were increased a further order of magnitude by IL-4. Both cytokines are produced by Th2 cells and suppress NOS. Activation of alternative pathways of arginine metabolism are demonstrated in two models of chronic inflammation. In a model of wound healing (s.c. implantation of a PVA sponge), initially PMNs were the predominant inflammatory cell and the NOS pathway was active. Subsequently, during M+ infiltration, ornithine and urea levels rose and the arginase pathway was triggered (Albina et al., 1990). In contrast, using a model of chronic granulomatous inflammation, induced in a murine air pouch by injection of Mycobacterium tuberculosis and croton oil, we have shown that the NOS pathway is active in the acute phase of inflammation and persists into the chronic phase up to Day 14, when M+s are the major inflammatory cells (Vane et al., 1994). While cytokines alone can induce NOS 11, bacterial products greatly enhance M+ production of NO. Cunha et al., (1993) have suggested that the dual stimulation by cytokines and bacterial products may ensure that elevated levels of NO are limited to pathogen invasion and are not in response to isolated cytokine signals. Therefore, in the chronic phase of the PVA sponge implant, in the absence of bacterial antigen, the arginase pathway is activated while NO is produced in the air pouch model in the presence of bacterial contamination. The presence of bacterial antigen and the switch from Thl to Th2-dependent cytokine production therefore may underlie the selective induction of arginase or NOS in inflammation. Another factor to consider is substrate availability. When arginine levels are low M+ production of NO is severely curtailed (Vodovotz et al., 1994). One hypothesis suggests that arginase may control NO production by substrate depletion (Granger et al., 1990). Interestingly, arginine is the only amino acid severly depleted in the later stages of wound healing (Gartner et al., 1991). Therefore, further investigation of the presence of bacterial antigen, the timing of the Th cell switch, the cellular population, and substrate availability may all elucidate pathways of arginine metabolism in inflammatory states. Corraliza et al. (1995) also reported that PGE2selectively induced arginase in murine bone marrow-derived M+s. In the murine air pouch model

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described above, inducible NOS activity peaks in the chronic phase at Day 7 then significantly declines. This reduction in NOS activity coincides with an increase in COX activity at Day 14 with PGEz being the predominant product (Appleton et al., 1994). Therefore, eicosanoids are also candidates for determining which pathway of arginine metabolism operates in inflammation. D. Nitric Oxide Production by Human Macrophages Induction of NO in human monocytes and M+s in vivo is controversial. Reports suggest that NO levels are low or nonexistent, and are confined to complex activation conditions or to certain M+ subsets. Monocyte-derived M+s are claimed to generate NO; and their tumoricidal activity is prevented by the arginine analogue P-mono-methyl-L-arginine (L-NMMA). However, the general concensus is that in vivo human M+s have low NOS I1 activity (Martin and Edwards, 1993; Keller et al., 1993; James and Nacy, 1993; Denis, 1994). E. Cell Types in Which Nitric Oxide Synthase Is Induced

In addition to PMNs and M+s numerous other cell types are capable of NOS I1 production. Providing that suitable triggering mechanisms prevail at the site of inflammation, they may contribute to total NO synthesis. The current list includes keratinocytes, respiratory, retinal and renal tubular epithelium, myoepithelium, mesothelium, hepatocytes, pancreatic islet cells, vascular endothelium, endocardium, mesangial cells, cardiac myocytes, vascular smooth muscle, uterine and fallopian tube smooth muscle, fibroblasts, chondrocytes, synoviocytes, osteoclasts, neurones, and astrocytes (see Nathan and Xie, 1994a). Recently, we identified NOS II in the epithelium of the rat fallopian tube, the activity of which is regulated during the estrus cycle (Bryant et al., 1993). In the majority of cases the contribution to tissue inflammation from NO synthesis by these cells remains to be determined. For example, NO synthesis by keratinocytes may be operative in the first line of defense against infection and may play a role in EGF-stimulated cellular proliferation in wound healing (Heck et al., 1992). F. Cytotoxicity and Tissue Damage

A large body of evidence supports murine M+ production of NO as a mediator of bacterial, parasitic, fungal, and tumor cell cytoxicity (James and Hibbs, 1990; Green et al., 1991; Nathan and Hibbs, 1991). Cytotoxic mechanisms by which invading pathogens are inactivated include the inhibi-

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tion of essential metabolic enzymes and the subsequent effects on cellular respiration, energy production, and reproduction in which they are involved (Stuehr and Nathan, 1989; Naxaki et al., 1990; Nathan and Hibbs, 1991). It is debatable whether NO accounts solely for this activity. Oxygen-free radicals produced by inflammatory cells can combine with N O to form peroxynitrite, which decomposes to form OH- and NO2-,the decomposition products being more toxic than N O itself (Beckman et al., 1990; Radi et al., 1991; see Laskin et al., 1994). In addition to NO, NOS can generate oxygen-freeradicals in suboptimal conditions of L-arginine or tetrahydrobiopterin (Mayer et al., 1991; Heinzel et al., 1992; Pou et al., 1992). Such conditions may arise in a healing wound, where arginine is the only amino acid to decrease with time (Gartner et al., 1991). Superoxide production in arginine depleted neurones was reported recently (Culcasi et al., 1994), with the suggestion that during ischemia or hypoxia, defects in the L-arginine transporter could occur accompanied by NO and superoxide production with ensuing neuronal tissue damage. There are conflicting reports of N O as an agent of both tissue protection and damage in inflammatory conditions. Undoubtedly the antipathogenic activity protects in nonspecific host defense, but the benefits of high levels of NO to combat infection may be detrimental when massive inappropriate production spills over into tissue damage. Large amounts of cytokineinduced NO contribute to the circulatory failure associated with shock due to sepsis (Joulou-Schaeffer et al., 1990; Thiemermann and Vane, 1991; Wright et al., 1992; Szabo et al., 1993). Others have reported that NO produced by M+s or pancreatic beta cells is capable of beta cell destruction contributing to insulin-dependent diabetes (Kolb and Kolb-Bachofen, 1992).

X. Nitric Oxide in Mechanisms of Acute Inflammation Acute inflammation is a short-lived resolvable event. Persistent irritation or infection precipitates a chronic response. Mechanical and thermal trauma or invasion of pathogens results in a sequential release of mediators of the acute phase: histamine, 5-HT, BK, thrombin, substance P, and activation of complement and PG production (Di Rosa et al., 1971).Platelets aggregate and PMNs infiltrate surrounding tissues in response to chemotactic signals. Thereafter, monocyte/M+ infiltration occurs. N O is involved at several stages of this process (see Fig. 2). Histamine, 5-HT, BK and, substance P reportedly trigger NO release from ECs resulting in an increase in vascular permeability. Recently, it was reported that N,-nitro-L-arginine methylester (L-NAME) had no effect on histamine and platelet activating factor (PAF)induced vascular permeability in mouse skin but instead attenuated that elicited by BK, 5-HT, and substance P. Vascular permeability elicited by PAF and histamine appeared to act independently of NO, whereas BK, 5-

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HT, and substance P have a NO-mediated component contributing to edema formation (Fujii et al., 1994). In a series of experiments, endothelium-derived NO was demonstrated to inhibit platelet aggregation and adhesion to the vessel wall and with PGI2 to regulate platelet/endothelium interactions (Radomski et al., 1987a-d; see Fig. 2).Therefore, in this capacity, an anti-inflammatory role may be ascribed to NO. Extravasation of inflammatory cells in response to a chemotactic signal is a characteristic of inflammation. By superfusing cat mesentery with arginine analogues, Kubes et a/. (1991) showed that there was a significant increase in leucocyte adhesion to endothelial cells suggesting that NO was acting as an anti-inflammatory molecule and an endogenous regulator of leukocyte adhesion (see Kubes, 1992 for review; see Fig. 3). A. Complement-Mediated and Cell-Mediated Inflammatory Models

Carrageenin-induced rat skin permeability and carrageenan and dextran-induced models of paw edema showed dose-dependent inhibition of vascular permeability and edema formation with arginine analogues suggesting that NO released at the inflammatory site was involved (Ialenti et al., 1992). The source of NO involved in these models may be from EC NOS I, an upregulation of NOS I1 in ECs or NOS I1 from inflammatory cells. Depending on the time course and type of inflammation, a combination of the above may be involved. We have measured the contribution of the inducible component to total NOS activity in a model of complement-mediated inflammation, the rat carrageenin-induced pleurisy (Tomlinson et al., 1994). Activity in pleural exudate cell pellets was assessed by the ability of NOS to convert [3H]-~arginine to [3H]-~-citrulline, with and without calcium (total and inducible activity respectively). High levels of total activity were recorded at 2 h, reaching maximum at 6 h before returning to baseline at 24 h. The profile of NOS I1 activity paralleled and almost entirely accounted for total NOS activity. Lung homogenates showed little NOS activity above baseline, but measurement in the vasculature alone was not possible. PMNs predominated initially in smears from the exudate cell pellets with M+ numbers increasing with time. In addition, mesothelial cells lining the pleural cavity are capable of producing N O in response to cytokines (Owens and Grisham, 1993). They represented 17% of the cell exudate in the first pleural washout. The relative contribution from the cells involved to the total NO produced remains unknown. However, these results suggest that N O from .NOS I1 activity is likely to be involved in vascular permeability and edema formation in complement-dependent inflammation.

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In an immune model of cell-mediateddelayed hypersensitivity,intrapleural challenge with Bordatella pertussis into a previously sensitized rat gives rise to an inflammatory response which persists for 72 h. We have shown that NOS activity over 48 h was predominantly from the inducible isoform (unpublished results).

B. Neurogenic Inflammation Topical application of mustard oil to rat skin releases Substance P from primary afferent nociceptive nerves. Neurogenically induced inflammation ensues characterized by vasodilatation and edema formation. The arginine analogue L-NAME attenuated cutaneous hyperemia; however, in contrast to complement-mediatedinflammation, it had no effect on plasma extravasation, indicating a lack of NO involvement in vascular permeability in this model (Lippe et al., 1993). However, plasma exudation was inhibited by arginine analogues in vagally induced inflammation in guinea pig airways (Kuo et al., 1992) and both components of inflammation were inhibited in a BK-induced rat blister model, with further attenuation in capsaicin-treated animals (Khalil and Helme, 1992). It is most likely that a proinflammatory role for NO can be ascribed in neurogenic inflammation. Taken together this body of work places NO directly in the frame as an in vivo mediator of acute inflammatory events.

XI. Nitric Oxide in Mechanisms of Chronic Inflammation

-

Subcutaneous injection of air into the dorsum of a rat or mouse produces a pouch, which 6 days later is lined with a tissue which has similarities to the synovium of joints (Edwards et al., 1981). Injection of croton oil in Freunds complete adjuvant into the air pouch produces a lining of granulomatous tissue with many similarities to the inflammatory tissue of rheumatoid joints (Al-Duaij et al., 1986). PMNs predominate up to 24 h with M+s dominant from Day 2 to 3. Fibroblasts and ECs migrate into the newly forming tissue resulting in a fibrotic and highly vascularized granuloma, which ultimately resolves. We have measured NOS activity in the acute and chronic phases of inflammation (Vane et al., 1994) and localized cellular expression of NOS (Tomlinson et al., 1992). Inducible NOS activity accounted for half of the total NOS activity in the acute inflammatory phase. At this point, PMNs and a small number of M+s were positively immunolabelled for NOS 11. From Days 3 to 7, the peak of chronic inflammation, there was an eightfold increase in NOS activity, >90% being inducible and correlating with labeled M+s. Activity was reduced substantially by Day 14 as the inflammation resolved. Temporal and spatial localization of cytokines in the granuloma

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(Appleton et al., 1993) showed a proinflammatory IL-1 and TNFa peak in the acute phase, both candidates for NOS induction. TGFP inhibits NOS and the highest levels of immunoreactivity of this cytokine coincided with reduced NOS activity between Days 7 and 14 in the chronic phase, suggesting that endogenous TGFP may regulate NO production. Prostanoid formation from constitutive and inducible isoforms of COX was measured because eicosanoids regulate NOS induction (Marotta et al., 1992; see Section XV for COX and NOS interactions). These results implicate inducible NOS activity in acute and chronic inflammation and indicate possible regulation by endogenous cytokines and prostanoids. Currently, we are assessing the contribution of NO and these regulators to the outcome of the inflammatory process. A study of the involvement of endogenous NO in granuloma formation, published at the time of writing, demonstrates that L-NAME reduces, in a dose-dependent manner, granulomatous tissue formation, cell infiltration, and NO; production in a model of carrageenin-soaked polyether sponges implanted in rats (Iuvone et al., 1994). A. Nitric Oxide in Angiogenesis

Development of new blood vessels, or angiogenesis, is essential for the maintenance of inflammatory tissue and tumor proliferation. Treatment with agents known to be angiostatic reduces the vascularity and tissue mass in experimental murine granulomas (Colville-Nash et al., 1993). The CAM is an in vivo model much used to study blood vessel development (Ausprunk et al., 1974). Sodium nitroprusside which generates NO and superoxide dismutase which inhibits the destruction of endogenous N O applied to the CAM inhibited thrombin-induced angiogenesis (Pipili-Synetos et al., 1994). Both the N O inhibitors L-NMMA and L-NAME stimulated new blood vessel growth. Taken together the data indicate that NO is an important regulator of angiogenesis under basal conditions and an inhibitor of promoters of the angiogenic response. Conversely, Ziche et al., (1994), evaluating the effects of NO donors and endogenous N O elicited by substance P on angiogenesis in the rabbit cornea, reported that NO potentiated the angiogenic response. Exposure of capillary ECs in vivo to substance P activated the calcium-dependent NOS, which results in low levels of NO release. NO donors promoted cell growth and mobilization of capillary ECs, events which were abolished by pretreatment with NOS inhibitors. Interestingly, NOS inhibitors had no effect on EC growth and migration elicited by bFGF, suggesting that other angiogenic mechanisms may be in operation. The contradictory findings from these two studies may be related to levels of N O generated. Substance P activated the calcium-dependent NOS

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which produces NO in small amounts whereas levels in the CAM may be higher and angiostatic. Obviously, further investigations are required. One note of caution is required in interpreting these results. An increase in vascular permeability as a consequence of N O production may result in the release of other angiogenic factors such as fibrin degradation products. If these potent angiogenic factors are driving the angiogenesis, the effects of NO therefore would be indirect.

XII. Nitric Oxide in Inflammatory Disease A. Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory autoimmune disease characterized by proliferation of the synovium lining the joint cavity forming an invasive inflammatory tissue termed pannus. M+derived IL-1 and TNFa in the joint contribute to cartilage erosion and eventual degradation. RA is a relapsing and remitting disease with reoccurrences accompanied by PMN influx into the synovium. PMNs, synoviocytes (macrophage and fibroblastlike cells), chondrocytes, and bone and endothelial cells are all capable of NO synthesis. Thus, any one or all may potentially contribute to production of N O in RA. The evidence for NO-mediation of RA is accumulating (for review, see Stefanovic-Racic et al., 1993). Elevated levels of NO2 and NO; have been detected in synovial fluids and serum from RA patients and are inferred to be produced by the inflamed synovium (Farrell et al., 1992). Human articular chondrocytes stimulated with IL-la and p, TNFa, or LPS in culture, release high levels of NO and show a substantial suppression of their proteoglycan synthesis (Haeuselmann et al., 1994). This suppression is only partially attenuated by L-NMMA, suggesting that IL-1 inhibition of the synthesis of cartilaginous matrix occurs by more than one mechanism. However, the evidence indicates an involvement of NO in cartilage breakdown. A number of animal models of inflammatory arthritis provide compelling data. MRL-lpr/lpr mice spontaneously develop autoimmune disease, including an inflammatory arthropathy. They excrete more urinary NO2/ NC& than normal and their peritoneal M+s possess increased capacity for NO and NOS synthesis. Oral administration of L-NMMAreduced the intensity of the inflammatory arthritis in this model (Weinberg et d., 1994). Adjuvant-induced arthritis in rats is exacerbated by L-arginine and suppressed by NOS inhibition. T lymphocyte proliferation and enhanced NO2 production by M+s in the arginine treated group was depressed by NOS inhibition. Cellular changes paralleled the severity of the arthritis (Ialenti et al., 1992). These data suggest NO as a mediator in this cell-mediated delayed hypersensitivityreaction. Similarly, streptococcal cell wall fragments

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injected into the synovial space of rats induce a cell-mediated response. Inflammatory cells invade the synovium and NO is elevated in the inflamed joints with ensuing destructive lesions. Reduction of joint swelling and changes in tissue morphology after treatment with L-NMMA implicate NO in the pathogenesis of this inflammatory arthritis (McCartney-Francis et al., 1993). Thus, the evidence for elevated NO production being involved in the pathogenesis of autoimmunity and inflammatory arthritis in particular seems persuasive.

B. Multiple Sclerosis Experimental allergic encephalomyelitis(EAE)is a model for the human central nervous system (CNS) demyelinating disorder, multiple sclerosis. It can be induced in susceptible animal strains by injecting foreign spinal cord, components of myelin basic protein, or T lymphocytes sensitized to myelin components. The mechanisms causing demyelination are not understood, but several studies have shown an involvement of NO. Signals characteristic of NO complexed with irodsulfur proteins have been detected in the spinal cords of EAE mice during M+ activation and sepsis, using electron paramagnetic resonance (EPR) spectroscopy (Lin et al., 1993). Activated lymphocytes sensitized to myelin basic protein induced NO synthesis in a murine M+ cell line, thereby providing a further line of evidence for NO involvement in this cell-mediated disorder (Cross et al., 1994). Aminoguanidine, a preferential inhibitor of NOS 11, attenuated the pathology of demyelination, axon necrosis, and inflammation in the spinal cords of sensitized mice. C. Graft-Versus-Host Reaction Several studies have provided evidence of a role for NO in the cellmediated rejection of allogeneic but not syngeneic transplants. Treatment with L-NMMAabolished mucosal pathology and reduced epithelial lymphocytic infiltration in mice with intestinal graft-vs-host reaction (Garside et al., 1992). Inhibition of N O also inhibited the enhanced activity of natural killer cells evident in this type of reaction, indicating a functional involvement of NO. EPR showed irodnitrosyl complexes in blood and tissues from vascularized rejected allografts of rat hearts (Lancaster et al., 1992) and in erythrocytes after orthotopic small bowel transplantation (Langrehr et al., 1992). Rats which acutely rejected allografts, or with graft-vs-host reaction, had elevated NO2/ND3in serum but levels were normal in those receiving immunosuppressive therapy.

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Studies performed by the same group demonstrated that adherent M+s retrieved from subcutaneous sponges seeded with splenocytes produced significantly more NO than M+s recovered from syngeneic grafts (Langrehr et al., 1993). In addition, mice receiving allogeneic heterotopic heart transplants had urinary nitrate levels and irodnitrosyl complexes in cardiac tissues peaking on rejection (Bastian et al., 1994). Thus, NO appears to be a mediator of graft-vs-host reaction.

D. Renal Inflammation Unstimulated rat mesangial cells produce NO2/N03 levels in culture which increase on stimulation with LPS (Schultz et al., 1991). This suggests a physiological role for NO and a potential involvement in glomerular inflammation. For an account of the role of N O in mesangial and glomerular physiology and pathophysiology see Raij and Schultz (1993). Immune complex (antibody/antigen) deposition in tissues causes activation of complement and recruitment and involvement of PMNs (Johnson and Ward, 1979).Animals with immune complex-induced glomerulonephritis excrete increased levels of urinary NO2 and their glomeruli synthesize NO ex vivo. Recently, the first direct evidence for in vivo induction implicating NO in the pathogenesis of this disorder was reported (Jansen et al., 1994). Mononuclear cells in the glomeruli and emigrating into the Bowman’s space in rat nephrotic kidneys were immunolabelled for NOS 11. MRL-lpr/lpr mice spontaneously develop immune complex glomerulonephritis. Oral administration of NOS inhibitor prevented the onset of the disease in this autoimmune model of systemic lupus erythematosus (Weinberg et al., 1994). Renal inflammation may also be exacerbated by a secondary action of NO. Induced NOS activity, recently described in the hydronephrotic kidney, in a rabbit model of ureteric obstruction leading to renal inflammation (Salvemini et al., 1994) appears to activate COX 2, resulting in the release of proinflammatory PGs. (See Section XV for COX/NOS interactions). E. Gastrointestinal Inflammation

NO is a NANC transmitter in the GI tract and appears to serve as the primary transmitter of enteric inhibitory motor neurones to the muscle. Stimulation of the nerves results in relaxation. (See Sanders and Ward, 1992; Brookes, 1993; McConalogue and Furness, 1994.) Inflammation of the GI tract is characterized by inflammatory cell infiltration and often involves motility disorders associated with toxic dilatation. It has been shown that granulocytes and unstimulated and activated mononuclear cells from human peripheral blood relax precontracted colonic circu-

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lar smooth muscle by the release of NO indicating its potential contribution to gut motility disorders (Middleton et al., 1993). Ulcerative colitis and Crohn’s disease are inflammatory disorders of differing origins in man. Colonic mucosa from ulcerative colitis patients had eightfold higher levels of NOS activity than controls, with similar levels in the surrounding musculature. The mucosal activity in Crohn’s disease did not differ from controls (Boughton-Smithet al., 1993a). Thus, induction of colonic NOS may be involved in the mucosal vasodilatation and increased vascular permeability of active ulcerative colitis and may contribute to the impaired gut motility found in this disorder. A variety of studies in animal models of acute GI inflammation have produced conflicting results. The release Qf NO from endogenous sources, or NO donors, appears to afford protection by antagonizing capillary leakage, PMN infiltration, and tissue damage to a greater or lesser extent depending on the model in use, the inflammatory stimulus, and the parameters measured (MacNaughton et al., 1989; Hutcheson et al., 1990; Boughton-Smith et al., 1992; MacKendrick et al., 1993; Miller et al., 1993). Conversely, NOS I1 activity in the intestine of endotoxin-treated rats was accompanied by increased vascular permeability, which was reduced by administration of LNMMA. These findings suggest that NOS induction is associated with vascular injury in this model (Boughton-Smithet al., 1993b). The aptly titled publication, “Nitric oxide: the Jekyll and Hyde of gut inflammation” (Miller et al., 1993), evaluated treatment with L-NAME in naive animals and guinea pigs with experimental ileitis. In naive pigs treatment with L-NAME resulted in a marked increase in PMN infiltration and conversion of the mucosa from an absorptive to secretory phase. A similar influx of PMNs and mucosal secretory response occurred in the animals with chronic ileitis; however, treatment with L-NAME was anti-inflammatory, reversing the responses. The authors concluded that intestinal NO is antiinflammatory under basal conditions, but is a mediator of gut injury in inflammation. It appears, therefore, that the source and levels of NO, the severity of inflammatory insult, and the animal models used have to be considered in evaluating the results in experimental gut inflammation.

F. Other Inflammatory Disease States It is not possible to cover in detail the involvement of NO in all inflammatory disease. However, NO is implicated in lung injury after immune complex deposition (Mulligan et al., 1992) and in neurogenic inflammation in guinea pig airways (Kuo et al., 1992); in hepatic inflammation (Billiar et al., 1992); in uveitis (Parks et al., 1994) and in persistent inflammatory events as risk factors fnr carcinogenesis (Grisham et al., 1992; Ohshima and Bartsch, 1994).

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XIII. Involvement of Nitric Oxide in Inflammatory Pain Pain is one of the fundamental signs of inflammation and both substance P from sensory fibers and BK a potent algogen stimulate the release of NO. Recent evidence shows that intracutaneous injections of NO solutions into the human forearm evoke pain (Holthusen et al., 1994).In animals, administration of L-NAME blocks the thermal hyperalgesia induced by carrageenin (Meller et al., 1994). Carrageenin injected into a rat hindpaw increases NADPH-diaphorase staining (indicative of NOS) in both ipsilateral and contralateral neurones of the lumar spinal cord (Traub et al., 1994). Tight ligation of LS and L6 spinal nerves produces symptoms of thermal hyperalgesia and mechanical allodynia purported to mimic the symptoms of painful neuropathies. In such a model, where tissue trauma is likely, dorsal root neurones were immunolabelled for NOS up to 2 weeks postligation suggesting that NO may play a part in maintenance of painful neuropathies (Steel et al., 1994). Activation of N-methyl-D-aspartate (NMDA) receptors in the spinal cord can induce hyperalgesia, either by N O and/or PG production. Intraperitoneal injection of LPS, thought to induce hyperalgesia by activating hepatic vagal afferents, has been shown to induce hyperalgesia by activating the NMDANO cascade at the level of the spinal cord (Wiertelak et al. 1994). Therefore the induction of NOS by inflammatory stimuli both peripherally and centrally is associated with pain processes. XIV. Conclusion

The evidence presented here firmly supports the involvement of NO in a variety of inflammatory events. However, the difficulty lies in ascribing a pro- or anti-inflammatory role for this molecule. What appears to be important is the encompassing nature of the inflammation, i.e., the inflammatory stimulus, the source, levels, and period of NO synthesis, the cellular environment and its state of activation, cytokines synthesized, the presence of bacterial antigen, and substrate availability. Thus, although NO is indubitably involved in inflammation its role cannot be defined as pro- or antiinflammatory but rather depends on the prevailing circumstances. XV. Interactions between the Nitric Oxide Synthase and Cyclo-Oxygenase Pathways

The products of the NOS and COX pathways as well as having potent effects on various cellular systems can also modulate the activity of their respective enzymes. Products of COX can affect the activity of NOS and

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similarly NO can affect COX activity. However, these effects are not clear cut as both stimulatory and inhibitory actions have been ascribed. In rat mesangial cells, endogenous production of PGE2 can inhibit the IL-1 stimulated induction of NOS (Tetsuka et a!., 1994). Similarly, exogenous PGEz and PGIz can inhibit LPS-induced NOS in 5774 macrophages (Marotta et al., 1992). In contrast endogenously formed PGE increases NO synthesis in LPS stimulated rat kupffer cells (Gaillard et al., 1992). Discrepancies in these results may depend on the stimulus used and the source of PGEz, i.e, exogenous or endogenous. The effects of NO on COX activity are just as complex and the information on the isoform of COX which results in changes in PG production is sparse. Corbett et al. (1993) have shown that NO can activate COX in rat islets of Langerhans. Inhibition of NOS using L-NAMEresults in a decrease in PGIz production in LPS treated rat lungs (Sautebin and Di Rosa, 1994), suggesting that N O can stimulate COX. A stimulatory role of N O on COX has also been shown in vivo. In the rat hydronephrotic kidney, the NOS inhibitor aminoguanidine inhibits the BK-inducedrelease of PGE2(Salvemini et a!., 1994). Evidence that NO can inhibit COX is also documented. Habib et al. (1994)have shown that NO inhibits PG production by specifically downregulating COX-2 in LPS stimulated rat peritoneal macrophages, while COX2-derived PGs will stimulate NO production. The stimulatory and inhibitory actions of NO on PG production may be explained by the relative concentrations of NO. This explanation is based on work using LPS stimulated 5774 macrophages. Low levels of NO may stimulate PG formation whereas high levels of NO derived from the NO donor sodium nitroprusside, inhibit PG production (Swierkoszet al., 1995). It is possible that during inflammation both of these interactions may exist due to the fluctuating activity of NOS and hence levels of NO. Studies on isolated enzyme systems have shown that NO has a weak binding capacity for heme of the ferric COX-1 but a strong affinity for the ferrous COX-1 under anaerobic conditions. The authors have concluded that there is no biochemical evidence for a direct stimulatory effect of NO on COX-1 under physiological conditions (Tsai et al., 1994).Thus although both stimulatory and inhibitory actions of NO on COX activity have been ascribed in vivo there seems to be no biochemical basis for these observations. It is therefore likely that many of the stimulatory effects of NO on COX are indirect possibly due to concentration effects or even to compensatory effects. In addition, NO can induce plasma extravasation, i.e., a component of acute inflammation, which will consequently result in the production of PGs. Thus, removal of NO will obviously indirectly result in a decrease in PG production. Furthermore, as many of the “selective” inhibitors of NOS such as L-NAME and L-NMMA can also inhibit COX (Peterson et al., 1992), this throws into doubt many of the inhibitory studies. Further work

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is therefore required before the exact nature of the interactions between these two enzyme systems can be completely elucidated.

Acknowledgments The authors are indebted to Mr. Derek Gilroy, Mrs. Nichola Brown, Mr. Dean Willis and Dr. Paul Colville-Nash for help in the preparation of this manuscript. Dr. Ian Appleton’s work is funded by a Royal Society, Smith and Nephew Research Fellowship. Research carried out in the Department of Experimental Pathology is in part funded by O N 0 Pharmaceutical Co., Osaka, Japan, The Hyal Research Foundation, Toronto, Canada and Institut de Recherches Servier, Paris, France.

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John A. Farmer Antonio M. Gotto, Jr.* Department of Medicine Baylor College of Medicine Ben Taub General Hospital Houston, Texas 77030

*

Department of Medicine Baylor College of Medicine The Methodist Hospital Houston, Texas 77030

Current and Future Therapeutic Approaches to Hyperlipidemia

1. Introduction

A dramatic and progressive decline in cardiovascular morbidity and mortality has occurred in the United States during the past several decades. This encouraging improvement is the result of multiple factors, including alteration of dietary and smoking habits. In addition, there have been major advances in pharmacologic therapy for dyslipidemic states combined with an explosion in knowledge about the role of lipoproteins in the pathogenesis and progression of atherosclerosis. This review focuses on the rationale for treating patients with elevated cholesterol and/or triglyceride levels and on the major lipid-lowering agents currently available (Table I), including their mechanisms of action, efficacy, and clinical trial data (Table 11). Although all these agents lower plasma cholesterol, they are classified here according to their predominant effect on the lipid profile. Therapeutic approaches that may be employed in the future (some of which are already under development) are also discussed. Advances in Phannacology, Volume 35

Copyright 0 1996 by Academic Press, Inc. All rights of reproduftion in any form reserved.

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TABLE I Hypolipidemic Drug Mechanisms and Effects Hypolipidemic agents

Mechanisms

Bile-acid sequestrants Cholestyramine (4-24 g/day)

LDL-C decreases 15-30% Decrease intrahepatic cholesterol by nonspecific HDL-C increases 3-5% binding of bile acids; increased activity of LDL receptors TG usually not affected; may increase

Colestipol (5-30 g/day)

Effects on lipids

Nicotinic acid Crystalline (1.5-6 g/day) Sustained-release (1-2 g/day)"

Decreased production of VLDL; decreased mobilization of free fatty acids from peripheral adipocytes

HMG-CoA reductase inhibitors: Fluvastatin (20-40 mg/day) Lovastatin (10-80 mg/day) Pravastatin (10-40 &day) Simvastatin (5-40mg/day)

Decrease in cholesterol synthesis caused by partial inhibition of HMG-CoA reductase

Fibric-acid derivatives Clofibrate (2 g/day) Gemfibrozil (1.2 g/day)

Increased activity of lipoprotein lipase; decreased release of free fatty acids from peripheral adipose tissue

LDL-C decreases 10-15% with high LDL-C; may increase with high TG HDL-C increases 10-15% TG decreases 20-50%

Probucol (1g/day)

Increased activity of LDL scavenger-receptor pathway; decreased oxidation of LDL

LDL-C decreases 5-15%

LDL-C decreases 10-25% HDL-C increases 15-35% TG decreases 20-50% LDL-C decreases 20-40% HDL-C increases 5-15% TG decreases 10-20%

HDL-C decreases 20-30% TG usually not affected

* Not generally recommended because of increased risk for hepatotoxicity.

HDL-C, high-density lipoprotein cholesterol; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; LDL, low-density lipoprotein; LDL-C, LDL cholesterol; TG, triglyceride; VLDL, verylow-density lipoprotein. Adapted from Farmer, J. A., and Gotto, A. M., Jr. (1995) Currently available hypolipidemic drugs and future therapeutic developments. Baillieres Clin. Endocrinol. Metab. 9, 825-847.

II. Agents That Predominantly Lower Cholesterol Elevated levels of cholesterol have been clearly linked to increased risk for coronary heart disease (CHD)in observational epidemiological, genetic, experimental, and interventional studies. For example, genetic conditions characterized by elevated cholesterol, such as familial hypercholesterolemia (FH), are marked by premature atherosclerosis, and, as described below, many clinical trials in which cholesterol was lowered by dietary or pharmacologic interventions have demonstrated reduced CHD incidence. Approximately 70% of plasma cholesterol is carried in low-density lipoprotein (LDL),which is the primary target of antidyslipidemic therapy in the guide-

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TABLE II Clinical Trials of Lipid-Regulating Agents Lipid-regulating agent(s)

Clinical triaP

Cholestyramine

LRC-CPPT NHLBI Type I1 Coronary Intervention Study STARS

Colestipol

+ Nicotinic Acid

FATS CLAS

Colestipol

+ Lovastatin

FATS

Lovastatin

MARS CCAIT

Pravastatin

PLAC I woscoPs

Simvastatin

MAAS 4s

Estrogen

PEPI

Probucol

+ Cholestyramine

PQRST

Nicotinic Acid

Coronary Drug Project Stockholm Ischaemic Heart Disease Secondary Prevention Study

Clofibrate

WHO Cooperative Trial Coronary Drug Project

Gemfibrozil

Helsinki Heart Study

CCAIT, Canadian Coronary Atherosclerosis Intervention Trial; CLAS, Cholesterol Lowering Atherosclerosis Study; FATS, Familial Atherosclerosis Treatment Study; LRC-CPPT, Lipid Research Clinics Coronary Primary Prevention Trial; MAAS, Multicentre Anti-Atheroma Study; MARS, Monitored Atherosclerosis Regression Study; NHLBI, National Heart, Lung, and Blood Institute; PEPI, Postmenopausal Estrogeflrogestin Interventions; PLAC I, Pravastatin Limitation of Atherosclerosis in the Coronary Arteries; PQRST, Probucol Quantitative Regression Swedish Trial; 4S, Scandinavian Simvastatin Survival Study; STARS, St Thomas’ Atherosclerosis Regression Study; WHO, World Health Organization.

lines of the second Adult Treatment Panel of the U.S. National Cholesterol Education Program (National Cholesterol Education Program, 1994). A. Bile-Acid Sequestrants

Bile-acid sequestrant therapy has been used to treat hypercholesterolemia for more than three decades, during which time extensive clinical experience has accumulated on both of the currently available agents, cholestyramine and colestipol. The bile-acid sequestrants are highly charged polycationic compounds that do not enter the plasma compartment from the gastrointestinal tract after oral administration.

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1. Mechanism of Action

The primary action of the bile-acid sequestrants is the nonspecific binding of bile acids and other anionic compounds within the lumen of the gastrointestinal tract through the exchange of a chloride ion for a negatively charged bile acid or other negatively charged compound. Normally, most of the bile acids are reabsorbed and recycled through the enterohepatic circulation, and only 3% are lost from the gastrointestinal tract. Bile-acid sequestrant administration interrupts the enterohepatic circulation of bile acids and increases their fecal excretion (Moore et al., 1968).Because cholesterol is a precursor of bile acids, the increased fecal loss of bile acids results in increased channeling of cholesterol to the production of bile acids through the rate-limiting enzyme of bile acid the activity of 7-alpha-dehydroxyla~e~ synthesis. Increased conversion of cholesterol to bile acids decreases the intrahepatic cholesterol level, which causes an upregulation of the B E receptor, thereby increasing the clearance of lipoproteins-LDL, intermediatedensity lipoprotein (IDL),and possibly very-low-density lipoprotein (VLDL) (Chappell et al., 1993)-which are removed by apolipoprotein (apo) B- or apo E-mediated recognition, binding, and internalization. The removal of these lipoproteins from the circulation causes an initial decrease in plasma cholesterol. However, the reduction in intrahepatic cholesterol also stimulates 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, the ratelimiting enzyme of cholesterol biosynthesis, resulting in a secondary increase in cholesterol production. Consequently,cholesterol may increase toward the pretreatment level with prolonged bile-acid sequestrant monotherapy. 2. Efpcacy

Bile-acid sequestrants are used predominantly to lower LDL cholesterol levels, although effects may also be seen on other lipid levels. Cholestyramine dosed at 4 to 16 g/day (maximum dosage 24 g/day) or colestipol dosed at 5 to 20 g/day (maximum dosage 30 g/day) may be expected to decrease LDL cholesterol 15 to 30%. HDL cholesterol may increase 3 to 5% through a mechanism that has not been clearly elucidated. Plasma triglyceride level is generally not affected by bile-acid sequestrant therapy; however, an increase in triglyceride may be noted, especially in patients who are hypertriglyceridemic before therapy. Compliance and efficacy may be improved by administering these agents in divided doses and by gradually titrating the dose up to allow adaptation. Twice-daily administration may be started at one packet and gradually increased to achieve maximum tolerated dose. 3. Side €fifeas and Drug Interactions

Patients receiving bile-acid sequestrants may have gastrointestinal complaints. The most frequent problem is constipation, which may be alleviated by ensuring adequate fluid intake and by following a high-fiber diet. An

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increase in soluble fiber may not only improve the gastrointestinal complaints but also have an independent, albeit modest, cholesterol-lowering effect. Stool softeners may also be used to alleviate constipation. Other nonspecific gastrointestinal complaints that may occur include bloating, nausea, heartburn, and abdominal pain. Theoretical concern has arisen about the potential carcinogenic effects of these agents because of the prolonged exposure of the colonic endothelium to bile acids with bile-acid sequestrant therapy. Cholestyramine was reported to be a cocarcinogen with 1,2-dimethylhydrazinein rats (Asano et af., 1975). However, no increase in carcinogenicity has been documented in large clinical trials with cholestyramine or colestipol. Because the bile-acid sequestrants are nonspecific anion binders, they may decrease the absorption of certain coadministered drugs. Dyslipidemic patients frequently have accompanying cardiovascular conditions that may be treated with medications such as digitalis preparations (Bazzano and Bazzano, 1972),thiazide diuretics (Hunninghake et af., 1982), beta blockers (Hibbard et al., 1984), and coumarin anticoagulants (Gallo et al., 1965); the absorption and plasma levels of these agents may be decreased by the bile-acid sequestrants. In patients with mixed hyperlipidemia, a bile-acid sequestrant to lower cholesterol may be combined with a fibric-acid derivative to lower triglyceride, but the absorption of the fibric-acid derivative may be decreased with concomitant administration (Forland et al., 1990). Susceptible agents should be taken at least 1 h before or 4 h after the bileacid sequestrant to ensure adequate absorption into the circulation. 4. Clinical Trials

The effects of bile-acid sequestrants administered as monotherapy or in combination with other lipid-lowering agents have been studied in trials with clinical and angiographic endpoints. a. Lipid Research Clinics Coronary Primary Prevention Trial Bile-acid sequestrant monotherapy was used in the Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT), which randomized 3806 dyslipidemic men without known CHD to receive cholestyramine at a prescribed dosage of 24 g/day or placebo for an average of 7.4 years (Lipid Research Clinics Program, 1984a). Subjects were considered at high risk for developing CHD because of a total cholesterol level of 265 mg/dl or greater and LDL cholesterol of 190 mgdl or greater. All were offered a moderate cholesterol-loweringdiet (cholesterol 400 mg/day, polyunsaturated fat :saturated fat ratio 0.8). In the group receiving diet plus placebo, total cholesterol decreased 5% and LDL cholesterol decreased 8%. Although many subjects in the group randomized to cholestyramine did not take the full dose, in part because of gastrointestinal side effects and poor palatability, total cholesterol was decreased 13%and LDL cholesterol was decreased 20% from baseline levels in the cholestyramine-treated group.

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The primary endpoint of the trial was the combined incidence of CHD death and nonfatal myocardial infarction (MI).After adjustments were made for baseline differences between subjects and for different lengths of followup, cholestyramine was estimated to reduce the primary endpoint 19%, which was a significant improvement. Other clinical coronary endpoints monitored in the trial showed similar reductions: new-onset angina decreased 20% and new positive exercise stress tests decreased 25%, both of which were significant reductions, and coronary bypass surgery incidence decreased 21%, which was not statistically significant. Although the LRC-CPPT was not designed to evaluate total mortality, total mortality was reduced 7% in the group randomized to cholestyramine, but this difference was not statistically significant. This group had a 24% reduction in definite CHD death, which was not a significant difference, but this improvement was largely counterbalanced by an increase in noncardiovascular death, particularly in violent and accidental death. Cancer mortality rates were similar in both groups. Among subjects randomized to cholestyramine, 32% had LDL cholesterol reductions of more than 25% (Lipid Research Clinics Program, 1984b). CHD incidence in this subgroup was reduced 64%, indicating a doseresponse relation between cholesterol lowering and CHD risk. On the basis of the LRC-CPPT results, a 1% decrease in total cholesterol is predicted to decrease CHD events 2%. b. National Heart, Lung, and Blood Institute Type I/ Coronary Intervention Study The National Heart, Lung, and Blood Institute (NHLBI) Type I1 Coronary Intervention Study was conducted in 143 men and women with angiographic evidence of CHD and an LDL cholesterol level above the 90th percentile of the general population after a low-fat, low-cholesterol diet (polyunsaturated fat :saturated fat ratio 2 : 1, cholesterol <300 mg/day) (Brensike et al., 1982,1984). All subjects were to continue on the diet and were randomized to receive cholestyramine at a prescribed dosage of 24 g/day or placebo for 5 years. Because this trial was designed before the availability of highly reproducible quantitative angiographic techniques, angiograms taken at baseline and at 5 years were evaluated visually to determine the primary endpoint, change in severity of CHD. Total cholesterol decreased 17% in the cholestyramine group and 1% in the placebo group; respective decreases in LDL cholesterol were 26% and 5%. HDL cholesterol increased 8% and 2% in the respective groups, and triglyceride increased 28% and 26%, respectively. Regression as the only angiographic change occurred in approximately 7% of both groups. However, progression as the only angiographic change was seen in 32% of the cholestyramine-treated subjects, compared with 49% of the placebo subjects, which was a statistically significant improvement, indicating that lipid lowering with cholestyramine stabilized lesions. The

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NHLBI Type I1 study was the first major prospective trial to evaluate angiographically the effect of lipid-lowering therapy on atherosclerotic lesions. c St Thomas’ Atherosclerosis Regression Study In the St Thomas’ Atherosclerosis Regression Study (STARS), 90 men with CHD were randomized to receive diet plus cholestyramine 16 g/day, the same diet aloqe, or usual care for an average of 39 months (Watts et al., 1992). The prescribed diet reduced total fat intake to 27% of total calories, saturated fat intake to 8 to 10% of total calories, and dietary cholesterol to 100 mg per 1000 kcal and increased omega-6 and omega-3 polyunsaturated fatty acid intake to 8% of total calories and intake of plant-derived soluble fiber to an equivalent of 3.6 g polygalacturonate per 1000 kcal. Total cholesterol, which averaged 280 mg/dl at baseline, decreased 25% in the group receiving diet plus cholestyramine, 14% in the group receiving diet alone, and 2% in the group receiving usual care. Respective decreases in LDL cholesterol were 36, 16, and 3%. The primary endpoint was change in the mean absolute width of coronary artery segments as evaluated by quantitative coronary angiography. This value increased 0.103 mm in the group receiving diet plus cholestyramine and 0.003 mm in the group receiving diet alone but decreased 0.201 mm in the group receiving usual care. Compared with the usual-care group, both of the other treatment groups showed significant improvement. Despite the modest changes in the anatomic appearance of the coronary arteries, lipid-lowering therapy with diet or diet plus cholestyramine significantly reduced the incidence of clinical cardiovascular events, defined as CHD death, MI, coronary surgery, angioplasty, or stroke, suggesting that stabilization of atherosclerotic lesions occurred. Of the 14 subjects for whom cardiovascular events were recorded, 1 (4%)was receiving diet plus cholestyramine, 3 (11%)were receiving diet alone, and 10 (36%)were receiving usual care. d. Familial Atherosclerosis Treatment Study Colestipol was used in combination with either lovastatin or nicotinic acid in the Familial Atherosclerosis Treatment Study (FATS), which selected 146 men not on the basis of total cholesterol or LDL cholesterol but on the basis of apo B (Brown et al., 1990). Entry criteria were an elevated plasma apo B level (>125 mg/dl), angiographic evidence of CHD, and a positive family history of CHD. After dietary counseling, subjects were randomized to receive a combination of lovastatin 40 to 80 mg/day and colestipol 30 g/day, a combination of nicotinic acid 4 to 6 g/day and colestipol 30 @day, or conventional therapy for 2.5 years. For ethical reasons, subjects in the conventional-therapy group whose baseline LDL cholesterol was higher than the 90th percentile for age (43%of conventional-therapy subjects) were given colestipol 30 g/day instead of placebo. The primary endpoint-mean change in percent stenosis

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caused by the worst atherosclerotic lesion in each of 9 proximal segmentswas assessed by quantitative coronary angiography. Both combination-drug regimens used in FATS are effective because of the complementary mechanisms of the two agents. With the combination of colestipol and lovastatin, the compensatory increase in cholesterol synthesis caused by bile-acid sequestrant monotherapy is partially inhibited by the concomitant use of an HMG-CoA reductase inhibitor. Similar complementarity is seen with the combination of nicotinic acid, which decreases the production of VLDL and consequently of LDL, its metabolic end product, and colestipol, which upregulates BE receptor activity to increase LDL removal from the circulation. LDL cholesterol decreased 46% in the group receiving lovastatin plus colestipol, 32% in the group receiving nicotinic acid plus colestipol, and 7% in the group receiving conventional therapy, and HDL cholesterol increased 15, 43, and 5 % in the respective groups. Quantitative coronary angiography demonstrated regression in both active-treatment groups: the mean percent stenosis for the 9 worst atherosclerotic lesions decreased 0.7 percentage points in the group receiving lovastatin plus colestipol and 0.9 percentage points in the group receiving nicotinic acid plus colestipol, which was a statistically significant improvement compared with an increase of 2.1 percentage points in the group receiving conventional therapy. Progression without comparable regression in at least 1 of the 9 worst proximal lesions was reported in 21,25, and 46% of the respective groups, and regression without comparable progression, which occurred significantly more often in the active-treatment groups, was reported in 32, 39, and 11%. Despite the modest angiographic improvement with combination-drug therapy, the active-treatment groups had a significant 73 % reduction in cardiovascular events, defined as death, MI, and need for peripheral or coronary bypass or angioplasty. Events were recorded in 3 subjects randomized to lovastatin plus colestipol, 2 subjects randomized to nicotinic acid plus colestipol, and 10 subjects randomized to conventional therapy. e. Cholesterol Lowering Atherosclerosis Study The Cholesterol Lowering Atherosclerosis Study (CLAS) evaluated the impact of combination-drug therapy on native coronary and saphenous vein bypass vessels in 162 nonsmoking men with total cholesterol of 185 to 350 mg/dl and progressive atherosclerosis who had undergone coronary bypass surgery (Blankenhorn et al., 1987). Subjects were randomized to receive either a combination of colestipol30 gday and nicotinic acid 3 to 12 gday or placebo for 2 years. Although both treatment groups were placed on cholesterol-lowering diets, the diet of the active-treatment group was more restrictive (cholesterol <125 mg/day; total fat 22%, polyunsaturated fat lo%, and saturated fat 4% of total calories) than that of the placebo group (cholesterol <250 mg/ day; total fat 26%, polyunsaturated fat lo%, and saturated fat 5% of total

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calories). The primary endpoint was a coronary global change score based on visual comparison of angiograms made at baseline and at 2-year follow-up. Diet and combination-drug therapy decreased total cholesterol 27%, LDL cholesterol 43%, and triglyceride 22% and increased HDL cholesterol 37%. Respective changes in the control group were 4, 5, 5, and 2%. The mean global change score was significantly smaller in the group receiving combination-drug therapy (0.3 compared with 0.8 in the group receiving placebo), indicating that aggressivetreatment reduced progression. Progression as defined by a positive global change score was seen in 39% of drug-treated subjects and 60% of placebo subjects; regression as defined by a negative global change score was seen in 16% and 4% of the respective groups, which was a statistically significant difference. Lesion stabilization was reported in 45% and 36% of the respective groups. Benefit was seen in both the native coronary arteries and the saphenous vein bypass grafts. Cardiovascular event rates were the same in both treatment groups. Risk factor analysis of the CLAS data supports a role for triglyceriderich lipoproteins in the pathogenesis of atherosclerosis. On multivariate analysis, the primary predictor of atherosclerotic progression in drug-treated subjects was the content of apo C-I11 in HDL, and the primary predictor of progression in placebo subjects was non-HDL cholesterol (Blankenhorn et al., 1990). HDL may function as a reservoir for the C apolipoproteins, including apo C-11, which stimulates lipoprotein lipase-the key enzyme in VLDL metabolism-and apo C-111, which is thought to inhibit lipoprotein lipase activity and to decrease removal of triglyceride-rich lipoproteins and their remnants by the liver. Sequestration of apo C-I11 into HDL may allow increased lipoprotein lipase activity, which would increase catabolism of triglyceride-rich lipoproteins and would decrease exposure of the endothelium to their potentially atherogenic remnant particles. In the 2-year extension of CLAS, which 103 subjects completed, lipid changes were maintained in drug-treated subjects (Cashin-Hemphill et al., 1990). At 4-year follow-up, significantly more drug-treated subjects had nonprogression or regression. Progression (positive global change score) was seen in 48% of drug-treated subjects and 85% of placebo subjects. Regression (negativeglobal change score) was seen in 18% and 6% of the respective groups, which was a statistically significant difference.

B. 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors The advent of the HMG-CoA reductase inhibitors has revolutionized the pharmacologic treatment of dyslipidemia because of the efficacy and tolerability of these agents. Fluvastatin, lovastatin, pravastatin, and simvastatin are currently available in the United States. The original HMG-CoA reductase inhibitor was compactin, a fungal derivative of Penicillium citri-

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num (Endo et al., 1976). Compactin was used in animal experiments but was never released for general clinical use for reasons that have not been clearly delineated but were presumably related to toxicity. 1. Mechanism of Action

The available agents share a common mechanism of action despite minor chemical differences. The HMG-CoA reductase inhibitors partially inhibit HMG-CoA, resulting in a decrease in cholesterol synthesis that is predominantly localized in the liver. Reduction in intrahepatic cholesterol causes an upregulation of the B E receptor, thus increasing recognition, binding, and internalization of the endogenous lipoproteins that contain apo B or apo E (VLDL,IDL, LDL) (Gianturco et al., 1993). HMG-CoA reductase inhibitors may also decrease the hepatic secretion of apo B-containing lipoproteins (Arad et al., 1990),but the potential role of this putative mechanism remains controversial. HDL cholesterol levels are increased by the reductase inhibitors through a mechanism that has not been completely elucidated. These agents also have nonlipid effects that may be beneficial in the treatment of atherosclerosis. Lovastatin (Isaacsohn et al., 1994) and pravastatin (Wada et al., 1993) have been reported to reduce significantly the level of plasminogen activator inhibitor 1 (PAI-1) in hypercholesterolemic patients; elevated levels of PAI-1 predispose to a hypercoagulable state, and a decrease would theoretically increase the efficacy of plasminogen activator whether endogenously produced or exogenously administered. Lovastatin has also been shown to shift endothelial function, which is impaired in hypercholesterolemic patients (Gilligan et al., 1994), toward vasodilation in response to acetylcholine (Treasure et al., 1995). 2. Eflcacy

Recommended dosages for the HMG-CoA reductase inhibitors are fluvastatin 20 to 40 mg/day, lovastatin 10 to 80 mg/day, pravastatin 10 to 40 mg/day, and simvastatin 5 to 40 mg/day. The primary effect of these agents is a 20 to 40% decrease in LDL cholesterol. The HMG-CoA reductase inhibitors achieve their major effect on LDL cholesterol at lower doses. Although the response continues to increase as the dose is increased, the increment of increase is less at higher doses. HDL cholesterol may be expected to increase 5 to 15%, and plasma triglyceride may be expected to decrease 10 to 20%. Despite the structural similarity between lipoprotein[a] (Lp[a]) and LDL, the HMG-CoA reductase inhibitors have not been shown to decrease Lp[a] level (Kostner et al., 1989). Synthesis of cholesterol exhibits a diurnal pattern with the peak activity of HMG-CoA reductase occurring at approximately midnight. Therefore, the HMG-CoA reductase inhibitors are administered at night to achieve maximum suppression of HMG-CoA reductase activity. Specifically, lovastatin is given with the evening meal, and pravastatin is given at bedtime.

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3. Side Effects and Drug Interactions

The HMG-CoA reductase inhibitors have proved to be safe and highly effective agents. Inhibition of certain other enzymes of cholesterol synthesis were reported to cause an accumulation of steroid metabolites, resulting in cataract formation. For example, the administration of triparanol, which inhibits an enzyme in the cholesterol synthetic pathway after the formation of the steroid nucleus, resulted in increased lens opacities in experimental animals (Kirby, 1967). However, the HMG-CoA reductase inhibitors have been studied extensively for the possibility of cataract generation, and no increase in lens opacities or cataract formation has been detected by slitlamp examination in clinical trials. The most serious clinical side effects of the HMG-CoA reductase inhibitors appear to be hepatic toxicity, myositis, and rhabdomyolysis. In the 1year Expanded Clinical Evaluation of Lovastatin (EXCEL)study, conducted in 8245 hypercholesterolemic men and women, hepatotoxicity as defined by a serum transaminase elevation greater than 3 times the upper limit of normal was seen in less than 2% of subjects randomized to maximumdosage (80 mg/day) lovastatin (Bradford etal., 1991).In the groups receiving lower dosages and in the placebo group, transaminase elevations of this magnitude occurred in less than 1%. The exact incidence of myositis with HMG-CoA reductase inhibitor use is difficult to establish because of problems in correlating muscular symptoms, elevated enzyme levels, and the effects of drug therapy. Creatine kinase, which is the predominant enzyme elevated in myositis, is highly sensitive to mild trauma. Routine measurement of creatine kinase is not recommended because of the frequency of elevation in individuals without muscular symptoms. For example, in the 977 men and women who continued the EXCEL study for a second year, creatine kinase levels above the upper limit of normal were observed in 54% of the placebo group and 50 to 67% of the groups receiving lovastatin at various dosages during the 2-year follow-up period (Bradford et al., 1994). Approximately 0.1% of individuals receiving an HMG-CoA reductase inhibitor as monotherapy have definite rhabdomyolysis as characterized by severe elevations of creatine kinase, myoglobinuria, and muscle pain (Tobert et al., 1990). The mechanism by which myopathy is induced by HMG-CoA reductase inhibitor use has not been clearly determined, although defects in membrane integrity or reduced intracellular levels of ubiquinone have been implicated (Grundy, 1991). Increased rates of myopathy may occur when an HMG-CoA reductase inhibitor is used in combination with erythromycin (Spach etal., 1991), cyclosporine (Corpier etal., 1988), nicotinic acid (Tobert, 1988),or a fibricacid derivative (Pierce et al., 1990), or when renal insufficiency is present (Grundy, 1991). The use of an HMG-CoA reductase inhibitor in combination with a fibric-acid derivative is not recommended because of the increased risk for

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rhabdomyolysis. However, monotherapy with either agent may not be sufficient in patients with mixed dyslipidemia. Combination therapy has been used in patients for whom the potential benefits of lipid lowering were expected to exceed the associated risks. Because fibric-acid derivatives increase the catabolism of VLDL (see below), the LDL cholesterol level may increase with fibrate monotherapy if BE receptor activity is inadequate to clear the increased number of LDL particles. Therefore, the addition of an HMG-CoA reductase inhibitor is theoretically attractive because the reductase inhibitors increase plasma clearance of LDL. Two recent studies have shown that this combination may be useful in highly selected patients. A retrospective analysis of 80 men and women with mixed dyslipidemia who had failed monotherapy with either lovastatin or gemfibrozil evaluated the effect of treatment with a combination of both drugs for an average of 21 months (Glueck et al., 1992). Rhabdomyolysis or myoglobinuria did not occur in these subjects; myositis attributable to combination therapy and requiring discontinuation of treatment occurred in 3 % of subjects. Pravastatin has also been used with a fibric-acid derivative without precipitating severe myopathy. In a prospective study, 290 men and women with primary hypercholesterolemia were randomized to receive pravastatin, gemfibrozil, a combination, or placebo for 12 weeks (Wiklund etal., 1993). Creatine kinase elevations of greater than 4 times the pretreatment level occurred in 1% of subjects receiving pravastatin alone, 3% of subjects receiving gemfibrozil alone, 5% of subjects receiving both pravastatin and gemfibrozil, and 1% of subjects receiving placebo. Although combination therapy decreased LDL cholesterol 37% and VLDL cholesterol 49% and increased HDL cholesterol 17%, compared with respective changes of 34, 22, and 6% with pravastatin monotherapy and 17, 49, and 15% with gemfibrozil monotherapy, the combination of an HMG-CoA reductase inhibitor and a fibric-acid derivative has potentially serious consequences and requires close observation and careful patient education. Central nervous system symptoms, including headache and sleep disturbances, have been reported with use of HMG-CoA reductase inhibitors. Despite concern that these symptoms may be more common with lovastatin and simvastatin, which are lipophilic, than with fluvastatin and pravastatin, which are hydrophilic, because the lipophilic compounds cross the bloodbrain barrier, comparative studies have not documented clinically significant differences. Sleep disturbances are relatively uncommon side effects with use of either lipophilic or hydrophilic agents (Illingworth and Tobert, 1994). 4. Clinical Trials

Although HMG-CoA reductase inhibitors have been in widespread use only during the past decade, a large body of clinical evidence has established the efficacy and tolerability of these compounds. Clinical trials using HMGCoA reductase inhibitors have evaluated their effect as monotherapy or in

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combination with other lipid-lowering agents on lipid parameters and on anatomic and clinical endpoints. a. Monitored Atherosclerosis Regression Study The Monitored Atherosclerosis Regression Study (MARS) used lovastatin as pharmacologic lipidlowering monotherapy in 270 men and women with angiographically documented CHD and total cholesterol of 190 to 295 mg/dl (Blankenhorn et al., 1993). Subjects were randomized to receive lovastatin 80 mg/day in divided doses or placebo for 2 years. All subjects were to follow a low-fat, low-cholesterol diet (cholesterol 5 2 5 0 mg/day; total fat 127%, saturated fat ~ 7 monounsaturated % ~ fat ~ 1 0 % and~polyunsaturated fat 1 1 0 % of total calories). In the lovastatin group, total cholesterol decreased 32%, LDL cholesterol decreased 45 %, and triglyceride decreased 22 %, and HDL cholesterol increased 8.59'0, all of which were significantly different from decreases of 2% and 3% and increases of 3.5% and 2% in respective lipid levels in the placebo group. Lovastatin treatment did not effect a significant improvement in the primary endpoint of mean per-patient change in percent diameter stenosis as determined by quantitative coronary angiography. Progression was the outcome in both groups, although there was less in the lovastatin group, in which mean percent diameter stenosis increased 1.6%, than in the placebo group, in which stenosis increased 2.2%. However, in lesions causing 50% or greater stenosis at baseline, significant improvement was seen in the lovastatin group, in which stenosis decreased 4.1 %, indicating regression, compared with the placebo group, in which stenosis increased 0.9%,indicating progression. By quantitative assessment of all lesions, progression was seen in significantly fewer lovastatin subjects (29%)than placebo subjects (41%), and regression was seen in significantly more lovastatin subjects (23%)than placebo subjects (12%). In addition to quantitative assessment, a global change score was determined by a panel of angiographers. Although both groups again showed progression by this assessment, significantly less progression was seen in the lovastatin group, which had an average global change score of 0.41, compared with the placebo group, which had an average score of 0.88. By visual assessment, progression (positive global change score) was seen in significantly fewer lovastatin subjects (47%)than placebo subjects ( 6 5 % ) , and regression (negative global change score) was seen in significantly more lovastatin subjects (23%)than placebo subjects (11%). Although fewer clinical coronary events, defined as MI, percutaneous transluminal coronary angioplasty, coronary artery bypass surgery, coronary death, and hospitalization for unstable angina, occurred in the lovastatin group (22) than in the placebo group (31), this difference was not statistically significant.

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b. Canadian Coronary Atherosclerosis Intervention Trial Lovastatin monotherapy was also evaluated in the Canadian Coronary Atherosclerosis Intervention Trial (CCAIT), which was a 2-year study conducted in 331 men and women with angiographically demonstrated diffuse CHD and total cholesterol of 220 to 300 mg/dl (Waters et al., 1994). All subjects received instruction on a Step I Diet (cholesterol <300 mg/day; total fat 530%, saturated fat 8 to lo%, polyunsaturated fat
c Pravastatin Limitation ofAtherosclerosis in the Coronary Arteries Pravastatin monotherapy was evaluated in the Pravastatin Limitation of Atherosclerosis in the Coronary Arteries (PLAC I ) trial, in which 408 men and women with angiographic evidence of CHD and LDL cholesterol of 130 to 190 mg/dl were randomized to receive pravastatin or placebo for 3 years (Pitt et al., 1995).The primary endpoint was change in mean diameter of 10 predefined coronary artery segments as determined by quantitative coronary angiography. In the pravastatin group, mean diameter decreased 0.02 mdyear, which was not significantly different from the placebo group. The group receiving pravastatin had a 19% decrease in total cholesterol, a 28% decrease in LDL cholesterol, and a 7% increase in HDL cholesterol. The pravastatin

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group also had significantly fewer events occurring after 90 days. Events analyzed were MI, nonfatal MI plus death, and nonfatal MI plus CHD death. d. MulticentreAnti-Atheroma Study The Multicentre Anti-Atheroma Study (MAAS)was an angiographically monitored trial of simvastatin monotherapy (MAAS Investigators, 1994). In this trial, 381 men and women with angiographic evidence of CHD were randomized to receive simvastatin 20 mg/day or placebo for 4 years. All subjects received dietary instruction according to the usual practice of each participating center. Compared with the placebo group, the simvastatin group demonstrated a 23% decrease in total cholesterol, a 31% decrease in LDL cholesterol, a 9% increase in HDL cholesterol, and an 18% decrease in triglyceride. Quantitative coronary angiography performed at 2- and 4-year followup was compared with baseline to evaluate the change in diffuse coronary atherosclerosis, as determined by the per-patient average of mean lumen diameter of all coronary artery segments, and the change in focal coronary atherosclerosis, as determined by the per-patient average of minimum lumen diameter of all segments that were atheromatous at baseline, follow-up, or both. At 4-year follow-up, progression of both diffuse and focal coronary atherosclerosiswas seen in both groups. The mean lumen diameter decreased 0.02 mm in the simvastatin group and 0.08 mm in the placebo group; the minimum lumen diameter decreased 0.04 mm and 0.13 mm in the respective groups. Both of these differences were statistically significant. Clinical event rates were not significantly different between treatment groups. Cardiac death occurred in 4 subjects in each group, and MI occurred in 11 subjects in the simvastatin group and 7 subjects in the placebo group. Percutaneous transluminal coronary angioplasty or coronary artery bypass surgery was required in 23 subjects in the simvastatin group and 34 subjects in the placebo group. e. Scandinavian Simvastatin Survival Study Simvastatin monotherapy was also evaluated in the Scandinavian Simvastatin Survival Study (4S), a large, multicenter trial conducted in 4444 men and women with a history of acute MI or angina pectoris, total cholesterol of 210 to 310 rng/dl, and triglyceride of 220 mg/dl or less after diet (Scandinavian Simvastatin Survival Study Group, 1994). All subjects received dietary instruction and were randomized to receive simvastatin, dosed to decrease total cholesterol to 115 to 200 mg/dl, or placebo. The initial simvastatin dosage of 20 mg/day was increased to 40 mg/day in 37% of simvastatin subjects and decreased to 10 mg/day in 2 subjects. Median time on trial was 5.4 years. The trial was specifically designed to evaluate total mortality, which was the primary endpoint. In the group receiving simvastatin, total cholesterol decreased 25%, LDL cholesterol decreased 35%, HDL cholesterol increased 8 %, and triglyceride

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decreased 10%. All these values increased in the group receiving placebo; respective changes were 1, 1, 1, and 7%. Total mortality was reduced 30% in the group receiving simvastatin, which was highly statistically significant. Much of this improvement was due to a 42% reduction in CHD mortality in the simvastatin group. No increase in noncardiovascular mortality was reported with lipid-lowering therapy. The secondary endpoint-incidence of major coronary events (coronary death, nonfatal MI, and resuscitated cardiac arrest)-was significantly reduced 34% in the simvastatin group. f: West of Scotland Coronary Prevention Study In the West of Scotland Coronary Prevention Study (WOSCOPS), 6595 hypercholesterolemic men (mean LDL cholesterol 192 mg/dl after diet) with no history of MI were randomized to receive pravastatin 40 mg/day or placebo for an average 4.9 years. In the pravastatin group, relative risk for nonfatal MI or CHD death as a first event was significantly reduced 31% compared with the placebo group (Shepherd et al., 1995).

C. Probucol Probucol is a bis-phenol compound that has structural similarities to the powerful antioxidant butylated hydroxytoluene. The chemical structure of probucol confers a high degree of lipid solubility, which allows probucol to be carried within lipoproteins, particularly LDL (Dachet et al., 1985). Therefore, at least part of the drug’s benefit in patients with CHD may be due to local, intracellular effects instead of its effect on circulating lipid levels. 1. Mechanism of action

Despite decades of clinical use, the precise mechanism of probucol and its role in hypolipidemic therapy have not been completely delineated. The decrease in LDL cholesterol obtained with probucol administration appears to be due to an increase in the fractional catabolic rate of LDL, not to a decrease in lipoprotein synthesis (Kesaniemi and Grundy, 1984). This increased clearance of LDL particles does not require the presence of functioning B E receptors but has been seen in patients with homozygous FH (Baker et al., 1982), in whom functioning B E receptors are lacking. Therefore, probucol appears to increase the removal of LDL by alternative pathways. In addition, the effect of probucol on lipid levels in patients with heterozygous FH was reported to be affected by apo E genotype (Eto et al., 1990). Probucol-induced reductions in total cholesterol and LDL cholesterol were significantly greater in patients with apo E4 than in patients without apo E4. The mechanism by which probucol decreases HDL cholesterol is also unclear. In experimental animals, probucol administration has been found to increase the fractional catabolic rate of apo A-I-one of the major apolipoproteins of HDL-and to decrease the rate of apo A-I synthesis (Ying et

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al., 1990). Probucol also increases the activity of cholesteryl ester transfer protein (CETP) (Franceschini et al., 1989),the enzyme that transfers triglyceride from the triglyceride-rich lipoproteins to HDL and LDL in exchange for cholesteryl ester. Increased CETP activity may explain, at least in part, the decrease in HDL cholesterol seen with probucol use. Although HDL is thought to be the vehicle of reverse cholesterol transport, the putative process by which cholesterol is returned to the liver from the periphery, xanthoma regression with long-term probucol use in patients with homozygous FH was directly related to the decrease in HDL cholesterol (Yamamoto et al., 1986). In addition to its lipid-lowering action, probucol has antioxidant properties that may confer clinical benefit. This antioxidant effect is maximized by probucol's localization within the LDL particle. Oxidized LDL is recognized by the scavenger receptor on macrophages, which does not bind native LDL. Unlike the B/E receptor, the scavenger receptor is not downregulated as intracelluar cholesterol accumulates, and these macrophages can become lipid-laden foam cells, which are thought to be the precursors of advanced atherosclerotic lesions. Probucol has been shown to decrease the rate of progression of atherosclerotic lesions in the Watanabe heritable hyperlipidemic rabbit, and this effect was attributed to the antioxidant effect of probucol rather than its lipid-lowering effect (Cared et al., 1987). Probucol has also been reported to decrease the vasoconstriction, thought to be caused by oxidized LDL, that occurs in experimental animals fed a high-cholesterol diet (Kaplan et al., 1990). 2. Emcacy

Probucol administered at a dosage of 500 mg twice a day decreases LDL cholesterol 5 to 15% and decreases HDL cholesterol 20 to 30%. The decrease in HDL cholesterol is of concern because of the inverse relation between HDL cholesterol level and CHD incidence (Gordon et al., 1977), but the clinical significanceof the probucol-induced decrease in HDL cholesterol is not known. Probucol usually has no effect on triglyceride level. Because probucol is highly lipophilic, its absorption is increased when taken with a fatty meal. Patients should be instructed to separate doses from meals and to adhere to a low-fat diet to prevent drug toxicity. 3. Side Effects and Drug Interactions

Probucol has few clinical side effects. Nonspecific gastrointestinal discomfort is the side effect most commonly reported, although it is seldom of sufficient severity to require discontinuation of the drug. Probucol has been shown to increase the electrocardiographic QT interval. In early animal experimentation, probucol caused an increase in sudden cardiac death that was thought to be attributable to ventricular arrhythmias (Buckley et al., 1989). However, these data are difficult to extrapolate to humans because of the possibility that the effect was species specific and

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because the drug was administered with an atherogenic diet. Probucol administration in humans has not been definitely associated with an increased risk for sudden cardiac death, although prolongation of repolarization may occur in as many as 50% of patients taking this drug (Dujovne et al., 1984). The QT interval may be prolonged in patients with a variety of conditions, including subarachnoid hemorrhage, mitral valve prolapse, and electrolyte disturbances, as well as in those following certain hypocaloric diets or receiving drugs such as tricyclic antidepressants, phenothiazines, or antiarrhythmic agents (quinidine, procainamide, disopyramide, amiodarone). Probucol should be used with great caution in these patients. 4. Clinical Trials a. Probucol Quantitative Regression Swedish Trial In the Probucol Quantitative Regression Swedish Trial (PQRST), 274 men and women with femoral atherosclerosis and hypercholesterolemia (total cholesterol >265 mgldl, LDL cholesterol >175 mgldl, and triglyceride 1350 mgldl) received dietary instruction (polyunsaturated fat :saturated fat ratio 0.5) and cholestyramine 8 to 16 glday in addition to either probucol 1 glday or placebo for 3 years (Walldius et al., 1994). The primary endpoint was the change in atheroma volume of the superficial femoral artery as calculated by comparing quantitative angiographic measurements of the lumen volume at baseline and at 3year follow-up. Compared with the group receiving cholestyramine alone, the group receiving probucol plus cholestyramine had a 17% decrease in total cholesterol, a 12% decrease in LDL cholesterol, and a 24% decrease in HDL cholesterol. In addition, LDL particles in the group receiving probucol plus cholestyramine were less susceptible to oxidation (Walldius et al., 1993). Despite the changes in lipid levels, the addition of probucol did not increase regression as assessed by the primary endpoint. The lumen volume increased in both treatment groups, indicating regression, but the combination of probucol and cholestyramine increased the lumen volume only O.6%, which was not significantly different from baseline, compared with a significant increase in lumen volume of 4.2% with cholestyramine alone. The investigators suggested that probucol is more effective in preventing progression than in inducing regression. Cardiovascular clinical events occurred more frequently in the group receiving probucol plus cholestyramine (39 events) than in the group receiving cholestyramine alone (29 events), but this difference was not statistically significant.

D. Estrogen-Replacement Therapy CHD risk in women increases substantially at menopause. For example, in the Framingham Heart Study, postmenopausal women were found to

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have more than twice the risk for a CHD event compared with premenopausal women, regardless of whether menopause was natural or surgical (Kannel, 1987).At least part of this increase in risk is thought to be due to the loss of estrogen. 1. Mechanism of Action

Although the mechanism by which estrogen decreases CHD risk is unclear, possibilities include improvement of coronary tone and alteration of platelet aggregation as well as the demonstrated effect of estrogen on lipid levels. In numerous clinical studies, estrogen induced a moderate increase in HDL cholesterol and a moderate decrease in LDL cholesterol (Granfone et al., 1992). In addition, animal studies have shown decreased accumulation of LDL in the arterial wall with estrogen administration (Wagner et al., 1992). 2. Efficacy

Orally administered estrogen (conjugated estrogen 0.625 mg/day or micronized estradiol 2 mg/day) typically increases HDL cholesterol up to 15% and decreases LDL cholesterol approximately 15 %. Triglyceride may increase, especially in women with elevated plasma triglyceride. Lp[a] levels may be reduced (Gotto, 1994), and a 50% decrease in Lp[a] has been reported with a combination of estrogen and progesterone (Soma et al., 1993).Estrogen administered transcutaneously or percutaneously is thought to have less effect on the lipid profile than estrogen administered orally. Estrogen-replacement therapy does not have a U.S. Food and Drug Administration indication for regulating Iipids or for reducing CHD risk. 3. Side Effects and Drug Interactions

Although estrogen-replacement therapy provides an attractive option to drugs in postmenopausal women with dyslipidemia, the cardioprotective effects of estrogen must be weighed against potential side effects. Estrogen appears to increase the risk for endometrial cancer and may increase the risk for breast cancer. Coadministration with progesterone may decrease these adverse effects, but the degree of protection is not known. 4. Clinical Trials a. Postmenopausal GtrogenlProgestin Interventions The Postmenopausal EstrogenProgestin Interventions (PEPI) trial randomized 875 healthy postmenopausal women, aged 45 to 64 years, to receive placebo, estrogen 0.625 rng/day alone, estrogen plus cyclic medroxyprogesterone acetate 10 mg/day for 12 dayshonth, estrogen plus consecutive medroxyprogesterone acetate, 2.5 mg/day, or estrogen plus cyclic micronized progesterone 200 mg/day for 12 dayshonth (Writing Group for the PEPI Trial, 1995). Results indicated that estrogen alone or in combination with any of the

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progesterone regimens improved lipid levels and decreased fibrinogen levels. In groups receiving estrogen, LDL cholesterol decreased approximately 20% compared with placebo. HDL cholesterol increased in all groups receiving estrogen, but the increase was less in groups also receiving progesterone. However, progesterone coadministration eliminated the increased risk for endometrial hyperplasia shown in the group receiving estrogen alone. Risk for breast cancer was not increased with estrogenreplacement therapy.

111. Agents That Predominantly Lower Triglyceride The role of plasma triglyceride in CHD risk assessment is controversial. Univariate analyses performed in prospective studies have consistently shown a direct relation between elevated triglyceride and CHD incidence; however, in multivariate analyses controlling for variables such as obesity, HDL cholesterol, and glucose intolerance, the predictive role of triglyceride is diminished (Austin, 1991). Among the possible explanations for the weakening of this association is the metabolic interrelation between the triglyceride-rich lipoproteins and HDL as well as interindividual and intraindividual variability in triglyceride measurements (Austin, 1989). However, in the Prospective Cardiovascular Munster (PROCAM) study, subjects with a combination of elevated triglyceride (2200 mg/dl) and a high LDL cholesterol :HDL cholesterol ratio (>LO) were at increased risk for CHD events; although this subgroup made up only 4% of the study population, it accounted for 25% of all CHD events (Assmann and Schulte, 1992). Similar findings were reported in the Helsinki Heart Study (see below). Triglyceride is typically measured after a 12-h fast, but postprandial lipemia has been shown to be predictive of CHD (Patsch et al., 1992). The inability to clear triglyceride-rich particles after a fatty meal may result in endothelial damage because remnants of these particles may be cytotoxic (Chung et al., 1989) and may increase delivery of cholesterol to the vascular wall (Zilversmit, 1979). The role of triglyceride in CHD risk is further complicated by the fact that hypertriglyceridemia is not a uniform clinical characteristic but is seen in a variety of conditions, not all of which are associated with increased incidence of CHD. For example, familial combined hyperlipidemia and dysbetalipoproteinemia (type I11 hyperlipidemia) confer increased risk for CHD, whereas hypertriglyceridemia occurring in familial hypertriglyceridemia in some kinships or in chylomicronemia does not appear to increase CHD risk. In addition, individual classes of triglyceride-rich particles are thought to differ in atherogenic potential. Chylomicrons and VLDL are not thought to be directly atherogenic, but chylomicron remnants and IDL may

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be. Further clarification is needed to define the role of elevated triglyceride in CHD risk assessment. Hypertriglyceridemia has also been linked to procoagulant conditions that may increase the risk for intravascular thrombosis. Elevated plasma triglyceride level has been associated with elevated plasma levels of prothrombin and of coagulation factors W, IX, and X (Constantino et d., 1977). In addition, triglyceride level has been correlated with PAI-1 level, suggesting that fibrinolytic capacity may be decreased in hypertriglyceridemia (Crutchley et al., 1989). A. Nicotinic Acid

Nicotinic acid is a B vitamin that can be obtained from dietary sources or converted from tryptophan. It functions as a coenzyme in carbohydrate metabolism as a component of the nicotinamide adenine dinucleotide coenzyme system, but in pharmacologic doses, nicotinic acid has an antidyslipidemic effect that is not related to its role in intermediary metabolism. 1. Mechanism of Action

The mechanism of nicotinic acid is complex, but the primary action appears to be a direct decrease in hepatic synthesis of VLDL, which carries the majority of endogenous triglyceride. Circulating levels of all lipoproteins in VLDL’s metabolic cascade, including IDL and LDL, are also decreased because of the reduction in precursor particles. In addition, nicotinic acid decreases the release of free fatty acids into the circulation. Because free fatty acids are the substrates for triglyceride synthesis, hepatic production of triglyceride is decreased by this peripheral effect. In addition to lowering triglyceride,nicotinic acid increases HDL cholesterol. Although the precise mechanism is not known, decreased catabolism of HDL with nicotinic acid administration has been reported (Shepherd et al., 1979). Nicotinic acid is the only hypolipidemic agent that has been shown to decrease Lp[a] (Carlson et al., 1989), but neither the mechanism involved nor the clinical significance of Lp[a] lowering is known. 2. Emcacy

Administration of nicotinic acid favorably alters levels of all circulating lipoproteins except chylomicrons and their remnants. Crystalline nicotinic acid dosed at 1.5 to 6 g/day may be expected to decrease LDL cholesterol 10 to 25%, increase HDL cholesterol 15 to 35%, and decrease triglyceride 20 to 50%. Although sustained-release preparations are available, their use is limited by increased side effects (see below) and a lack of safety and efficacy data. Because lipid lowering with nicotinic acid does not involve upregulation of the B/E receptor, nicotinic acid therapy is effective in familial defective

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apo B-100 (Schmidt et al., 1993), in which the number of LDL particles is increased because abnormal apo B- 100 prevents their recognition and removal by the BE receptor. Nicotinic acid is useful in all dyslipidemiasexcept those characterized by elevated circulating levels of chylomicrons. 3. Side Effects and Drug Interactions

The widespread clinical use of nicotinic acid has been hampered by side effects that range from mild clinical irritations to life-threatening toxicity. Crystalline nicotinic acid is rapidly absorbed after oral administration, which may account at least in part for its side effect profile. Flushing is the most common side effect and occurs in almost all patients treated with nicotinic acid. The flushing is caused by vasodilation that is secondary to endothelial release of prostaglandin and may be of sufficient severity to cause systemic hypotension. Prostaglandin inhibitors, such as aspirin, given prior to nicotinic acid may decrease flushing. Other dermatologic side effects are pruritus and, rarely, acanthosis nigricans. Another adverse effect seen with nicotinic acid use is hepatic dysfunction, which may be partially explained by the high first-pass extraction of the drug by the liver. Nicotinic acid-induced liver toxicity ranges from mild, asymptomatic elevations of liver enzymes to fulminant hepatic failure (Mullin et al., 1989).Hepatic toxicity appears to be more common with sustainedrelease preparations (Rader et al., 1992).Transaminase elevations may occur in as many as 5% of patients taking more than 3 g/day of nicotinic acid (Brown et al., 1991) and is not in itself an indication for drug cessation. However, if liver enzymes increase to three times the normal level or greater, nicotinic acid should be discontinued. Other gastrointestinal problems reported with nicotinic acid administration include activation of peptic ulcer disease (Charman et al., 1972). Metabolic abnormalities that may occur with nicotinic acid use include hyperuricemia and decreased glucose tolerance. Nicotinic acid should only be used with caution in patients with diabetes mellitus or a predisposition to diabetes. Myopathy has been reported with nicotinic acid monotherapy (Litin and Anderson, 1989),and rhabdomyolysis has been reported in combination therapy with an HMG-CoA reductase inhibitor (Reaven and Witztum, 1988). Ophthalmologic side effects may rarely occur with nicotinic acid use and include decreased visual acuity (Gass, 1973).Cystic maculopathy occurs in approximately 0.7% of patients taking more than 3 g/day (Millay et al., 1988). 4. Clinical Trials a. Coronary Drug Project The Coronary Drug Project randomized 8341 men with previous MI to receive one of several lipid-lowering agents or

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placebo (Coronary Drug Project Research Group, 1975). In one of the active-treatment arms, 1119 subjects received nicotinic acid 3 glday for 5 years. Total cholesterol decreased 10% and triglyceride decreased 26%, after correction for lipid changes in the placebo group. Compared with the placebo group, incidence of nonfatal MI in the group receiving nicotinic acid was reduced 27%, which was a significant improvement. There was no significant difference in total mortality or CHD mortality between these treatment groups at the end of the trial. At 15-year follow-up, the group that had received nicotinic acid demonstrated a highly statistically significant reduction in total mortality of 11% compared with the placebo group (Canner et al., 1986). b. Stockholm lschaemic Heart Disease Secondary Prevention Study The Stockholm Ischaemic Heart Disease Secondary Prevention Study randomized 555 men and women with prior MI to combination therapy with nicotinic acid 3 glday and clofibrate 2 glday or to a control group (Carlson and Rosenhamer, 1988). The study was nonblinded. After 5 years, total cholesterol decreased 13% and triglyceride decreased 19% in the group receiving nicotinic acid plus clofibrate compared with the control group. CHD mortality was significantly reduced 36% and total mortality was significantlyreduced 26% in the group receiving combinationdrug therapy compared with the control group. In retrospective subset analysis, the decrease in CHD mortality was directly related to the decrease in triglyceride. In the subgroup whose triglyceride decreased 30% or more, CHD mortality was 10%; CHD mortality in the control group was 26%.

B. Fibric-Acid Derivatives The fibric-acid derivatives available in the United States are clofibrate, which is little used, and gemfibrozil. Fenofibrate is approved but is not yet available. Other fibric-acid derivativesused in other countries are bezafibrate and ciprofibrate. 1. Mechanism of Action

Although the lipid-lowering mechanisms of the fibrates are complex and not completely understood, their major action appears to be an increase in the activity of lipoprotein lipase, thereby enhancing the catabolism of triglyceride-rich lipoproteins (Nikkila et al., 1977). This increased catabolism is thought to be responsible not only for decreasing triglyceride but also for increasing HDL cholesterol, because of the transfer of surface components from catabolized triglyceride-rich lipoproteins to HDL (Simpson et al., 1990). A postulated peripheral action of the fibrates is a reduction in plasma levels of free fatty acids (Levy et al., 1976). Although some early

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studies reported decreased cholesterol synthesis with fibric-acid derivative administration (Berndt et al., 1978), more recent investigations have been unable to document an increase in urinary mevalonic acid with fibric-acid derivative therapy, indicating that the biosynthetic capacity of cholesterol is not altered (Beil et al., 1990). Instead, LDL cholesterol appears to be decreased because of increased B E receptor activity in response to decreased intracellular cholesterol in hepatocytes (Illingworth, 1991). Fibric-acid derivatives have also been reported to alter the composition of circulating lipoproteins. Fenofibrate has been shown to increase the apo C-I1 content of VLDL in hypertriglyceridemic patients (Franceschini et al., 1985), which is consistent with increased lipoprotein lipase activity. Bezafibrate (Eisenberg et al., 1984) and gemfibrozil (Tilly-Kiesi and Tikkanen, 1991) have been shown to decrease circulating levels of small, dense LDL particles, which have been associated with a threefold-increased risk for MI (Austin et al., 1988), thereby shifting LDL subclass pattern to a potentially less atherogenic phenotype although LDL cholesterol level may not be decreased. Fibrates may also have beneficial nonlipid effects in patients with atherosclerosis, Gemfibrozil has been shown to decrease platelet aggregability and reactivity in response to epinephrine (Todd and Ward, 1988) and to decrease clotting factor VII-phospholipid complex level (Andersen et al., 1990), and the other fibrates decrease plasma levels of fibrinogen (Davignon, 1994). 2. Eflcacy

Fibric-acid derivatives are used predominantly to decrease triglyceride and to increase HDL cholesterol. Clofibrate dosed at 2 g/day or gemfibrozil dosed at 1.2 g/day typically decreases LDL cholesterol 10 to 15%. Occasionally in patients with marked hypertriglyceridemia, LDL cholesterol levels may increase, possibly because of an inability of the B E receptors to remove the increased LDL generated by enhanced VLDL catabolism. Fibrate therapy decreases triglyceride 20 to 50% and increases HDL cholesterol 10 to 15%. 3. Side Effects and Drug Interactions

Serious side effects with fibric-acid derivative administration are uncommon. Increased lithogenicity of bile has been reported with clofibrate (Coronary Drug Project Research Group, 1975) but has not been conclusively linked with the other fibrates. Mild, nonspecific gastrointestinal complaints may occur in as many as 5% of patients receiving fibric-acid derivatives but generally do not necessitate drug cessation (Illingworth, 1991 ). Transaminase levels may occasionally be increased but serious liver dysfunction is uncommon (Sirtori et al., 1992). Myositis with fibric-acid derivative monotherapy is uncommon (Langer and Levy, 1968), but the incidence of this potentially serious complication may be increased when fibrates are combined with other agents, especially

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HMG-CoA reductase inhibitors, as noted above. The coadministration of a fibrate increases the anticoagulant activity of warfarin and therefore warrants monitoring of the prothrombin time. As noted above, concomitantly administered bile-acid sequestrants may decrease the absorption of fibricacid derivatives into the plasma compartment. Other drug interactions with fibric-acid derivatives are uncommon. 4. Clinical Trials a. World HeaM Organization Cooperative Trial The World Health Organization (WHO) Cooperative Trial randomized more than 10,000 men without known CHD who were in the upper tertile of total cholesterol distribution to receive clofibrate 1.6 g/day or placebo for an average of 5.3 years (Committee of Principal Investigators, 1978). The original trial results were not analyzed on an intent-to-treat basis. In the group receiving clofibrate, total cholesterol decreased 9% from baseline. Nonfatal MI incidence decreased 25% and CHD incidence decreased 20% compared with placebo; both reductions were statistically significant. However, CHD mortality was not improved with clofibrate treatment, and total mortality increased significantly in the group receiving clofibrate. Almost 8 years after the trial had ended, the excess in total mortality, which was 47% during the trial, had decreased to 11% for the entire 13-year follow-up period and was no longer statistically significant (Committee of Principal Investigators, 1984).

b. Coronary Drug Project In the 1103 subjects in the Coronary Drug Project randomized to receive clofibrate 1.8 g/day, total cholesterol decreased 6.5% and triglyceride decreased 22%, after correction for lipid changes in the placebo group (Coronary Drug Project Research Group, 1975). Compared with the placebo group, combined incidence of CHD death and nonfatal MI was 9% lower in the clofibrate group, but this difference was not statistically significant. Total mortality and CHD mortality were similar between these treatment groups. c. Helsinki Heart Study In the Helsinki Heart Study, 4081 men with non-HDL cholesterol greater than 200 mg/dl and no known CHD were randomized to receive gemfibrozil 1.2 g/day or placebo for 5 years (Huttunen et al., 1991). All subjects received dietary counseling. In the gemfibrozil group, total cholesterol decreased lo%, LDL cholesterol decreased 11%, HDL cholesterol increased 11%, and triglyceride decreased 35% compared with the placebo group. CHD events, defined as fatal and nonfatal MI and cardiac death, were significantlyreduced 34% in the group receiving gemfibrozil. Total mortality was higher in the gemfibrozil group because of an increase in noncardiovascular deaths that was primarily due to accidents or violence or to intracranial

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John A. Farmer and Antonio M. Gotto, Jr.

hemorrhage, but the difference was not statistically significant (Frick et al., 1987). On reanalysis, it was determined that CHD risk was reduced 71% in the subgroup with triglyceride greater than 200 mgldl and an LDL cholesterol :HDL cholesterol ratio greater than 5; this subgroup accounted for approximately 10% of the study population (Manninen et al., 1992). C. Fish Oil In epidemiological studies, an inverse relation has been found between CHD incidence and consumption of omega-3 polyunsaturated fatty acids (Bang and Dyerberg, 1972). For example, in the Multiple Risk Factor Intervention Trial (MRFIT), separate analysis of the usual-care control group demonstrated an inverse relation between estimated dietary intake of the omega-3 fatty acids and 10.5-year CHD mortality rate (Dolecek, 1992). In autopsy studies, Alaskan natives, who consume large quantities of omega3 fatty acids, were shown to have less extensive coronary atherosclerosis than nonnatives (Newman et al., 1993). The mechanism by which consumption of omega-3 fatty acids confers protection against CHD has not been totally elucidated, although several potential mechanisms have been proposed. Triglyceride appears to be decreased by a suppression in VLDL production (Nestel et al., 1984). In addition to decreasing circulating triglyceride, fish oil has been reported to increase the proportion of HDL2,which is larger and contains more cholesterol, to the smaller HDL3 (Abbey et al., 1990), thereby increasing the cholesterol-carrying capacity of HDL and so potentially increasing the efficacy of the posited reverse cholesterol transport. Nonlipid parameters may also be improved by these compounds. A recent meta-analysis of 31 placebo-controlled trials enrolling a total of 1356 subjects found that omega-3 fatty acids had a mild but statistically significant dose-response effect on blood pressure; this hypotensive effect was strongest in subjects with hypertension, hypercholesterolemia, or CHD at baseline (Morris et al., 1993). Bleeding time has been shown to be prolonged with fish oil administration, but the alteration of platelet function and hemostasis appears to be less than early reports suggested (Braden et al., 1991). In hamsters, fish oil administration decreased the binding of leukocytes to the endothelium during reperfusion after pressure-induced ischemia; this decrease appears to be secondary to the displacement of arachidonic acid, which is the precursor of leukotriene B4-a potent adhesion promoter-with eicosapentaenoic acid, which is the precursor of the less potent leukotriene BS (Lehr et al., 1991). Neutrophil adhesion in acute ischemic syndromes may be an important manifestation of MI. Although consumption of fish containing a high content of omega-3 fatty acids is essentially a risk-free intervention, its benefit has not been definitely demonstrated in randomized, blinded, controlled clinical trials.

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Fish oil supplements or capsules cannot be recommended until the mechanisms involved have been clarified and clinical benefit established.

IV. Combination-Drug Therapy In patients whose dyslipidemia is refractory to pharmacologic monotherapy, a second agent may be necessary to reduce elevated lipid levels (Table 111). Combination-drug therapy may increase the degree of lipid lowering, reduce cost and side effects, and increase compliance. Combining drugs with synergistic mechanisms of action often allows each drug to be administered at lower dosages. However, caution is required in combining an HMGCoA reductase inhibitor with a fibric-acid derivative, because of the increased risk for myopathy, or in combining an HMG-CoA reductase inhibitor with nicotinic acid, because of possible increased risk for myopathy and hepatitis.

V. Future Developments Future advances in antidyslipidemictherapy may focus on refining existing pharmacologic agents. For example, the potency of bile-acid sequestrants may be increased and their palatability improved to increase patient compliance. More potent HMG-CoA reductase inhibitors such as atorvastatin have been developed and may become available for clinical use in the future. Atorvastatin 80 mg/day has been reported to decrease LDL cholesterol 61% and to decrease triglyceride 43% (Black, 1994). TABLE 111 Combination-Drug Therapy in Adults: National Cholesterol Education Program Recommendations ~

Hyperlipidemia

Combination Drug

Elevated LDL cholesterol and triglyceride <200 mg/dl

Bile-acid sequestrant + HMG-CoA reductase inhibitor Bile-acid sequestrant + nicotinic acid HMG-CoA reduaase inhibitor + nicotinic acid"

Elevated LDL cholesterol and triglyceride 200-400 mg/dl

Nicotinic acid + HMG-CoA reductase inhibitof HMG-CoA reductase inhibitor + gemfibrozilb Nicotinic acid + bile-acid sequestrant Nicotinic acid + gemfibrozil

Possible increased risk for myopathy or liver dysfunction. Increased risk for myopathy; must be used with caution. From National Cholesterol Education Program, (1994). HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; LDL, low-density lipoprotein.

a

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In addition to the existing agents whose primary action is to lower LDL cholesterol or triglyceride, new agents may be developed specifically to increase HDL cholesterol. Another potential target for pharmacologic therapy is Lp[a], which has been identified as an independent CHD risk factor in a number of studies (Loscalzo, 1990). Future pharmacologic agents may alter the activity of other enzymes involved in lipoprotein metabolism besides lipoprotein lipase. An inhibitor of CETP would be expected to increase HDL cholesterol levels, although the clinical implications of this mechanism have not been delineated. Acyl coenzyme A :cholesterol acyltransferase (ACAT) activity, which leads to increased levels of cholesteryl ester in the intracellular compartment, might be decreased by an agent that shunts cholesterol into the bile acid pool (Sliskovic and White, 1991) but does not cause the compensatory increase in cholesterol production that occurs with the bile-acid sequestrants. An ACAT inhibitor administered to rabbits with endogenous hypercholesterolemia decreased LDL cholesterol 43% and decreased VLDL cholesterol 62% compared with control rabbits with endogenous hypercholesterolemia (Krause et al., 1994). Agents may be developed to inhibit other enzymes of cholesterol biosynthesis besides HMG-CoA reductase (Fig. 1). Squalene synthase inhibitors (Bergstrom et al., 1993) may upregulate the BE receptor, thereby enhancing th.e removal of LDL from the plasma compartment. Squalene epoxidase inhibitors (Hidaka et al., 1991) would prevent the conversion of squalene to cholesterol, but the effect of the resulting increase in squalene concentration is not known. In addition to altering concentrations of circulating lipids, future therapy may be directed at protecting the arterial wall against atherogenesis, for example, by preventing LDL oxidation or by increasing prostacyclin production. Additionally, agents may be designed to decrease the production of growth factors associated with atherogenesis, such as platelet-derived Acetyl coenzyme A

5.

3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) 4 < -HMG-CoA reductase inhibitor Mevalonic acid Ubiquinones, dolichols c 1 Farnesyl pyrophosphate + Heme 4 < -Squalene synthase inhibitor Squalene 4 < -Squalene epoxidase inhibitor Cholesterol

FIGURE I Cholesterol production may be decreased by the inhibition of various enzymes of cholesterol biosynthesis.

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growth factor, and to increase the production of growth factors that improve vascular tone, such as endothelium-derived relaxing factor. Ex vivo gene therapy has been used to introduce functioning BE receptors in a patient with homozygous FH (Grossman et al., 1994), although it has been suggested that more stringent criteria be applied to evaluate the success of this intervention (Brown et al., 1994). The ex vivo procedure requires hepatic resection, but adenoviruses (Li et al., 1993) may be utilized in future in vivo techniques (Kozarsky et al., 1994) that may enable the therapeutic gene to be administered like a conventional pharmacologic agent. Currently, however, the usefulness of adenoviruses is limited by the transitory effect on gene expression with this delivery vector (Lemarchand et al., 1993). VI. Conclusion Pharmacologic therapy in patients with dyslipidemia can be tailored to the precise underlying lipid disorder by selecting among drugs with different mechanisms of action. Available agents decrease elevated levels of circulating lipoproteins by decreased synthesis, enhanced catabolism, and increased plasma clearance. Pharmacologic agents that alter lipoprotein levels have also demonstrated nonlipid effects, including alteration of oxidative potential and of coagulation parameters, that may play a major role in the decrease in CHD risk obtained with these agents in clinical trials. References Abbey, M., Clifton, P., Kestin, M., Belling, B., and Nestel, P. (1990). Effect of fish oil on lipoproteins, lecithin :cholesterol acyltransferase, and lipid transfer protein activity in humans. Arteriosclerosis 10, 85-94. Andersen, P., Smith, P., Seljeflot, I., Brataker, S., and Arnesen, H. (1990).Effects of gemfibrozil on lipids and haemostasis after myocardial infarction. Thromb. Haemost. 63, 174-1 77. Arad Y., Ramakrishnan, R., and Ginsberg, H. N. (1990). Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: Implications for the pathophysiology of apoB production. J. Lipid Res. 31, 567-582. Asano, T., Pollard, M., and Madsen, D. C. (1975). Effect of cholestyramine on 1,2dimethylhydrazine-induced enteric carcinoma in germfree rats. Proc. SOC. Exp. Biol. Med. 150,780-785. Assmann, G., and Schulte, H. (1992). Relation of high-density lipoprotein cholesterol and triglycerides to incidence of atherosclerotic coronary artery disease (the PROCAM experience). Am. J. Cardiol. 70, 733-737. Austin, M. A. (1989). Plasma triglyceride as a risk factor for coronary heart disease: The epidemiologic evidence and beyond. Am. /. Epidemiol. 129, 249-259. Austin, M. A. (1991). Plasma triglyceride and coronary heart disease. Arterioscler. Thromb. 11, 2-14.

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Austin, M. A., Breslow, J. L., Hennekens, C. H., Buring, J. E., Willett, W. C., and Gauss, R. M. (1988).Low-density lipoprotein subclasspatterns and risk of myocardial infarction. JAMA 260,1917-1921. Baker, S. G., Joffe, B. I., Mendelsohn, D., and Seftel, H. C. (1982).Treatment of homozygous familial hypercholesterolemia with probucol. S. Afr. Med. J. 62, 7-11. Bang, H. O., and Dyerberg, J. (1972).Plasma lipids and lipoproteins in Greenlandicwest coast Eskimos. Acta Med. Scand. 192, 85-94. Bauano, G., and Bazzano, G. S. (1972).Digitalis intoxication. Treatment with a new steroidbinding resin. JAMA 220, 828-830. Beil, F. U., Schrameyer-Wernecke,A., Beisiegel, U., Greten, H., Karkas, J. D., Liou, R., Alberts, A. W., Ekkardt, H. G., and Till, A. E. (1990). Lovastatin versus bezafibrate: Efficacy, tolerability, and effect on urinary mevalonate. Cardiology 77, Suppl. 4,22-32. Bergstrom, J. D., Kurtz, M. M., Rew, D. J., Amend, A. M., Karkas, J. D., Bostedor, R. G., Bansal, V. S., Dufresne, C., VanMiddlesworth, F. L., Hensens, 0. D., Liesch, J. M., Zink, D. L., Wilson, K. E., Onishi, J., Milligan, J. A., Bills, G., Kaplan, L., Nallin Omstead, M., Jenkins, R. G., Huang, L., Meinz, M. S., Quinn, L., Burg, R. W., Kong, Y. L., Mochales, S., Mojena, M., Martin, I., Pelaez, F., Diez, M. T., and Alberts, A. W. (1993). Zaragozic acids: A family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase. Proc. Natl. Acud. Sci. U.S.A. 90, 80-84. Berndt, J., Gaumert, R., and Still, J. (1978). Mode of action of the lipid-lowering agents, clofibrate and BM15075, on cholesterol biosynthesis in rat liver. Atherosclerosis 30, 147-152. Black, D. (1994).Atorvastatin: A step ahead for HMG-CoA reductase inhibitors. Atherosclerosis 109, 88-89. [Abstract] Blankenhorn, D. H., Nessim, S. A., Johnson, R. L., Sanmarco, M. E., Azen, S. P., and CashinHemphill, L. (1987).Beneficial effects of combined colestipol-niacintherapy on coronary atherosclerosis and coronary venous bypass grafts. ]AMA 257, 3233-3240. Blankenhorn, D. H., Alaupovic, P., Wickham, E., Chin, H. P., and Azen, S. P. (1990).Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts: Lipid and nonlipid factors. Circulation 81, 470-476. Blankenhorn, D. H., Azen, S. P., Kramsch, D. M., Mack, W. J., Cashin-Hemphill, L., Hodis, H. N., DeBoer, L. W. V., Mahrer, P. R., Masteller, M. J., Vailas, L. I., Alaupovic, P., Hirsch, L. J., and the MARS Research Group. (1993). Coronary angiographic changes with lovastatin therapy: The Monitored Atherosclerosis Regression Study (MARS).Ann. Intern. Med. 119, 969-976. Braden, G. A., Knapp, H. R., and FitzGerald, G. A. (1991).Suppressionof eicosanoid biosynthesis during coronary angioplasty by fish oil and aspirin. Clrcukation 84, 679-685. Bradford, R. H., Shear, C. L., Chremos, A. N., Dujovne, C., Downton, M., Franklin, F. A., Gould, A. L., Hesney, M., Higgins, J., Hurley, D. P., Langendorfer, A., Nash, D. T., Pool, J. L., and Schnaper, H. (1991).Expanded Clinical Evaluation of Lovastatin (EXCEL) Study results. I. Efficacy in modifying plasma lipoproteins and adverse event profile in 8245 patients with moderate hypercholesterolemia. Arch. Intern. Med. 15 1, 43-49. Bradford, R. H., Shear, C. L., Chremos, A. N., Dujovne, C. A., Franklin, F. A., Grillo, R. B., Higgins, J., Langendorfer,A., Nash, D. T., Pool, J. L., and Schnaper, H. (1994).Expanded Clinical Evaluation of Lovastatin (EXCEL) study results: Two-year efficacy and safety follow-up. Am. J. Cardiol. 74, 667-673. Brensike, J. F., Kelsey, S. F., Passamani, E. R., Fisher, M. R., Richardson, J. M., Loh, I. K., Stone, N. J., Aldrich, R. F., Battaglini, J. W., Moriarty, D. J., Myrianthopoulos, M. B., Detre, K. M., Epstein, S. E., and Levy, R. I. (1982). National Heart, Lung, and Blood Institute Type I1 Coronary Intervention Study: Design, methods, and baseline characteristics. Controlled Clin. Trials 3, 91-111. Brensike, J. F., Levy, R. I., Kelsey, S. F., Passamani, E. R., Richardson, J. M., Loh, I. K., Stone, N. J., Aldrich, R. F., Battaglini, J. W., Moriarty, D. J., Fisher, M. R., Friedman, L.,

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Friedewald, W., Detre, K. M., and Epstein, S. E. (1984).Effects of therapy with cholestyramine on progression of coronary arteriosclerosis: Results of the NHLBI Type 11Coronary Intervention Study. Circulation 69, 313-324. Brown, G., Albers, J. J., Fisher, L. D., Schaefer, S. M., Lin, J.-T., Kaplan, C., Zhao, X.-Q., Bisson, B. D., Fitzpatrick, V. F., and Dodge, H. T. (1990).Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. N. Engl. J. Med. 323, 1289-1298. Brown, M. S., Goldstein, J. L., Havel, R. J., and Steinberg, D. (1994). Gene therapy for cholesterol [letter]. Nut. Genet. 7, 349-350. Brown, W. V., Howard, W. J., and Field, L. (1991). Nicotinic acid and its derivatives. In “Drug Treatment of Hyperlipidemia” (B. M. Rifkind, Ed.), pp. 189-213. Marcel Dekker, New York. Buckley, M. M.-T., Goa, K. L., Price, A. H., and Brogden, R. N. (1989).Probucol: Areappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia. Drugs 37,761-800. Canner, P. L., Berge, K. G., Wenger, N. K., Stamler, J., Friedman, L., Prineas, R. J., and Friedewald, W., for the Coronary Drug Project Research Group. (1986). Fifteen year mortality in Coronary Drug Project patients: Long-term benefit with niacin. J. Am. Coil. Cardiol. 8, 1245-1255. Carew, T. E., Schwenke, D. C., and Steinberg, D. (1987).Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in uiuo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc. Natl. Acad. Sci. U.S.A. 84, 7725-7729. Carlson, L. A., and Rosenhamer, G. (1988).Reduction of mortality in the Stockholm Ischaemic Heart Disease Secondary Prevention Study by combined treatment with clofibrate and nicotinic acid. Actu Med. Scand. 223, 405-418. Carlson, L. A., Hamsten, A., and Asplund, A. (1989). Pronounced lowering of serum levels of lipoprotein Lp(a) in hyperlipidaemic subjects treated with nicotinic acid. J. Intern. Med. 226,271-276. Cashin-Hemphill, L., Mack, W. J., Pogoda, J. M., Sanmarco, M. E., h e n , S. P., and Blankenhorn, D. H. (1990).Beneficial effects of colestipol-niacin on coronary atherosclerosis: A 4-year follow-up. JAMA 264, 3013-3017. Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., and Pladet, M. W. (1993).Low density lipoprotein receptors bind and mediate cellular catabolism of normal very low density lipoproteins in uitro. J. Biol. Cbem. 268, 25487-25493. Charman, R. C., Matthews, L. B., and Braeuler, C. (1972).Nicotinic acid in the treatment of hypercholesterolemia: A long term study. Angiology 23, 29-35. Chung, B. H., Segrest, J. P., Smith, K., Griffin, F. M., and Brouillette, C. G. (1989). Lipolytic surface remnants of triglyceride-rich lipoproteins are cytotoxic to macrophages but not in the presence of high density lipoprotein: A possible mechanism of atherogenesis? J. Cltn. Invest. 83, 1363-1374. Committee of Principal Investigators. (1978). A co-operative trial in the primary prevention of ischaemic heart disease using clofibrate. Br. Heart J. 40, 1069-1118. Committee of Principal Investigators. (1984). WHO cooperative trial on primary prevention of ischaemic heart disease with clofibrate to lower serum cholesterol: Final mortality follow-up. Lancet 2, 600-604. Constantino, M., Merskey, C., Kudzma, D. J., and Zucker, M. B. (1977). Increased activity of vitamin K-dependent clotting factors in human hyperlipoproteinaemia-association with cholesterol and triglyceride levels. Tbromb. Huemost. 38, 465-474. Coronary Drug Project Research Group. (1975).Clofibrate and niacin in coronary heart disease. JAMA 231, 360-381.

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Corpier, C. L., Jones, P. H., Suki, W. N., Lederer, E. D., Quinones, M. A., Schmidt, S. W., and Young, J. B. (1988). Rhabdomyolysis and renal injury with lovastatin use. Report of two cases in cardiac transplant recipients. JAMA 260, 239-241. Crutchley, D. J., McPhee, G. V., Terris, M. F., and Canossa-Terris, M. A. (1989). Levels of three hemostatic factors in relation to serum lipids. Monocyte procoagulant activity, tissue plasminogen activator, and type-1 plasminogen activator inhibitor. Arteriosclerosis 9,934-939. Dachet, C., Jacotot, B., and Buxtorf, J. C. (1985).The hypolipidemic action of probucol: Drug transport and lipoprotein composition in type IIa hyperlipoproteinemia. Atherosclerosis 58,261-268. Davignon, J. (1994).Fibrates: A review of important issues and recent findings. Can. J. Cardiol. 10, SUPPIB, 61B-71B. Dolecek, T. A. (1992).Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial. Proc. SOL. Exp. Biol. Med. 200, 177-182. Dujovne, C. A., Atkins, F., Wong, B., DeCoursey, S., Krehbiel, P., and Chernoff, S. B. (1984). Electrocardiographic effects of probucol. A controlled prospective clinical trial. Eur. J. Clin. Pharmacol. 26, 735-739. Eisenberg, S., Gavish, D., Oschry, Y., Fainaru, M., and Deckelbaum, R. J. (1984).Abnormalities in very low, low, and high density lipoproteins in hypertriglyceridemia: Reversal toward normal with bezafibrate treatment. J. Clin. Invest. 74, 470-482. Endo, A., Kuroda, M., and Tsujita, Y. (1976). ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J. Antibiot. (Tokyo) 29, 1346-1348. Eto, M., Sato, T., Watanabe, K., Iwashima, Y., and Makino, I. (1990). Effects of probucol on plasma lipids and lipoproteins in familial hypercholesterolemic patients with and without apolipoprotein E4. Atherosclerosis 84, 49-53. Forland, S. C., Feng, Y., and Cutler, R. E. (1990).Apparent reduced absorption of gemfibrozil when given with colestipol. J. Clin. Pharmacol. 30, 29-32. Franceschini, G., Sirtori, M., Gianfranceschi, G., Frosi, T., Montanari, G., and Sirtori, C. R. (1985).Reversible increase of the apo CIYapo CIII-1 ratio in the very low density lipoproteins after procetofen treatment in hypertriglyceridemic patients. Artery 12, 363-38 1. Franceschini, G., Sirtori, M., Vaccarino, V., Gianfranceschi, G., Rezzonico, L., Chiesa, G., and Sirtori, C. R. (1989).Mechanisms of HDL reduction after probucol: Changes in HDL subfractions and increased reverse cholesteryl ester transfer. Arteriosclerosis 9,462-469. Frick, M. H., Elo, O., Haapa, K., Heinonen, 0. P., Heinsalmi, P., Helo, P., Huttunen, J. K., Kaitaniemi, P., Koskinen, P., Manninen, V., Maenpaa, H., Miilkonen, M., Manttari, M., Norola, S., Pasternack, A., Pikkarainen, J., Romo, M., Sjoblom, T., and Nikkila, E. A. (1987). Helsinki Heart Study: Primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N. Engl. J. Med. 317, 1237-1245. Gallo, D. G., Bailey, K. R., and Sheffner, A. L. (1965).The interaction between cholestyramine and drugs. Proc. SOL. Exp. Biol. Med. 120, 60-65. Gass, J. D. (1973).Nicotinic acid maculopathy. Am. J. Ophthalmol. 76, 500-510. Gianturco, S. H., Bradley, W. A., Nozaki, S., Vega, G. L., and Grundy, S. M. (1993).Effects of lovastatin on the levels, structure, and atherogenicity of VLDL in patients with moderate hypertriglyceridemia. Arterioscler. Thromb. 13, 472-481. Gilligan, D. M., Guetta, V., Panza, J. A., Garcia, C. E., Quyyumi, A. A., and Cannon, R. O., 111. (1994).Selective loss of microvascular endothelial function in human hypercholesterolemia. Circulation 90, 35-41. Glueck, C. J., Oakes, N., Speirs, J., Tracy, T., and Lang, J. (1992). Gemfibrozil-lovastatin therapy for primary hyperlipoproteinemias. Am. J. Cardiol. 70, 1-9.

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Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B., and Dawber, T. R. (1977).High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am. J. Med. 62, 707-714. Gotto, A. M., Jr. (1994).Postmenopausal hormone-replacement therapy, plasma lipoprotein[a], and risk for coronary heart disease [editorial]. J. Lab. Clin. Med. 123, 800. Granfone, A., Campos, H., McNamara, J. R., Schaefer, M. M., Lamon-Fava, S., Ordovas, J. M., and Schaefer, E. J. (1992).Effects of estrogen replacement on plasma lipoproteins and apolipoproteins in postmenopausal, dyslipidemic women. Metabolism 41, 11931198. Grossman, M., Raper, S. E., Kozarsky, K., Stein, E. A., Engelhardt, J. F., Muller, D., Lupien, P. J., and Wilson, J. M. (1994). Successful ex vim gene therapy directed to liver in a patient with familial hypercholesterolaemia. Nut. Genet. 6, 335-341. Grundy, S. M. (1991).HMG CoA reductase inhibitors: Clinical applications and therapeutic potential. In “Drug Treatment of Hyperlipidemia” (B. M. Rifkind, Ed.), pp. 139-167. Marcel Dekker, New York. Hibbard, D. M., Peters, J. R., and Hunninghake, D. B. (1984). Effects of cholestyramine and colestipol on the plasma concentrations of propranolol. Br. J . Clin. Phannacol. 18,337-342. Hidaka, Y., Hotta, H., Nagata, Y., Iwasawa, Y., Horie, M., and Kamei, T. (1991). Effect of a novel squalene epoxidase inhibitor, NB-598, on the regulation of cholesterol metabolism in Hep G2 cells. J. Biol. Chem. 266, 13171-13177. Hunninghake, D. B., King, S., and LaCroix, K. (1982). The effect of cholestyramine and colestipol on the absorption of hydrochlorothiazide. Int. J. Clin. Phannacol. Ther. Toxicol. 20,151-154. Huttunen, J. K., Manninen, V., Manttari, M., Koskinen, P., Romo, M., Tenkanen, L., Heinonen, 0. P., and Frick, M. H. (1991).The Helsinki Heart Study: Central findings and clinical implications. Ann. Med. 23, 155-159. Illingworth, D. R. (1991). Fibric acid derivatives. In “Drug Treatment of Hyperlipidemia” (B. M. Rifkind, Ed.), pp. 103-138. Marcel Dekker, New York. Illingworth, D. R., and Tobert, J. A. (1994).A review of clinical trials comparing HMG-CoA reductase inhibitors. Clin. Ther. 16, 366-385. Isaacsohn, J. L., Setaro, J. F., Nicholas, C., Davey, J. A., Diotalevi, L. J., Christianson, D. S., Liskov, E., Stein, E. A., and Black, H. R. (1994).Effects of lovastatin therapy on plasminogen activator inhibitor-1 antigen levels. Am. J. Cardiol. 74, 735-737. Kannel, W. B. (1987).Metabolic risk factors for coronary heart disease in women: Perspective from the Framingham Study. Am. Heart J. 114,413-419. Kaplan, R., Aynedjian, H. S., Schlondorff, D., and Bank, N. (1990). Renal vasoconstriction caused by short-term cholesterol feeding is corrected by thromboxane antagonist or probucol. J. Clin. Invest. 86, 1707-1714. Kesaniemi, Y.A., and Grundy, S. M. (1984).Influence of probucol on cholesterol and lipoprotein metabolism in man. J. Lipid Res. 25, 780-790. Kirby, T. J. (1967). Cataracts produced by triparanol. Trans. Am. Ophthalmol. SOC.65, 493-543. Kostner, G. M., Gavish, D., Leopold, B., Bolzano, K., Weintraub, M. S., and Breslow, J. L. (1989). HMG CoA reductase inhibitors lower LDL cholesterol without reducing Lp(a) levels. Circulation 80, 1313- 1319. Kozarsky, K. F., McKinley, D. R., Austin, L. L., Raper, S. E., Stratford-Perricaudet, L. D., and Wilson, J. M. (1994).In uiuo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses. I. B i d . C h m . 269, 13695-13702. Krause, B. R., Pape, M. E., Kieft, K., Auerbach, B., Bisgaier, C. L., Homan, R., and Newton, R. S. (1994). ACAT inhibition decreases LDL cholesterol in rabbits fed a cholesterol-

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free diet: Marked changes in LDL cholesterol without changes in LDL receptor mRNA abundance. Arterioscler. Thromb. 14, 598-604. Langer, T., and Levy, R. I. (1968). Acute muscular syndrome associated with administration of clofibrate. N. Engl. J. Med. 279, 856-858. Lehr, H. A., Hubner, C., Nolte, D., Kohlschutter, A., and Messmer, K. (1991). Dietary fish oil blocks the microcirculatory manifestations of ischemia-reperfusion injury in striated muscle in hamsters. Proc. Natl. Acud. Sci. U.S.A. 88, 6726-6730. Lemarchand, P., Jones, M., Yamada, I., and Crystal, R. G. (1993).In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ. Res. 72, 1132-1 138. Levy, R. I., Morganroth, J., and Rifkind, B. M. (1976).Treatment of hyperlipidemia. N. Engl. J. Med. 290, 1295-1301, Li, Q., Kay, M. A., Finegold, M., Stratford-Perricaudet, L. D., and Woo, S. L. (1993).Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum. Gene Tber. 4,403-409. Lipid Research Clinics Program. (1984a).The Lipid Research Clinics Coronary Primary Prevention Trial results. I. Reduction in incidence of coronary heart disease.JAMA 251,351-364. Lipid Research Clinics Program. (1984b).The Lipid Research Clinics Coronary Primary Prevention Trial results. II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 251, 365-374. Litin, S. C., and Anderson, C. F. (1989).Nicotinic acid-associated myopathy: A report of three cases. Am. J. Med. 86, 481-483. Loscalzo, J. (1990).Lipoprotein(a): A unique risk factor for atherothrombotic disease. Arteriosclerosis 10, 672-679. MAAS Invesrigators. (1994). Effect of simvastatin on coronary atheroma: The Multicentre Anti-Atheroma Study (MAAS). Lancet 344, 633-638. Manninen, V., Tenkanen, L., Koskinen, P., Huttunen, J. K., Manttari, M., Heinonen, 0. P., and Frick, M. H. (1992). Joint effects of serum triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study: Implications for treatment. Circulation 85, 37-45. Millay, R. H., Klein, M. L., and Illingworth, D. R. (1988).Niacin maculopathy. Ophthalmology 95, 930-936. Moore, R. B., Crane, C. A., and Frantz, I. D., Jr. (1968).Effect of cholestyramine on the fecal excretion of intravenously administered chole~terol-4-~~C and its degradation products in a hypercholesterolemic patient. J. Clin. Invest. 47, 1664-1671. Morris, M. C., Sacks, F., and Rosner, B. (1993).Does fish oil lower blood pressure? A metaanalysis of controlled trials. Circulation 88, 523-533. Mullin, G. E., Greenson, J. K., and Mitchell, M. C. (1989). Fulminant hepatic failure after ingestion of sustained-release nicotinic acid. Ann. Intern. Med. 111, 253-255. National Cholesterol Education Program. (1994).Second report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel 11). Circulation 89, 1329-1445. Nestel, P. J., Connor, W. E., Reardon, M. F., Connor, S., Wong, S., and Boston, R. (1984). Suppression by diets rich in fish oil of very low density lipoprotein production in man. J. Clin. Invest. 74, 82-89. Newman, W. P., Middaugh, J. P., Propst, M. T., and Rogers, D. R. (1993). Atherosclerosis in Alaska Natives and non-natives. Lancet 341, 1056-1057. Nikkila, E. A., Huttunen, J. K., and Ehnholm, C. (1977). Effect of clofibrate on postheparin plasma triglyceride lipase activities in patients with hypertriglyceridemia. Metabolism 26,179-186. Patsch, J. R., Miesenbock, G., Hopferwieser, T., Muhlberger, V., Knapp, E., Dunn, J. K., Gotto, A. M., Jr., and Patsch, W. (1992).Relation of triglyceride metabolism and coronary artery disease: Studies in the postprandial state. Arterioscler. Thromb. 12, 1336-1345.

Current and Future Therapeutic Approaches to Hyperlipidemia

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Pierce, L. R., Wysowski, D. K., and Gross, T. P. (1990). Myopathy and rhabdomyolysis associated with lovastatin-gemfibrozil combination therapy. JAMA 264, 71-75. Pitt, B., Mancini, G. B. J., Ellis, S. G., Rosman, H. S., Park, J. -S., and McGovern, M. E., for the PLAC I Investigators. (1995).Pravastatin Limitation of Atherosclerosis in the Coronary Arteries (PLAC I) reduction in atherosclerosis progression and clinical events. J. Am. CON. Cardiol. 26, 1133-1139. Rader, J. I., Calvert, R. J., and Hathcock, J. N. (1992). Hepatic toxicity of unmodified and time-release preparations of niacin. Am. J. Med. 92, 77-81. Reaven, P., and Witztum, J. L. (1988).Lovastatin, nicotinic acid, and rhabdomyolysis [letter]. Ann. Intern. Med. 109, 597-598. Scandinavian Simvastatin Survival Study Group. (1994).Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4s).Lancet 344, 1383-1389. Schmidt, E. B., Illingworth, D, R., Bacon, S., Russell, S. J., Thatcher, S. R., Mahley, R. W., and Weisgraber, K. H. (1993). Hypolipidemic effects of nicotinic acid in patients with familial defective apolipoprotein B-100. Metabolism 42, 137-139. Shepherd, J., Packard, C. J., Patsch, J. R., Gotto, A. M., Jr., and Taunton, 0. D. (1979). Effects of nicotinic acid therapy on plasma high density lipoprotein subfraction distribution and composition and on apolipoprotein A metabolism. J. Clin. Invest. 63, 858-867. Shepherd, J., Cobbe, S. M., Ford, I., Isles, C. G., Lorimer, A. R., Macfarlene, P. W., McKillop, J. H., Packard, C. J., for the West of Scotland Coronary Prevention Study Group. (1995). Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N.Engl. 1.Med. 333,1301-1307. Simpson, H. S., Williamson, C. M., Olivecrona, T., Pringle, S., Maclean, J., Lorimer, A. R., Bonnefous, F., Bogaievsky, Y., Packard, C. J., and Shepherd, J. (1990). Postprandial lipemia, fenofibrate and coronary artery disease. Atherosclerosis 85, 193-202. Sirtori, C. R., Calabresi, L., Werba, J. P., and Franceschini, G. (1992). Tolerability of fibric acids. Comparative data and biochemical bases. Pharmacol. Res. 26, 243-260. Sliskovic, D. R., and White, A. D. (1991).Therapeutic potential of ACAT inhibitors as lipid lowering and anti-atherosclerotic agents. Trends Pharmacol. Sci. 12, 194-199. Soma, M. R., Osnago-Gadda, I., Paoletti, R., Fumagalli, R., Morrisett, J. D., Meschia, M., and Crosignani, P. (1993).The lowering of lipoprotein[a] induced by estrogen plus progesterone replacement therapy in postmenopausal women. Arch. Intern. Med. 153, 14621468. Spach, D. H., Bauwens, J. E., Clark, C. D., and Burke, W. G. (1991).Rhabdomyolysis associated with lovastatin and erythromycin use. West J. Med. 154, 213-216. Tilly-Kiesi, M., and Tikkanen, M. J. (1991).Low density lipoprotein density and composition in hypercholesterolaemic men treated with HMG CoA reductase inhibitors and gemfibrozil. J. Intern. Med. 229, 427-434. Tobert, J. A. (1988).Efficacy and long-term adverse effect pattern of lovastatin. Am. J. Cardiol. 62,285-345. Tobert, J. A., Shear, C. L., Chremos, A. N., and Mantell, G. E. (1990). Clinical experience with lovastatin. Am. J. Cardiol. 65, 23F-26F. Todd, P. A., and Ward, A. (1988).Gemfibrozil: A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in dyslipidaemia. Drugs 36, 314-339. Treasure, C. B., Klein, J. L., Weintraub, W. S., Talky, J. D., Stillabower, M. E., Kosinski, A. S., Zhang, J., Boccuzzi, S. J., Cedarholm, J. C., and Alexander, R. W. (1995).Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N. Engl. 1. Med. 332,481-487. Wada, H., Mori, Y., Kaneko, T., Wakita, Y., Nakase, T., Minamikawa, K., Ohiwa, M., Tamaki, S., Tanigawa, M., Kageyama, S., Deguchi, K., Nakano, T., Shirakawa, S., and Suzuki, K. (1993).Elevated plasma levels of vascular endothelial cell markers in patients with hypercholesterolemia. Am. 1.Hematol. 44, 112-1 16.

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Wagner, J. D., St Clair, R. W., Schwenke, D. C., Shively, C. A., Adams, M. R., and Clarkson, T. B. (1992).Regional differencesin arterial low density lipoprotein metabolism in surgically postmenopausalcynomolgus monkeys. Effects of estrogen and progesterone replacement therapy. Arterioscler. Thromb. 12, 717-726. Walldius, G., Regnstrom, J., Nilsson, J., Johansson, J., Schnfer-Hinder, L., Moelgaard, J., The role of lipids and antioxidative Hadell, K., Olsson, A. G., and Carlson, L. A. (1993). factors for development of atherosclerosis.The Probucol Quantitative RegressionSwedish Trial (PQRST).Am. J. Curdiol. 71, ISB-19B. Walldius, G., Erikson, U., Olsson, A. G., Bergstrand, L., Hidell, K., Johansson, J., Kaijser, L., Lassvik, C., Molgaard, J., Nilsson, S., Schafer-Elinder,L., Stenport, G., and Holme, I. (1994).The effect of probucol on femoral atherosclerosis: The Probucol Quantitative Regression Swedish Trial (PQRST). Am. J. Cardiol. 74, 875-883. Waters, D.,Higginson, L., Gladstone, P., Kimball, B., Le May, M., Boccuzzi, S. J., Lesptrance, M., and the CCAIT Study Group. (1994).Effects of monotherapy with an HMG-CoA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantitative arteriography: The Canadian Coronary Atherosclerosis Intervention Trial. Circulation 89, 959-968. Watts, G. F., Lewis, B., Brunt, J. N. H., Lewis, E. S., Coltart, D. J., Smith, L. D. R., Mann, J. I., and Swan, A. V. (1992).Effects on coronary artery disease of lipid-lowering diet, or diet plus cholestyramine, in the St Thomas’ AtherosclerosisRegression Study (STARS). Lancet 339,563-569. Wiklund, O., Angelin, B., Bergman, M., Berglund, L., Bondjers, G., Carlsson, A., Lindtn, T., Miettinen, T., Odman, B., Olofsson, S.-O., Saarinen, I., Sip&, R., Sjbstrom, P., Kron, B., Vanhanen, H.,and Wright, I. (1993). Pravastatin andgemfibrozilalone and incombination for the treatment of hypercholesterolemia. Am. J. Med. 94, 13-20. Writing Group for the PEP1 trial. (1995).Effects of estrogen or estrogedprogestin regimens on heart disease risk factors in postmenopausal women: The Postmenopausal Estrogen/ Progestin Interventions (PEPI) trial. JAMA 273, 199-208. Yamamoto, A., Matsuzawa, Y., Yokoyama, S., Funahashi, T., Yamamura, T., and Kishino, B. -I. (1986).Effects of probucol on xanthomata regression in familial hypercholesterolemia. Am. J. Curdiol. 57, 29H-35H. Ying, H., Saku, K., Harada, R., Takami, N., Sasaki, N., Saito, Y., and Arakawa, K. (1990). Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits. Biochim. Biophys. Acta 1047,247-254. Zilversmit, D. B. (1979). Atherogenesis: A postprandial phenomenon. Circulation 60,473-485.

Jean F. Borel Gotz Baumann Ian Chapman Peter Donatsch Alfred Fahr Edgar A. Mueller Jean-Marie Vigouret Sandoz Pharma AG, Preclinical Research Division, CH-4002 Basel Switzerland

In Vivo Pharmacological Effects of Ciclosporin and Some Analogues

1. Introduction and Summary

The former use of unspecific cytostatic or lymphocytotoxic agents to suppress the immune response is today being replaced by more specific, i.e., immunopharmacological, approaches. The powerful immunosuppressiveactivity of ciclosporin (CS), which is apparently restricted at the cellular level of certain T lymphocytes, is recognized as a breakthrough in immunopharmacology (Borel et al., 1989, 1995). The chemical structure of CS is shown in Fig. 1. Because of its potent inhibition of the antibody- and cell-mediated immune response, CS is now being used as the mainstay in clinical immunosuppression (Evans et al., 1993). After a difficult learning process in which the efficacy and the side effects of the drug were explored, CS has proved to be of permanent clinical value and has, in consequence, revitalized the field of organ transplantation. Advances in Pharmacology, Volume 35 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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10

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9

11

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t

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D-Ala-

Ala

8

7

3

:

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FIGURE I Structure of ciclosporin (cyclosporin A, a cyclic undecapeptide)corresponding to the confirmation observed in the crystal. (From Prog. Allergy 38,48, 1986. By permission).

CS is a potent inhibitor of chronic or immune-mediated inflammatory reactions, but has no effect in models of acute inflammation. Its importance for exploring the pathogenesis and treatment of some acute and chronic inflammatory diseases, generally grouped as suspected autoimmune.diseases, is clearly emerging in the clinic. The use of CS in the treatment of several such diseases has revealed its potential to counteract the imbalance of the immune system by improving the condition of both experimental animals and patients. CS can induce and maintain remission in many autoimmune diseases, but discontinuation of therapy usually results in a relapse, because CS does not apparently modify the underlying immunopathogenic mechanism causing autoimmunity. At the present time, the efficacy and the clinical benefit of CS therapy have been conclusively demonstrated for severely affected patients in four diseases: autoimmune uveitis, psoriasis, idiopathic nephrotic syndrome, and rheumatoid arthritis (Feutren, 1992; Borel et al., 1994). In contrast to classical immunosuppressants, CS exerts a specific action on lymphocytes but is not detrimental to the functions of phagocytes or hemopoietic stem cells. It is neither lymphocytotoxic, as its action is revers-

In Vivo Pharmacological Effects of Ciclosporin and Some Analogues

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ible, nor mutagenic. Nephrotoxicity, its major side effect, can mainly be minimized by drug combinations, especially in the early stages, or by dose reduction during the maintenance phase. The prospects of refining protocols further and thus improving the results and avoiding the other more trivial side effects remain reasonably optimistic and widen the scope of immunosuppression (Feutren and von Graffenried, 1992; Feutren and Mihatsch, 1992; Kahan 1993). Besides being an immunosuppressive drug, CS is also widely used as an experimental probe in basic research. Thus, for immunologists, it has become an important pharmacological tool for defining in vitro the respective roles of cell interactions and mediators in lymphocyte activation, analyzing the cell regulatory mechanism of the genes, and studying the different steps involved generally in an immune response (Bierer 1994; Bore1 et al., 1989). The resultant fairly coherent, though still incomplete, working model that is emerging from in vitro studies essentially proposes that CS causes immunosuppression by preventing the activation of resting lymphocytes at an early stage of the cell cycle (Goto GI transition), thus inhibiting primarily the production and release of interleukin-2 and other lymphokines by helper T cells (cf. reviews by Fruman et al., 1994a; Liu, 1993; Baumann 1992; Baumann et al., 1991). The details are presented in Figs. 2 and 3 and described in Section I1 (Molecular mechanism of immunosuppression). Immunophilin:

Cyclophilin \

Ciclosporin

lmmunophilin binding domain

Effector domain

Effector:

Calcineurin (+ Calmodulin, Ca++)

4

Inhibition of ‘Early’ Gene Transcription

FIGURE 2 by CS.

Dual domain concept for the molecular mechanism of immunosuppression

APC

+ RNA pol II

‘Early’ Genes

3

r

T Cell FIGURE 3 General overview of T cell signaling pathways following ligation of the T cell receptor (TCR)or the IL-2 receptor (IL-2R). Mode of action of CS. In the presence of costimulatory signals provided by multiple cell surface receptorfligand interactions (e.g., CD28/B7-1, 2; LFA-l/ICAM; CD2LFA-3; CD45/CD22; IL-1ML-I) antigen (AG) presented by an antigenpresenting cell (APC) in the context of major histocompatibility complex (MHC) proteins induces a T cell differentiation process that results ultimately in lymphokine secretion (Goto GI transition of cell cycle) and proliferation (GI to S) of the antigen-specific T cell. Signal transduction involves a number of biochemical events, including phosphorylation and dephosphorylation (P) of tyrosine, threonine, or serine residues of intracellular signaling proteins, leading to gene transcription of “early” genes like IL-2. CS exerts its immunosuppressive effects downstream from the very early membrane-associated events, such as activation of phospholipase C-y (PLC-y) and recruitment of tyrosine kinases (e.g., LCK, FYN, CSK, ZAP70) and SHC to the “Immunoreceptor Tyrosine-based Activation Motifs” (ITAM) in the E and I;-chains of the T cell receptor (TCR)/CD3complex. It is hypothesized that cyclophilin) phosphatase activity of calcineurin (CN-A, bound CS inhibits the calcium-dependent (CaZ+ -B, and Cam, Calmodulin) as a crucial step in the activation ( * ) and nuclear translocation of cytoplasmic transcription factor subunits like NF-AT, and/or NF-KBrequired for “early” gene transcription (see text for details). IL-2R-mediated signaling processes involving activation of JAK-kinases, STATs (SignalTransducers and Activators of Transcription), RAFTS(Rapamycin ~ ~not ) affected. and FKBP Target), and p70 S6 kinase ( ~ 7 0 ”are

In Vivo Pharm'acological Effects of Ciclosporin and Some Analogues

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While evidence for this concept is compelling, various phenomena in vivo are not readily explained by this model. This contradiction first led Klaus and Chisholm (1986) to ask the seminal question: Does CS act in vivo as it does in vitro? Several reports have clearly underlined the importance of this unresolved problem (e.g., Prud'homme and Vanier, 1993; Black et al., 1988; Fukuzawa and Shearer, 1989; Lim et al., 1987; Klaus and Dongworth, 1982). This review will show that in vitro several pathways can be used to induce gene transcription in lymphocytes. Whether the alternative pathways, i.e., those bypassing the T-cell receptor, are also relevant in vivo remains another question. However, only the pathways involving mobilization of intracellular calcium are CS-sensitive. In addition, it seems reasonable to anticipate that, if such pathways are functional in cells other than T cells, they might also be susceptible to CS, as will be discussed in this review. It has become evident that several findings obtained in vitro under well-defined conditions do not directly apply to the extremely complex situation encountered in an animal. Although necessary and often helpful, ex vivo results may also be misleading, because the experimental reactions are occurring outside the organism, i.e., under conditions which differ widely from those existing within a living organism, where CS as well as many other crucial factors may be present. We are fully aware of these major, almost insurmountable difficulties; therefore, wherever necessary, we have also considered related in vitro results and compared them with in vivo or ex vivo findings. We shall nevertheless present a tentative hypothesis which tries to unify a number of results that have been discussed in the literature (for review cf., Borel, 1989). This scheme (Fig. 4) should be considered as food for further thought and not as a conclusive statement. We are concentrating our attention on data obtained from experiments relevant to long-term immunosuppression, as for example those used for studying allograft protection and treatment of autoimmunity. This review presents more recent findings and we shall arrive at the conclusion that these findings do strengthen this hypothesis. The main interest in CS was at first almost exclusively focused on its immunosuppressive potential. However, the pharmacological spectrum of CS, the 25 natural cyclosporins, and their some 2000 derivatives is astonishingly widespread. This fact was recognized already in the later 70s as shown in Fig. 5. In spite of the very rudimentary knowledge of the pharmacological properties of many of these compounds at that time, it became soon evident that, surprisingly, none of the cyclosporins and their analogues possessed significantly greater immunosuppressive potency than CS in either in vitro tests or in vivo models. Only few natural cyclosporins, namely CS, (Thrz)-CS(cyclosporin C), ( Valz)-CS (cyclosporin D), (Nvalz)-CS (cyclosporin G), and (Nva2,7-CS (cyclosporin M), have been found to exert potent in vivo effects, even though several derivatives

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Jean F. Bore1 et al. function of APC (MHC+AG)

4 4

priming TH

TS

activation (MHCexpr., IL-2R) rH

).

S '

proliferation T'CP

activation

4 TC

s +W k rfunction 4\ TH memory

proliferation

4

' e

TDTHp

function (Z'response)

activation

&

function

T

activation

4

B'

Ab-production (1' response)

6 TI-1 4 k

cytolysis

4

NK cell activity

Z0 response

FIGURE 4 Mechanism of action of ciclosporin in vivo: A unifying hypothesis. APC, antigenpresenting cell; MHC, major histocompatability complex; AG, antigen; T,, T helper cell; T,, T suppressor cell; IL-2R, interleukin-2 receptor; T', B', activated cells; Tcp,cytotoxic T precursor; T,, cytotoxic T cell; TDmp precursor of TDmcell; TDW,T cell mediating delayed type hypersensitivity; Ab, antibody; TD, thymus-dependent antigens; TI, thymus-independent antigens; IL, interleukin; NK,natural killer cell. Black boxes indicate CS-sensitive steps; all other steps are presumably CS resistant. (From Phannacol. Rev. 41,283, 1989. By permission.)

do elicit appreciable in vitro activity. It is known today that the CS molecule acts via two regulatory domains: an immunophilin-binding domain, which includes the unusual C9-amino acid (MeBmt chain), and

Side Etteas (Organ dysfunctions)

c3

Fungicidal A

Anti-parasitic

U

Pharmacologlcal

Effects of Cyclosporin A

Humoral and CellMediated Immunity

Anti-inflammatory Chronic, i.e. ImmuneMediated Inflammation

FIGURE 5 Ciclosporin exhibits different pharmacological properties. However, its most potent and biologically unique activity is the immunosuppressive effect mediated through inhibition of immunocompetent T lymphocytes. (From Transplant. Proc. 15, 1882, 1983. By permission.)

In Vivo Pharmacological Effects of Ciclosporin and Some Analogues

I21

TABLE I Pharmacological Spectrum of the Cyclosporins: Immunosuppressive Activity and Other Biological Effects Linked with it Inhibition of gene transcription for several lymphokines Anti-inflammatory effects in chronic inflammation Prolactin Antagonism Major side effects: nephrotoxicity and hypertension

an effector domain, which is intimately involved with suppression (Fig. 2). Since our intensive and long-lasting quest for an analogue with much higher biological potency and significantly less side effects, in particular nephrotoxicity and hypertension, has been disappointing, it would appear from our numerous collected data that immunosuppression might be, at least to a major extent, linked with these side effects. Both the natural (Nvalz)-CS(cyclosporin G or SDZ OG 37-325) (for review cf. Hiestand et al., 1994) and the hydroxyethyl derivative of (D-serineE)-CS(SDZ IMM 125), which is of similar potency as CS, but shows a clearly reduced toxicity in the rat (Hiestand et al., 1992), were also tested clinically. However, their development was discontinued mainly due to lack of superiority over CS or unexpected hepatotoxicity in man. With our present knowledge of the wide pharmacological spectrum of the cyclosporins, the basic concept of Fig. 5 can be extended by considering on the one hand the immunosuppressive activity proper and additional effects which are directly or seemingly associated with it, and on the other hand all the other effects which appear to be separable and independent from the immunosuppressive activity (Tables I to 111). This distinction of the varied biological properties of this rich class of compounds reveals their

TABLE II Pharmacological Spectrum of the Cyclosporins: Biological Effects Which may be Correlated with the Immunosuppressive Activity Development of suppressor cells Therapeutic effects in psoriasis Therapeutic effects in asthma? Prevention of chronic rejection Inhibition of apoptosis Effects on hair follicles

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usefulness as research tools as well as their potential for further developments in several therapeutic areas. We will review the available evidence in support of each of these different claims. Since a high variability among different patients is observed in clinical use of the current market form SandimmunB (CS),despite careful monitoring of blood levels and similar histoincompatibility, Masy et al. (1994) have compared the immune response of peripheral blood mononuclear cells from healthy subjects in the presence of CS. They observed marked individual differences in sensitivity when measuring in vitro the inhibition of lymphocyte proliferation and interleukin-2 production. The half inhibitory concentrations (ICs0)varied from less than 200 ng/ml in the responder group (61% of subjects) to sometimes over 1 pg/ml in nonresponders (19%). Although these findings would rather favor a correlation between in vitro and in vivo, it should be noted that much smaller CS concentrations, compared with the blood levels from CS-treated patients, would suffice in vitro to completely abrogate the same lymphocyte functions. Batiuk et al. (1995b)have recently made the important observation that the induction of cytokine expression measured ex vivo in lymphocytes of patients on CS was not blocked at therapeutic drug levels, despite the ability of CS to inhibit calcium-triggered signal transduction in isolated lymphocytes. Finally, Batiuk et al. (1995a) have reported that in peripheral blood lymphocytes, recovery from CS was slow and limited in vitro, but rapid in vivo, where CS equilibrates among a complex set of extralymphocytic binding sites. In addition, they observed

TABLE 111 Pharmacological Spectrum of the Cyclosporins: Other Biological Effects Which Are Separable from the Immunosuppressive Activity Effects on nonlymphoid cells Antigen-presentingcells Granulocytes Keratinocytes Other cells Effects on various cellular functions Reversal of multidrug resistance Selective formylpeptide receptor antagonism Inhibition of exocytosis Blocking of cyclophilin isomerase activity Antibiotic effects Fungicidal effect Insecticidal effect ( ? ) Antiparasitic effect Inhibition of HIV-1 replication

In Vivo Pharmacological Effects of Ciclosporin and Some Analogues

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that CS inhibition of leukocyte calcineurin was at least an order of magnitude less in whole blood than in culture medium (Batiuk et al. 1995b). This would explain why patients on CS can still mount immune responses which may be desirable for preventing viral infections but not in view of the danger of graft rejection. It seems that the maintenance dosing of CS is at the threshold for signaling by marginal stimuli, while still leaving the potential for vigorous responses to strong stimuli. Clinical observations confirm the assumption that the transplanted patients are, at least during the early posttransplant period, not optimally immunosuppressed with the present protocols, in which CS is the major immunosuppressive drug, because the rate of acute rejection at 1 year for renal allograft recipients is as high as 65% (Lindholm et al., 1993; Tesi et al., 1994). The same rate varies in liver transplant patients between 50 and 76% (Eur. FK506 Multicenter Liver Study Group 1994 andU.S. Multicenter FK506 Liver Study Group 1994, respectively) and in cardiac transplants it peaks at 87% (Grattan et al., 1990). However, an increase of the CS dose is not practicable in patients due. to the limiting side effects, although CS itself would be potent enough as can be demonstrated in many species, such as rodents, pigs, dogs, monkeys etc., in which these side effects, in particular nephrotoxicity, are not encountered. Therefore, the missing immunosuppressive potency has to be supplemented by the addition of a compatible and efficacious adjunctive agent; however, this remains a major challenging clinical problem. A major progress has recently been achieved with the development of a novel galenical formulation for oral administration of CS (Sandimmun Neoral). This new formulation is based on the microemulsion technology and should significantly improve dose linearity in CS exposure as well as provide a more consistent absorption profile compared with the current market form of CS. Thus, the risk of over- and underdosing patients should be much reduced and there should also be a stronger correlation between trough CS levels and total drug exposure (Kovarik et al., 1994c,d,e; Mueller et al., 1994a,c,d). This is critical for a drug with a narrow therapeutic window and which has to be administered long term. The previous major review by Borel et al. (1989) summarized the state of the art on CS up to the end of 1989. The goal of the present review is to focus mainly on more recent in vivo biological effects and to address unresolved problems and new developments with the cyclosporins in basic research as well as in selected clinical indications (cf., Moller, 1988; Borel et al., 1989). However, this review is not restricted to CS and immunosuppression, but rather includes derivatives and many of their various biological properties which may or may not be linked with immunosuppressive effects. Emphasis is given to recent advances concerning the use of cyclosporins either as biological probes or as potential therapeutic compounds.

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Jean F. Bore1 et at.

We have attempted to bring many different and often contradictory contributions into a coherent concept, while at the same time trying to avoid an exhaustive compilation of the literature. The first author wishes also to acknowledge the helpful and constructive criticism from some of his colleagues, in particular Dr. Francis Loor (reversal of multidrug resistance), Mr. Peter Hiestand (discussionof many in vivo results), Dr. Michael Dreyfuss (antifungal effects), and Dr. Roland Wenger (chemical structures). We refer those readers who need more information to Table IV which lists all sources containing the proceedings of congresses, symposia, and workshops devoted to CS. In addition, and independently of meetings, several major reviews on CS have been published (Borel, 1986; Thomson, 1989; Borel et al., 1989; 1994; Faulds et al., 1993). By the end of March 1995 more than 22,500 publications related to CS had been recorded, of which about 8500 were experimental papers. Since there is much confusion about the spelling of this compound, we decided to use throughout this publication the generic name ciclosporin (WHO), abbreviated as CS, which stands for the old cyclosporin A, the active agent in SANDIMMUN (see also p. 11 in Borel, 1986).

II. Molecular Mechanism of Immunosuppression Early immunological studies revealed that CS blocks activation of T cells and that this, in part, results from inhibition of transcription of lymphokines, most notably interleukin-2, the main growth factor for T cells (Kronke et al., 1984). The current concept of the mechanism of immunosuppression by CS suggests that, by inhibiting interleukin-2 expression in T cells, CS prevents helper T cells from orchestrating a response to foreign antigens. Because of the specific effects of CS on lymphokine transcription in T cells, and the important role of T cells in graft rejection, research on the mechanism of action has focused mainly on the role of CS in regulating gene expression in T lymphocytes (Bierer, 1994). At least one of the intracellular targets for CS has been identified and found to have an enzymatic activity. This main intracellular binding protein for CS has been identified as cyclophilin A, a cytoplasmic peptidyl-prolyl cis-trans isomerase, which enyzymatic activity facilitates protein folding. It belongs to the increasingly diverse family of general and tissue-specific immunosuppressant-binding proteins, the immunophilins (for review see Fruman et al., 1994b; Galat and Metcalfe, 1995). Cyclophilin A was shown to bind with high affinity (Kd [CS] = 6 nM) to residues 1 , 2 , 9 , 10, and 11 of the CS molecule, residues which were recognized to be essential for the immunosuppressive activity of CS early on (Handschumacher et al., 1984; Quesniaux et al., 1988). To define the structure-activity relationship of the CS molecule in more detail, several cell-permeable,cyclophilin-binding, but

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In Viw Pharmacological Effects of Ciclosporin and Some Analogues

TABLE IV List of Major Congresses and Symposia Concerned with CS and Reference of Proceedings Date

Place

Main topic: Reference of proceeding

Sep 1981

Cambridge, UK

May 1983

Houston, TX, USA Basel. CH

Exp. and clin. research: White, D. J. G. (ed.). Cyclosporin A Elsevier Biomed. 1982. Exp. and clin research: Transplant. Proc. 15, Suppl. 1 and 2), 1983. Autoimmunity: Schindler, R. (ed.). Ciclosporin in autoimmune diseases. Springer-Verlag, 1985. Renal side effects: Transplant. Proc. 17, Suppl. 1, 1985. Exp. nephrotoxicity: Thiel, G. (ed.). Clin. Nephrol. 25, Suppl. 1, 1986. Clin. renal transplant.: Ponticelli, C., and de Vecchi, A. (eds.). Contrib. Nephrol. 51, Karger, 1986. Clin. renal transplant.: Transplant. Proc. 18, Suppl. 1, 1986. Pharmacology: Transplant. Proc. 18, Suppl. 5, 1986. Mech. of action: Transplantation 46, Suppl., 1988. Exp. and clin. research: Transplant. Proc. 20, Suppl. 2,3,4, 1988. Clin. research: Transphnt. Proc. 21, Suppl. 1, 1989. Cardiac transplant.: Transplant. Proc. 22, Suppl. 1, 1990. Drug monitoring: Transplant. Proc. 22, 1097-1361, 1990. Dermatology: J. Am. Acud. Dennatol. 23, 1241-1334, 1990. Dermatology: Br. J. Dennatol. 122, Suppl. 36, 1990. Dermatology: Z . Hautkrankheiten 66, Suppl. 1,1991 Drug monitoring: Clin. Biochem. 24, 1-111, 1991 Clin. renal transplant.: I. Nephrol. 3, Suppl. 1, 1990. Nephrotic syndrome: Clin.Nephro1. 35, Suppl. 1, 1991. Clin. transplant.: Transplant. Int. 5, Suppl. 1, 1992 Exp. and clin. research: Transplant. Proc. 24, Suppl. 2, 1992 Rheumatoid arthritis: Sand. J. Rheumatol. 21, Suppl. 95, 1992.

Mar 1985 Mar 1985 Apr 1985

San Juan, Puerto Rico Basel, CH

Oct 1985

Milano, I

Nov 1985

Palm Beach, FL, USA Hardangervidda, N

Jul 1986 Jun 1987 Nov 1987

Bad Schauenburg, CH Washington, DC,

Nov 1988

Penang, Malaysia

Nov 1989

Barcelona, E

Jan 1990 Jan 1990

Hawk's Cay, FL, USA Orlando, FL, USA

Feb 1990

Loja, Granada, E

Mar 1990

Dasseldorf, D

May 1990

Minaki Lodge, CAN

Jun 1990

Porto Conte, Sardinia, I

Nov 1990

Nice, F

Oct 1991

Maastricht, NL

Nov 1991

Basel, CH

Jun 1992

Malm6, UK

(continues)

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Jean F. Bore1 et al.

TABLE IV (continued) Date

Place

Main topic: Reference of proceeding

Jun 1992

Paris, F

JuI 1992

Marlow, UK

Apr 1993

Taipei, ROC

May 1993

Wien, A

Mar 1994

Sevilla, SP

Aspects on clin. use: Thirapie 47, 299-342, 1992. Rheumatoid arthritis: Br. /. Rheumatol. 32, Suppl. 1, 1993. Clin. transplant.: Transplant. Proc. 25, Suppl. 3, 1993. Dermatology:J. Dermatol. Treatment 5, Suppl. 1, 1994. Exp. and clin. research:Transplant. Proc. 26, 2469-3092, 1994.

nonimmunosuppressive CS derivatives were analyzed in comparison to CS (Baumann et al., 1992). These CS-analogues with modifications in residues not contributing to cyclophilin A binding antagonize CS-mediated inhibitory effects as assessed by interleukin-2 promoter-driven reporter gene expression in a stably transfected T-cell line. Effective inhibition of the cis-trans isomerase activity of cyclophilin A by antagonistic CS analogues in vitro provides compelling evidence that inhibition of the enzymatic activity of cyclophilin is either irrelevant or at least insufficient for explaining the mode of action of CS. It appears now that the CS molecule acts via two regulatory domains: a cyclophilin-binding domain and an effector domain which is intimately involved in the immunosuppressive activity of CS (Fig. 2). The cyclophilid CS complex acts as a single effector and has been shown to directly bind and inhibit the calcium- and calmodulin-dependent serinelthreonine phosphatase calcineurin ( Friedman and Weissman, 1991; Liu et al., 1991).Using molecular modeling and mutagenesis, Milan et al. (1994) have defined a region in the B-subunit of calcineurin which is used to allosterically activate calcineurin A as the target of the dual complex. As CS reveals specificity for cellular activation pathways that induce an increase in intracellular calcium concentration, such as that emanating from the T-cell receptor on antigen recognition, and as the phosphatase activity of calcineurin reveals a strict requirement for calcium, calcineurin most likely is the common key element in all CS-sensitive cells regulating the modification of intracellular signaling components (Fig. 3 ) . It has been demonstrated in vitro that the activity of calcineurin toward phosphorylated peptides is strongly inhibited in the presence of the cyclophilidCS complex. Moreover, the pharmacological relevance of the inhibition of calcineurin phosphatase activity by CS is further supported by experiments in which overexpression of the catalytic subunit A of calcineurin in T cells rendered these cells more resistant to the effects of CS as measured by inhibition of interleukin-2 promoter driven transcription (O’Keefe et al., 1992; Clipstone and Crabtree, 1992). Thus,

In Vim Pharmacological Effects of Ciclosporin and Some Analogues

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it appears that CS functions as an immunosuppressant by altering the activity of calcineurin to its substrates in T cells. These substrates are most likely cytoplasmic components of the signal transduction pathway which finally results in “early” gene transcription including the lymphokines, e.g., interleukin-2. Potential candidates that are essential for interleukin-2 gene activation are the antigen-inducible transcription factors NF-AT (nuclear factor of activated T cells) and NF-KB (nuclear factor of immunoglobulin K light chain in B cells) which were both reported to be affected by CS in their DNA-binding to the interleukin-2 promoter (Emmel et af., 1989; Baumann et af., 1991). The cytoplasmic component of NF-AT, NF-AT,, has recently been cloned and shown to be a substrate of calcineurin in vitro (McCaffrey et af., 1993). Modulation of NF-AT, phosphorylation, and therefore presumably of NF-AT, activity would explain the sensitivity to CS for interleukin-2 transcription as well as for interleukin-3, interleukin-4, granulocytemacrophage colony stimulating factor and tumor necrosis factor-a transcription. It is likely that NF-AT DNA-binding sites also exist in other promoters of CS-sensitive genes. Cytoplasmic localization of another transcription factor involved in lymphokine transcription, NF-KB, a heterodimeric protein consisting of p50 and p65 subunits, is stabilized by the binding of a third protein IKB.It appears to be phosphorylated in its bound form. Dephosphorylation of IKBleads to the release of NF-KBand its subsequent translocation to the nucleus and to degradation of IKB;it is hypothesized that calcineurin may catalyze the dephosphorylation of IKB.Other substrates of the ubiquitously expressed calcineurin are likely to exist in a number of different cells and tissues and may, in general, contribute not only to the immunosuppressive activity of CS but also to the side effect profile of the drug. The relatively specific and pronounced effects of CS for T lymphocytes may be due to the low levels of calcineurin in these cells. In addition, other phosphatases might be able to substitute for calcineurin in many tissues, whereas dephosphorylation of critical T-cell substrates might only be catalyzed by calcineurin. In summary, recent data on the molecular mechanism of some immunosuppressive drugs provide strong support for the fascinating postulate that CS and tacrolimus (FK 506) work by binding to immunophilins and then, as a drug-immunophilin complex, inhibiting the calcium-activated protein phosphatase calcineurin. This inhibition could result in an altered modification pattern of the cytoplasmic components of transcription factors, thereby disturbing their nuclear translocation, which is a prerequisite for proper interleukin-2 transcription. It looks as if, with the immunosuppressive microbial metabolites as molecular probes, the pieces of this complex signal transduction puzzle are starting to fit together. Once the details of the cascade of events along the T-cell signaling pathways are known, the molecular structures involved will possibly provide new tools to be used in the search for the rational design of novel and improved therapeutic agents.

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111. Suppressive Effects on Cell-Mediated Immunity Numerous in vitro studies on the mechanism of action of CS have revealed that its immunosuppressive activity was due to the inhibition of gene transcription for several lymphokines (see Section 11). We will first review how CS affects in vivo and ex vivo the various functions of the T cells and, where necessary, compare these results with in vitro findings.

A. T Cell-Mediated Cytotoxicity In the first paper mentioning CS (Borel, 1976), it was reported that, firstly, CS exerted a profound and dose-dependent ex vivo inhibition of allogeneic target cell lysis when administered to mice during the sensitization period, and that the drug depressed both primary and secondary responses by preventing the development of effector cytotoxic T lymphocytes (CTL). Secondly, when CS was given postimmunization to fully sensitized mice the existing CTL cells were not affected (ex vivo assay). Thirdly, CS failed to suppress cell-mediatedcytotoxicity after in vitro addition to sensitized mouse spleen cells. These results clearly indicated that CS was only suppressing the development of CTL, but failed to modify the cytolytic function of these cells both in vivo and in vitro. Further studies, especially in allografted animals, confirmed and extended these early findings. Cells recovered from kidney allografts in CStreated host rats, while largely retaining nonspecific cytotoxicity, were very deficient in donor-specific activity (Mason and Morris, 1984). These results may partly explain the antirejection effect of CS, since it was demonstrated that specific CTL were those playing an essential role in allograft rejection in the rat (Bradley et al., 1985). These observations were substantiated in bone marrow (Deeg et al., 1983) as well as in lung transplanted dog experiments (Norin et al., 1986). Using the ex vivo mixed lymphocyte reaction assay, it was found that lymphocytes from unresponsive lung allograft recipient dogs had a diminished ability to generate donor-specific CTL cells, but retained normal cytolytic responses to third-party allogeneicstimulator cells, thus demonstrating specificity of the unresponsive state. In contrast, high levels of specific CTL activity were detected in cell preparations from lung allografts undergoing rejection (Norin et al., 1987). Rejection phenomena observed after termination of CS therapy were reversed by resumption of CS treatment, which caused also a decrease in intragraft CTL activity (Norin, 1988; Norin et al., 1986).While studying in vitro the growth characteristics of renal allograft infiltrating lymphocytes from patient biopsies, Kirk et al. (1992) have demonstrated that irreversible allograft rejection was the result of an inadequately suppressed T-cell population which was capable of amplifying its responsivenessthrough secretion of interleukin-2. These cells mediating irreversible kidney damage differed from those clinically suppressed

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only in their lymphokine-secretion abilities but not in their surface-phenotype or cytotoxic abilities. Using the local graft-versus-host reaction (also called local lymph node weight assay or Simonsen test) in rats, Marwick et al. (1979) have demonstrated the efficacy of CS to inhibit both the graft-versus-host response and the host-versus-graft reaction, which was elicited by injecting F1 hybrid spleen cells in rats of the parental strain. However, the popliteal lymph node enlargement was never completely abolished and splenic lymphocytes from CS-treated recipients showed no significant reduction in their response to donor-strain lymphocytes in ex vivo mixed lymphocyte reactions, indicating that clonal deletion had not occurred. Since the increase in cell number in the involved lymph node derives from a combination of specific lymphocyte proliferation (clonal expansion) and a nonspecific retention of recirculating lymphocytes arrested in their normal migration through the node as a consequence of lymphokines released by activated cells, these results suggested that CS had little effect on the activation and proliferation of responding T cells within the node but did inhibit the local lymphokine release responsible for lymphocyte recruitment to the node. Chisholm and Bevan (1988) have examined in detail the effect of CS on a systemic graft-versus-host reaction, cardiac allograft rejection, and a local host-versus-graft reaction in the rat. In the systemic graft-versus-host model the activation and proliferation of donor cells was not affected in the lymph nodes, but their subsequent release into the circulation was completely prevented. These animals showed no clinical sign of disease for as long as CS was given, but withdrawal of the drug resulted in accelerated lethal graft-versus-host disease. Although CS did not inhibit early activation of T cells in the cardiac allograft model, the drug did prevent the later consequences of T-cell activation and, ultimately, graft destruction. The effects of CS in the local host-versus-graft assay essentially confirmed those observed by Marwick et al. (1979). Taken together, these studies provide clear evidence that therapeutic levels of CS did not inhibit the early stages of lymphocyte activation, but did prevent maturation of the immune response to full effector function such as graft rejection and graft-versus-host disease. These data agree with others demonstrating that in unresponsive rats bearing cardiac or skin allografts cytotoxic T cells against donor targets are found residing in lymph nodes but not among lymphocytes taken from the peripheral circulation (White and Limy 1988). Kroczek et al. (1987) have used a local graft-versus-host assay in the mouse and shown that CS administration in vivo had no effect on alloantigen-induced increases in cell size, percentage of cells expressing the interleukin-2 receptor, the spontaneous or interleukin-2-driven proliferation of freshly explanted cells, or the induction of CTL activity (ex vivo). However, Granelli-Piperno (1990),also using the same assay in mice, has shown that allogeneic cells as well as several other mitogens induced in untreated animals the expression of interleukin-

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Jean F. Borel et al.

2 and y-interferon mRNAs in popliteal lymph node T cells and that these transcripts were, after in vivo administration of CS, markedly reduced, although not as completely as after in vitro addition of the drug. To the contrary of the local graft-versus-host model, CS treatment of mice in the concanavalin A model was capable of inhibiting not only interleukin-2 receptor induction but also proliferative responses with and without interleukin-2 (Black et al., 1988). Some reduction in cell size was also evident at the highest doses used. Further work by Pereira et al. (1990) confirmed that the mechanism of action of CS in vivo was probably identical to that seen in vitro as CS effectively suppressed the induction of CTL activity and interleukin-2 mRNA expression in lymph node cells from alloantigen primed mice; nevertheless, the T cells from these same CS-treated animals appeared to have undergone both priming and differentiation. In addition, freshly explanted cells from CS-treated mice manifested in vitro an enhanced secondary response to the priming alloantigen which suggested that they had undergone clonal expansion in vivo. They concluded that even in the presence of high drug concentrations in vivo T cells could be primed to alloantigens, and proliferate and differentiate to precytotoxic T cells. Hodgkin et al. (1985) have demonstrated that lymphocytes recognizing class I alloantigens were able to lyse appropriate target cells and to release lymphokines in vitro. Since CS inhibits lymphokine release from class Ispecific CTL but has no effect on their cytotoxic activity, the in vivo function of class I-specific CTL was analyzed in two models: the local graft-versushost reaction induced by transfer of sensitized CTL cells to the footpad in mice and the islet allograft rejection model in the mouse induced by the passive transfer of sensitized CTL. CS inhibited the in vivo functions of the transferred sensitized CTL in both systems; hence, these functions appeared to be lymphokine dependent. Similar findings were also obtained in a murine virus model. CS inhibited the function of transferred, influenza-specific,class I-restricted cytotoxic T cells which normally lead to clearance of virus in the lungs of infected mice (Schiltknechtand Ada, 1985a).The results strongly suggested that the in vivo clearance of influenza virus by class I-restricted CTL involved a lymphokine mechanism. Using an alloreactive T-cell clone producing y-interferon and tumor necrosis factor but not interleukin-2 and -3, Hao et al. (1990) have demonstrated in the murine islet allograft model that the latter lymphokines were not required for the rejection process. However, this process was inhibited by CS but treatment of islet tissue with y-interferon prior to grafting, which increases the density of class I antigen, abolished this sensitivity to CS, indicating that in islet graft rejection both lymphokines and cytotoxic activity are acting cooperatively. Black et al. (1990)have investigated the effect of topical application of CS on site-specific suppression of cell-mediated immunity in a dual skin

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allograft model in rats. Anti-inflammatory efficacy and prolonged skin allograft survival were observed in the presence of topically administered CS, while the contralateral vehicle-treated control graft underwent vigorous rejection. Interestingly, systemic T cell-mediated immunity appeared unaffected or possibly activated even with concomitant CS treatment but CS serum levels were quite low. This study indicates that topical CS is capable of locally suppressing a strong T cell-mediatedresponse after an initial shortterm systemic dose of CS. More recently, Llull et al. (1995) have reported that a synergistic efficacy could be induced by site-specific immunomodulation with CS in combination with topical hydrocortisone during the maintenance phase. It should be noted that potentially useful topical formulations, such as a nonionic liposomal system (Novasome l), have recently been experimentally studied (Niemiec et al., 1994). Hooten et al. (1990) have studied the effects of CS on the in vitro generation of CTL from their precursors and observed that even in the presence of exogenously added interleukin-2, CS blocked the generation of CTL at an activated, intermediate stage, referred as “precursor-effector CTL.” However, when such cells, which were generated in a 5-day mixed lymphocyte culture containing CS but not interleukin-2, were washed and recultured in the presence of added interleukin-2 there was a rapid conversion to alloantigen-specific cytotoxic effector cells. Similarly, Kiziroglu and Miller (1990a) have confirmed that CS inhibits the development of the cytotoxic response in a primary mixed lymphocyte reaction and shown that exogenous interleukin-2 could not restore this inhibition. CS seems to interfere with CTL induction in a primary mixed lymphocyte reaction by acting directly on CD8+ lymphocytes known to include precursor CTL. Kiziroglu and Miller (1990b) provided experimental evidence that CS also inhibits the induction of CTL in a secondary mixed lymphocyte reaction and that addition of exogenous interleukin-2 can only partially overcomethis effect. The CS-resistant cells represent an activated population of memory precursor CTL that require only lymphokines (interleukin-2 and/or -4) for clonal growth and that kill targets of the original stimulator type specifically. Recent findings by Smyth and Ortaldo (1993) indicate that activated human peripheral blood CD4 and CD8 T-cell effectors can lyse target cells by at least two distinct mechanisms: (1)a CS-sensitive redirected lysis of sheep erythrocytes that correlates with exocytosis and presumably occurs via membrane lesions, and (2)a CS-insensitiveredirected lysis of nitrophenylmodified nucleated target cells that does not appear to involve exocytosis and is metabolically distinct in activated CD4 and CD8 T-cell effector subsets. Kaiser et al. (1993) have used CS in vitro to block the transcription of early T cell activation genes, including the gene coding for interleukin-2, and anti-interleukin-2 neutralizing antibody to specifically block interleukin2 bioactivity in cultures of anti-CD3-stimulated murine T cells in order to study the role of interleukin-2 in the induction of cytolytic activity and

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cytotoxic cell proteinases 1and 2 expression. Their results provided evidence that anti-CD3-activated CTL function and cytotoxic cell proteinases 1 and 2 expression are not dependent on the synthesis of interleukin-2, but rather are regulated by a CS-sensitive mechanism. Resting T cells can be activated by selected pairs of anti-CD2 monoclonal antibodies in the presence of accessory cells which can be replaced by antiCD28. Van Goo1 et al. (1995) have shown that CD28 ligation can provide a costimulatory signal for anti-CD2-induced CTL generation. They found that interleukin-2 production and CTL generation in the CD2/CD28 pathway were at least partly CS-resistant, although the CS-resistant CTL generation appears almost completely interleukin-2 dependent. Since CD28 triggering affects cytokine gene enhancer activity and occurs through a calciumindependent pathway, it does, therefore, escape inhibition by CS (cf., Linsley and Ledbetter 1993). Both the calcium-independent and calcium-dependent pathways seem to be initiated by protein tyrosine kinase activity following CD28 triggering and it is well known that generally only the calciumdependent pathways are sensitive to CS. Orosz et af. (1988a,b,c) have further investigated the effect of CS on CTL modulation by attempting to correlate results from both in vitro and in vivo models. Under limiting-dilution culture conditions, CS was shown to block terminal CTL clonal expansion. These results would suggest that the signaling system for proliferation and for cytolytic function in CTL are separable and differentially sensitive to CS, and also imply that CS has an inhibitory effect on CTL which is independent of helper T-cell dysfunction (Orosz et af., 1988a,b). By studying the effect of CS on the alloantigenand/or lymphokine-driven proliferation of murine CTL clones, it was observed that lymphokine-driven proliferation of CTL clones continues in the presence of CS, but that alloantigen does not synergistically increase lymphokine-driven proliferation, if CS is present (see also Paetkau et al., 1988). The sponge matrix allografts used to study in vivo immunologic events associated with allograft rejection accumulate both donor-reactive CTL in an active cytolytic state and donor-reactive alloantibodies within ) the fluids several days of allograft implantation (Orosz et al., 1 9 8 8 ~and did also allow measuring the concentration of drug. CS interfered with the accumulation of donor alloantigen-reactive CTL in sponge matrix allografts by markedly reducing their number in a dose-dependent manner as determined by limiting dilution analysis. However, CS little affected the number of donor-reactive CTL in the peripheral blood. Consequently, it appeared that the donor-reactive CTL were available but were unable to accumulate in the graft under the influence of CS (Orosz et al., 1988b). Further experiments demonstrated that CS could differentiallyimpair the accumulation of donorreactive CTL cells, but not donor-specific alloantibodies in sponge matrix allografts (Orosz et af., 1 9 8 8 ~ ) .

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Orosz et al. (1989b)have developed a modified limiting dilution analysis technique which can differentially enumerate CTL that have responded in vivo to graft alloantigens. This modified technique as well as the conventional technique have been used in conjunction to examine the effects of allograft implantation on alloactivation and redistribution of graft-reactive CTL in allograft recipient mice. In general, CS appears to act on the donor-reactive CTL in allograft recipients by interfering with limiting dilution analysisdetectable activation of CTL at the regional lymph node and by interfering with the accumulation of both alloactivated and precursor CTL at the graft site (Orosz et al., 1989b). They also observed that none of the numerous donor-specific CTL in the peripheral blood of sponge or skin allograft recipients demonstrated limiting dilution analysis evidence of prior contact with graft antigen, despite the prominent accumulation of donor-reactive, alloantigen-conditioned CTL cells at the graft site, and to a lesser extent, the draining lymph nodes. This could either reflect the functional inactivation of CTL traveling in the peripheral blood, or the absolute lack of alloantigenconditioned CTL, due presumably to rapid and efficient acquisition by the graft site. Finally, their studies have demonstrated that the role of alloantigen in graft-induced T lymphocyte activation and redistribution is complex in that graft alloantigens apparently initiate both local and systemic mechanisms that influence precursor and/or alloantigen-conditioned CTL accumulation and/or activation at the graft site (Orosz et al., 1989a). Vaessen et al. (1994) have used limiting dilution analysis to study CS sensitivity and differential avidity of committed, donor-specific, graft-infiltrating CTL and their precursors from endomyocardial biopsies of heart transplant patients. Almost all antigen-primed, committed CTL present in the graft of patients with rejections were CS resistant, whereas in most patients of the nonrejector group a substantial part of these cells could be inhibited by CS. However, the cytotoxic precursors in both groups were predominantly CS sensitive. The predominant subpopulation in the graft of rejectors was a CS resistant, committed CTL with high avidity for donor antigens, while in the graft of most nonrejectors CTL with low avidity dominated. The CS resistant, high avidity, committed CTL were already detectable in the graft before myocytolysis was observed and may be an early indication of an approaching rej ection.

B. T-cell Functions for Help, Memory, and Delayed-Type Hypersensitivity (DTH) It is evident that CS inhibits both the induction and the effector phases of the DTH response in several species, including man, and that this suppressive effect is independent of the type of antigens used (chemicals, proteins, virus, or spleen cells) (for review, cf., Bore1 et al., 1989). Both the primary and the secondary DTH reactions were abrogated by appropriate treatment with

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CS and the secondary response was suppressed independently of whether the primary response had been modulated by the drug or not (Bore1 et al., 1977). If the DTH reaction was elicited by injection of activated, antigenspecific, cloned T-helper cells, CS given up to 10 h later was efficacious in preventing the reaction, but not when given after 15 h (Herrmann et al., 1986).In contrast, when the challengewas induced with antigen, the effector phase was inhibited under the condition that CS was given before and/or simultaneously with antigen. It is worth noting that topical CS application at the site of antigen administration also prevented the effector phase of the DTH reaction (Aldridge et a/., 1985; Nakagawa et al., 1988). The suppression by CS is reversible and of short duration, since a secondary challengeperformed 96 h after the primary one did elicit a positive DTH response (Rullan et al., 1984). The target cells for the action of CS in the DTH response are those mediating the specific or immunologic component of the reaction, because CS is known to have no direct effects on the nonspecific, acute inflammatory component (Anderson and Groth, 1985; Dunn and Miller, 1986; Nakagawa et al., 1988). Various studies have demonstrated that the DTH reaction could be adoptively transferred by CDS nonadherent T lymphocytes which are the cells affected by CS (Attridge and Kotlarski, 1985; Langer et al., 1985). These cells are also found to release lymphokines in vitro when cultured with the relevant antigen. CS interferes with the DTH reaction by presumably suppressing the release of lymphokines and other mediators of inflammation, because addition of interleukin-2 or of supernatants from activated antigen-specific T-helper clone cultures reversed the inhibition by CS (Xue et al., 1986; Herrmann et al., 1986). Of particular interest is the finding that CS did not interfere with the clonal expansion of antigen-specific DTH-mediating cells in vivo (Milon et al., 1984). This implies that DTH precursors are not sensitive to CS inhibition, since cells recovered from 6-day sheep erythrocyte-stimulated spleen cell cultures in the presence of CS were able to induce a DTH reaction to sheep erythrocytes after transfer to naive mice (Shidani et al., 1987). The authors suggested that only the effector functions of the T-helper and T lymphocytes mediating DTH were suppressed by CS, but that their proliferative capacity was resistant to the drug (Truffa-Bacchi,1987).This was clearly confirmed in other transfer experiments which showed that the induction of T-effector cells was insensitive to CS, whereas the effector function of this T subpopulation mediating DTH was CS-sensitive (Braida and b o p , 1986). The results obtained by Palestine et al. (1985) in uveitis patients also seem to indicate that the peripheral blood lymphocytesretained a normal proliferative response (ex vivo) under CS therapy, while the DTH immune function was suppressed. Motta et al. (1991) have determined the effect of CS on the induction of T-helper cells during an in vitro antigenic stimulation of mouse spleen

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cells and have shown in a limiting dilution analysis that the frequency of the T-helper cells recovered from CS-treated cultures was very significantly increased compared with naive spleen cells. This highly increased frequency, the sensitivity to irradiation of the generation of these T-helper cells, and the inhibition of lymphokine gene transcription for interleukin-2 and -4 demonstrate that T-helper cell precursors proliferate in the presence of CS. In experiments on the effects of CS on antibody production in mice, Klaus and Kunkl(l983) and Klaus (1988)observed that in vivo CS could dissociate effector function (primary help) from T-helper cell proliferation (priming). Additional evidence supporting this contention was obtained in lung allotransplanted tolerant dogs. Unresponsiveness was a selective phenomenon in that donor alloantigens in mixed lymphocyte culture induced a strong proliferative response of T cells from unresponsive recipients ex vivo, but the donor-specific CTL activity was very low compared with that of thirdparty alloantigens (Norin et al., 1987). During infection of mice with lymphocytic choriomeningitis virus, interleukin-2 is produced by spleen lymphocytes with a time course corresponding to that of T-cell activation and proliferation. Kasaian and Biron (1990a,b) have reported that proliferation of both CD4 and CD8 T-cell subsets were affected in vivo by CS treatment. In addition, CS extensively inhibited virus-induced interleukin-2 gene transcript levels and blocked interleukin-2 production. Decreased levels of interleukin-2 receptor p55 a-chain gene transcripts and loss of interleukin-2 responsiveness by freshly isolated cells were also observed. Charan et al. (1986)have used mice injected with vesicular stomatitis virus and demonstrated that the initial IgM antibody response, which is T-cell independent, was CS-resistant, whereas the IgG antibodies, which are thymus dependent, were completely suppressed in the primary response. It appeared that the switch from IgM to IgG was prevented by CS, an observation also made by Schildknecht and Ada (1985b) in a different mouse model using influenza A virus. However, once the switch to IgG in a primary response had occurred, the IgG response became refractory to CS. Moreover, the secondary IgG response was highly resistant to CS. Roost et al. (1990) have extended these experiments by using serologically distinct viral serotypes and they confirmed the evidence in support that priming occurs in spite of CS treatment and that primed or memory T and B cells are quite resistant to the suppressive effect of CS. The effect of CS on the expression of the interleukin-2 receptor on T cells is controversial, both in vitro and in vivo. Hkmar and Dautry-Varsat (1990) have investigated this question by using a human T-cell line which constitutively expresses such receptor chains and also spontaneously secretes interleukin-2. While they had previously shown that CS prevented the constitutive transcription of the interleukin-2 in these cells, they further demonstrated that as soon as 4 h after CS addition the transcription of the gene encoding for the a-chain of the interleukin-2 receptor was inhibited. How-

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ever, no decrease of the receptor a-chain on the cell surface could be detected after 48 h in the presence of CS. In addition, CS did not seem to block the receptor a-chain mRNA through inhibition of interleukin-2 synthesis. The high stability of the a-chain may account for the observed discrepancy on the effect of CS as probed at the mRNA or protein level. Foxwell et al. (1990) have independently studied the sensitivity of interleukin-2 receptor expression to CS by using a combination of ligand binding and interleukin2 responsiveness analysis. Whereas inhibition of human lymphocyte proliferation (ICs0)occurred at 10 ng/ml CS after cell activation via CD3 or CD2, higher CS concentrations (300 ng/ml) were necessary to inhibit lectinmediated (PHA, ConA) cell activation. The interleukin-2 receptor expression of the a-chain was found to be CS-sensitive in T cells activated by either the CD3 or the CD2 pathways but only at higher concentrations (300 ns/ml). However, it remained unclear whether inhibition of interleukin2 production by CS provided a secondary mechanism of inhibition of interleukin-2 receptor expression in addition to a primary effect on the antiCD3 activation signal to the interleukin-receptor gene. In contradistinction, the signal provided by interleukin-2 via its receptor to the interleukin-2 receptor gene appears to be CS resistant. The same group (Foxwell et al., 1995)further showed that CS inhibited interleukin-2 receptor a-chain (p.55) gene transcription and protein expression after anti-CD3 or ionomycin stimulation, although at a twofold higher concentration than that required to inhibit interleukin-2 gene transcription. The upregulation of interleukin-2 receptor mediated by phorbol ester (PMA) or interleukin-2 was resistant to CS. It appears, therefore, that drug resistance and sensitivity to interleukin2 receptor upregulation is correlated with the dependence on de novo protein synthesis of a given activation pathway. Li et al. (1992) also explored the effect of CS on the induction of interleukin-2 receptor a- and &chains in normal human T cells at the levels of mRNA, protein, and function. The T cells were stimulated either with crosslinked anti-CD3 and anti-CD2 monoclonal antibodies or with sn-l,2-dioctanylglyceroland ionomycin. Their results showed that CS at 100 ng/ml inhibited partially the induction of the receptor a-and P-chains (in contrast to complete inhibition of interleukin-2 synthesis) by a direct effect on T cells and that the inhibitory activity was detectable at the pretranslational level, since CS significantly reduced the induction of mRNA encoding interleukin-2 receptor a- and @chains. The ability of CS to interrupt the emergence of interleukin-2 receptors on the surface of normal human T cells may be due to the drug (1 pg/ml) affecting the receptor expression, at least that of the a-chain, by inhibiting the interaction of transacting factors to &-like sequences following mitogen (PHA) activation, as suggested by the work of Brini et al. (1990). The induction of interleukin-2 receptor expression has also been examined in vivo in response to injection of concanavalin A into the footpad of mice. More than 70% of all cells from the draining lymph nodes were

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interleukin-2 receptor positive at the peak of the response. Black et al. (1988) demonstrated that in this in vivo model treatment of the animals with CS was inhibiting both interleukin-2 receptor expression and induction of spontaneous proliferation. Granelli-Piperno ( 1990) has reported confirming results indicating that in vivo stimulation of mice with allogeneic cells or mitogen induced the activation of the receptor a-chain and that this induction was partially inhibited by CS treatment. On the other hand, Yoshimura et al. (1991) investigated the effects of CS administered orally to renal transplant patients on the capacity of peripheral blood mononuclear cells to express interleukin-2 receptor gene at the level of mRNA after mitogen stimulation in vitro. Interestingly, there were no differences in the percentage of interleukin-2 receptor positive cells among the groups of normal individuals, azathioprine-prednisolonetreated, and CS-prednisolone treated recipients, using labeled monoclonal anti-a chain antibodies. However, in a study of Northern blotting using anti-a chain specific cDNA as probe, both the 3500 and 1400 base families of interleukin-2 receptor mRNA were remarkably decreased in the peripheral lymphocytes from CS-prednisolone treated recipients compared with the two other groups. This study demonstrated that CS could inhibit interleukin-2 receptor expression at the level of mRNA at physiological concentrations (trough levels between 100 and 200 ng/ml as determined by radioimmunoassay using polyclonal antibodies). Finally, when Kay et al. (1994)used CS therapy to treat chronic severe asthma, they detected a significantly lower serum concentration of soluble interleukin-2 receptors on CS therapy as compared with placebo. In spite of all the possible methodological reasons for the prevailing confusion, one might conclude that there is accruing evidence for CS exerting a partial or complete direct inhibitory effect both in vitro and in vivo on the genes coding for interleukin2 receptor a- and possibly &chains. C. Cell-Mediated Suppressor Function

There is much experimental evidence suggesting that donor-specific unresponsiveness and tolerance may be both induced and maintained by suppressor cells of one form or another, as suppression could be adoptively transferred to a syngeneic recipient with mononuclear cells from the tolerant animal. However, the failure to identify and clone a separate population of suppressor cells (as it is feasible with helper or cytotoxic cells) has led to skepticism about the existence of a separate subset of cells whose sole function is suppression (Morris et a]., 1993). Bore1 et al. (1989) have reviewed the effect of CS on cell-mediated suppressor function in transplantation (ibid. pages 311-314) and in autoimmune models (ibid. pages 337338). Part of the maintenance of unresponsivenessseen in CS-treated animals may be due to sparing or activation, or both, of T-suppressor cells (for review see Kupiec-Weglinski et al., 1984). Generation of suppressor lymphocytes in

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vitro and in the presence of CS has been repeatedly reported (e.g., DosReis and Shevach, 1982; Hess et al., 1983) (see also Fig. 4). However, Pawelec (1990), while studying in vitro the requirements for induction of suppression in human peripheral blood mononuclear cells, has observed that the inclusion of CS in the induction culture essentially abrogated the generation of suppressor-inducer T-cell clones in a similar fashion as it inhibited the generation of helper T cells. Wang et al. (1982)have demonstrated that mice sensitized with alloantigens and treated with CS were incapable of generating CTL ex vivo; this effect was long-lasting and specific because lymphocytes from these animals could not be reactivated on subsequent exposure to the same alloantigens in vitro, but their response to a third party remained intact. The suppressor cells appeared to be T lymphocytes because treatment with anti-Thy-1,2 antibody and complement abrogated their suppressor activity. Furthermore, these suppressor T cells are radioresistant, cyclophosphamide sensitive, and undetectable in animals receiving CS only, i.e., without alloantigens, implying that CS does not induce, but rather permits the development of suppressor cells generated by allosensitization (Haug et al., 1987; Kupiec-Weglinski et al., 1984).Thymic extirpation in prospective CS-treated mouse recipients prior to transplantation of skin allografts invariably results in rejection once CS is being discontinued (Lems et al., 1980). However, thymectomy after grafting and completion of the CS regimen does not influence indefinite graft acceptance. This finding emphasizes the close interdependence between the “central” thymus and “peripheral” allograft. Thus, it appears that suppressor T cells are “schooled” in the thymus and then accumulate within the graft to ensure its acceptance by the host (Kupiec-Weglinski et al., 1985). Adoptive transfer studies were performed to investigate the functional significance of the T-cell subsets mediating suppression. Survival of test grafts was prolonged significantly, when cells infiltrating grafts and spleen cells were transferred at 7 days posttransplantation. Before that period, test graft survival was shortened in a second-set manner (Araujo et al., 1985). Prolongation of graft survival was also achieved with inocula of peripheral blood cells obtained from the CS treated recipients bearing long-standing well-functioning grafts. Thornburg et al. ( 1988) have confirmed suppressor activity in a rat skin allograft model and have shown that effector function, increasing after discontinuation of maintenance CS, can be again inhibited by suppressor T-cell activity after reinstitution of the drug. Nisco et al. (1995) have also demonstrated by using adoptive transfer experiments the presence in the spleen of antigen-restricted, radiosensitive suppressor cells capable of preventing allograft rejection by naive as well as sensitized cells in vivo. The use of thymectomized rats revealed that the establishment and maintenance of tolerance occurred peripherally, i.e., independently of the thymus. Utilizing a combination of perioperative injection of donor antigen

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and three cycles of CS in a rat renal allograft model, Yoshimura and Kahan (1986)have demonstrated the occurrence of splenic and peripheral suppressor T cells. The presence of suppressor T cells both in the spleen and in situ in renal allografts with prolonged survival in primary hosts suggested that local mechanisms may augment systemic elements to control generation of alloimmunity (Yoshimura and Kahan, 1986; Ito et al., 1990; Didlake et al., 1988).Other groups using similar adoptive transfer models arrived at similar conclusions (see references in Bore1 et al., 1989). Lancaster et al. (1985) have examined the properties of spleen-derived rat suppressor T cells and concluded from their results that this subpopulation was anti-idiotypic, with specificity for the idiotypes carried by syngeneic T cells stimulated by the kidney allograft. Ishii et al. (1990)and Aoki et al. (1990)have found different types of T cells acting as suppressor cells and shown that they were all CSresistant in their DTH mouse models, but that the DTH mediating T cells, which proliferate and secrete interleukin-2, had their effects blocked by CS. Hall et al. (1985a,b), using adoptive transfer assays and irradiated rat recipients, purified lymphocyte subpopulations mediating suppression and showed that radiosensitive CD4 T cells of the helperhnducer subclass, when injected alone, failed to restore rejection and were also able, when injected with normal lymph node cells or the CD4 cells separated from them, to prevent these cells from effecting rejection. CD8 T cells of the cytotoxic/ suppressor subclass, B cells, and serum from rats with long-survivingcardiac grafts all failed to inhibit the allograft responsiveness of normal lymph node cells, and thus were not identified as mediators of the state of specific unresponsiveness. More recent results (Hall et al., 1990) showed that the CD4 suppressor cell was capable of overriding the capacity to effect rejection of the CD4 and activated CD8 cells that were present in the CS-treated host shortly after transplantation. The normal response of T cells from unresponsive animals in both proliferative and effector graft-versus-host assays showed that cells with the potential to respond to the specific donor alloantigens and mediate tissue damage are present in unresponsive animals but are prevented from mediating rejection (Pearce et al., 1990). However, when comparing two different rat strain combination, it was observed that not only did the ease of the induction of long-term graft survival differ between strains, but also that increasing immunosuppression did not necessarily lead to tolerance induction in certain strains (Ilano et al., 1991). Pearce et al. (1993a,b) have demonstrated in a combination of in vivo and in vitro studies that the CD4 suppressor cell from CS-treated rats with long-surviving grafts was short-lived and that its survival was dependent on contact with specific alloantigens and interleukin-2 (see below Section III., D.). Although Pearce et al. (1993a)were unable to demonstrate the presence of blocking antibodies or factors in the sera from CS-treated rats, Nelson et al. (1990) have reported that human T lymphocytes, which are induced in vitro by heat-inactivated allostimulator cells in the presence of CS, release

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a soluble suppressive factor which seems not to be antigen specific or major histocompatibility complex-restricted and which is not identical with the suppressivecytokine transforming growth factor p. Yoshimura et al. (1988) have also claimed that their suppressor T-cell population mediated their suppressor effect by soluble factor(s), since culture supernatants from these cells inhibited in vitro cytotoxic T lymphocyte generation. Yahata et al. (1994) have, furthermore, cloned human suppressor T cells and shown that they inhibited the mixed lymphocyte reaction (but not mitogen-induced proliferation) by secreting alloantigen nonspecific and major histocompatibility complex nonrestricted suppressor factors. These factors affected the proliferation of cytolytic cells by blocking interleukin-2 synthesis as well as the expression of adhesion molecules. It also seems that the type of suppressor cells generated under CS treatment may vary with different models. Suzuki et al. (1985) have studied the development of suppressor cells in the peripheral blood of recipient dogs receiving heterotopic cardiac allografts and being treated daily with CS. Deeg et al. (1987) have investigated the development of specific tolerance and immunocompetence in recipient dogs of marrow grafts from DLAhaploidentical littermates or completely allogeneic unrelated donors. The time course of appearance and disappearance of suppressor cells in the dog, however, was similar to that observed by Tutschka (1987) in the rat model. There is controversy in the literature concerning the nature of the mononuclear cells effecting suppression, their expression of cell surface phenotype, the specificity of their suppressive activity, and their sensitivity to cyclophosphamide and irradiation (reviewed by Borel et al., 1989). However, it seems generally accepted that antigen-specific suppressor cells generated against alloreactive effector cells are spared and resistant to the action of CS. It further appears difficult to envisage that such cells developing in models of autoimmune diseases should be at variance with those occurring in allograft reactivity, especially in the antigen-induced autoimmune models in which self-antigens are manipulated so as to virtually be seen as alloantigens (Cohen, 1986). Several observations suggest that besides antigen-specific suppressor T cells, as found under allograft conditions, other types of suppressor cells or mechanisms may prevail in experimental models of autoimmunity. Their respective susceptibility to CS may not necessarily be the same. It is well documented that rats susceptible to experimental allergic encephalomyelitis (EAE) will spontaneously recover from paralysis and subsequently become resistant to further attempts to induce the disease (Hinrichs et al., 1981). Spleen, lymph node, and thymic cells obtained from such recovered rats contain suppressor T cells which, on transfer into syngeneic hosts, are able to render them refractory to renewed induction of this disease (Sun et al., 1988; Chabannes and Borel, 1991). Discontinuation of prophylactic CS therapy, especially after short-term administration, may usually be followed by a bout of EAE, suggesting that the rats did not build up a

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state of resistance during drug treatment (Bore1et al., 1986; Fredane et al., 1983; Feurer et al., 1988). However, the experiment of Ellerman et al. (1988),in which CS was used in vitro to allow the generation of a suppressor T-cell line invalidates the concept that CS negatively affects existing suppressor T cells. Similar experiments were reproduced to generate in vitro suppressor T cells in rat experimental allergic myasthenia gravis (McIntosh and Drachman, 1987) and in normal nonobese diabetic mice (Formby et al., 1988). Some of the suppressor T-cell lines release a soluble factor which is effective in vitro but not in vivo (McIntosh and Drachman, 1987).Whitham et al. (1990) using a model of chronic EAE in mice have described the generation in vitro with CS and myelin basic protein of antigen-nonspecific suppressor macrophages as well as lymphoid suppressor cells which were antigen specific. In contrast to the EAE model, prophylactic treatment of rats with CS to prevent the development of experimental allergic uveitis (Fujino et al., 1988; Mochizuki et al., 1988), guinea pigs to prevent onset of experimental allergic orchitis (Hojo and Hiramine, 1985), and mice to inhibit production of autoantibodies in experimental erythrocyte autoimmunity (Cox et al., 1983), resulted in each model in the prevention of the disease and in the generation in vivo of suppressor cells which, in adoptive transfer experiments, would effectively protect naive syngeneic recipients from disease induction. There is some evidence that in all three models the suppressionmediating cells might have been antigen-specific lymphocytes. Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self-tolerance is maintained in mature T-cell populations. Hiruma and Gress ( 1992) have identified reproducible veto cell activity in marrow cells derived from athymic NCr-nu mice and investigated the effect of CS in such cultures. CS inhibited veto cell-mediated suppression of cytotoxic T-cell responses, and this inhibition correlated with a lack of clonal deletion of precursor CTL by veto cells in the presence of the drug. In addition, CS exerted its effect through precursor CTL and not through veto cells, indicating that precursor CTL may play an active role in their own deletion by veto cells. In the context of suppressor functions, the multifunctional cytokine transforming growth factor /3, which is also a secretory product of T cells, should be considered because it acts as a potent inhibitor of T-cell growth and differentiation. This factor exerts also marked anti-inflammatory effects in experimental models of autoimmune disease (Kuruvilla et al., 1991). Li et al. (1991) have reported that the steady-state level of the transforming growth factor /3 mRNA in the stimulated T cells, in contrast to that of interleukin-2 mRNA, was increased by CS and that this effect was also appreciable at the level of production of functionally transforming growth factor /3 protein. Observations by Khanna et al. (1994) and Prashar et al. (1995) further support the notion that CS, but not the nonimmunosuppres-

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sive (D-MeVal)" -CS (cyclosporin H), may regulate cell growth through a transforming growth factor P-dependent mechanism. Using epithelial cell lines that neither secrete nor respond to interleukin-2, their system permitted exploration of the antiproliferative activity of CS in absence of the interleukin-2 mechanism. Interestingly, their findings advance a mechanism that links, via the transforming growth factor which also augmented endothelin production, the beneficial (immunosuppression) and the harmful (fibrosis, hypertension) consequences of CS usage. According to Wolf et al. (1995) CS stimulates the expression of the transforming growth factor P also in renal cells. Although belonging into another context, the only other lymphokine possibly directly upregulated by CS is interleukin-6. Williamson et al. (1994)have reported that the likely mechanism for CS-induced gingival overgrowth may be caused by the upregulation of interleukin-6 gene expression, as determined by in situ hybridization in gingival tissues from patients taking CS. In conclusion, there is substantial evidence that in vivo CS might dissociate between priming and proliferation on the one hand, and some major effector functions on the other (cf., Fig. 4). Therefore, antigen priming, cellular activation, and proliferation remain apparently normal and may not be modulated in sensitized and CS-treated animals. In contrast, CS effectively inhibits in vivo effector functions of T cells such as the synthesis and release of many lymphokines (but not all), help, cytotoxicity, and mediation of DTH. Generally, it seems that activated effector lymphocytes are not released into the periphery nor are they present or being activated de novo in an allografted organ under CS treatment. However, there obviously exist situations in which effector CTL are present at the periphery (Orosz et al., 1988b,c; Black et al., 1990)and even CS-resistant CTL may be found in the graft (Vaessen et al., 1994). It is of interest to note that ex vivo experiments demonstrate that in allografted but unresponsive rodents their alloreactive T cells remain silent, whereas on in vitro challenge with the same alloantigens they respond almost normally. The question arises as to whether there is a difference in immunogenicity between the strong in vitro antigens and the weak antigens present in a longstanding graft in vivo. In contrast, CS allows priming and full development of memory and suppressor functions. Moreover, proliferation of precursor helper and DTH inducing T cells also seems CS resistant. Not reviewed here (cf., Bore1 et al., 1989, pp. 260-269) is the antibody response by B cells to thymusdependent and thymus-independent type 2 antigens, which is strongly inhibited by CS both in vitro and in vivo, although at relatively higher doses than those needed to suppress cell-mediated immunity. Although not shown in Fig. 4, it has been repeatedly observed in several experimental models that CS exerts rather little effect on IgM synthesis, whereas it clearly inhibits the switching from IgM to IgG antibody production. Recently, Goodlad and Macartney (1995) have examined the effects of differently timed doses

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of CS on the regulation of murine germinal center cell (mostly B cell) proliferation in vivo. When CS was given prior to antigen, the day +4 germinal center cell birth rate was significantly suppressed, most likely due to the inhibition of the initial T-cell dependent B-cell activation. When CS treatment began one day after antigen, the cell birth rate reached a normal day + 4 peak, indicating that recruitment of germinal center precursor cells took place and supporting the concept that precursor B cells were triggered to divide within 24 hours of exposure to antigen. Finally, since all these results and observations do widely concur, there is no compelling evidence suggesting that the immunosuppressive mode of action of CS differs significantly, depending on whether they were obtained from transplantation or from autoimmune models. D. Induction of Antigen-Specific Hypoor Unresponsiveness

Operational or antigen-specific functional unresponsiveness-often misnamed as tolerance-can be defined by three criteria: (1) maintenance of a grafted organ with normal functions, (2) lack of chronic immunosuppression, and (3) specific unresponsiveness of the recipient’s T cells to donor antigens but not to third party. This state of unresponsiveness may depend on clonal deletion and/or clonal anergy and/or active suppression (Brent, 1991; Morris et al., 1993; Kirkberg et al., 1993). Experimentally, induction of specific unresponsivenesshas been attempted in several allograft models, but has been mainly successful in rodents. Various procedures have been used, such as donor-specific transfusions, injection of donor antigens or living replicating cells (e.g., spleen or bone marrow cells), suppressor T-cell stimulation, total lymphoid irradiation, or histocompatibility matching in addition to immunosuppressive agents or combinations thereof (see also George, 1993; Thomson, 1994). There are clearly much fewer cells infiltrating an allograft with CS treatment and the infiltrate shows a preponderance of CD4 T cells over CD8 lymphocytes. T cells expressing suppressor functions play a pivotal role in maintaining unresponsiveness in CS-modified allograft recipients. The state of unresponsivenessis dependent on the persistence of alloantigens. Likewise, major histocompatibility complex class I antigens may be more important than the class I1 products which are more easily depressed by CS. Since enhanced major histocompatibility complex product expression correlates with rejection in transplantation and relapse in autoimmune diseases, the ability of CS to effectively downregulate their expression must be implemental in the induction and maintenance of unresponsiveness. Moreover, CS does not inhibit activation of lymphocytes in the lymph nodes, but it does inhibit their release into the circulation. Surprisingly, lymph node cells do not exert donor-specific suppression when added ex vivo in a mixed

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lymphocyte reaction; however, there is a donor-specific hyporeactivity of lymphocytes in the peripheral circulation. This hyporesponsivenesspertains only to the cytotoxic reactivity but not to the proliferative capacity (for review cf., Bore1 et al., 1989, pp. 307-314). Tolerance induction in connection with CS treatment has been investigated in several transplantation models, mainly in the rodent, canine, and porcine species. Donor-specific unresponsiveness has also been observed in transplant patients. In the former decade, fundamental observations were made while in the more recent publications emphasis has shifted toward the mechanisms operating in the generation of donor-specific unresponsiveness. We shall proceed stepwise starting with the extensive work performed in various rat models. White and Lim (1988) have summarized their studies on the induction of tolerance by CS in the rat heart allograft model. When recipient rats of heterotopic cardiac allografts, which were treated for only 14 days with CS, were challenged with an additional skin graft from the heart donor strain, a substantial donor-specific protective effect was observed by Week 16 postcardiac transplantation. They also found that the administration of CS during the first 2 weeks postgrafting was not optimal for all animals. The best results (i.e., 100% long-term survivorship) were obtained if the drug was given during the risk periods in this model which were determined to be the first, fifth, and eighth week postransplant. In addition, class I or minor histoincompatibilities alone were insufficient to cause cardiac rejection in this model, but CS treatment generated long-term heart allograft survivors. When testing such long-term survivors for their ability to prolong donor skin graft survival, only the CS-treated, heart-allografted recipients were able to prolong skin survival, showing that CS treatment and the presence of an allograft were required for the induction of donor-specific tolerance. The alloreactive repertoire of long-term tolerant rats showed surprisingly that lymph node lymphocytes were generating cytotoxic T cells against donor targets in a mixed lymphocyte reaction similar to those made against third party cells and comparable with those from naive controls. In contrast, the alloreactivity of peripheral blood lymphocytes revealed a donor-specific hyporeactivity. Similar results were reported for the peripheral blood mononuclear cells from renal allografted tolerant dogs in that the alloreactive precursors of donor-specificcytolytic T cells remained silent in mixed leukocyte culture assay (Davies et al., 1988). Taken together, these data indicate that an active immunological process, which is not only CS resistant but may actively require the action of CS, is involved in keeping the allografts resident for long term in their nonimmunosuppressed recipients. Finally, Lim etal. (1990)have reported that the addition of steroids to a CS tolerizing schedulewas detrimental to the induction of tolerance in the above rat model. The inducti,on of tolerance in vivo by CS has been demonstrated during persistant Borna disease virus infection in rats (Stitz et al., 1989). Further

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studies by Stitz (1992) reproduced antigen-specific tolerance induction in vivo by CS in mice infected intracerebrally with lymphocytic choriomeningitis virus. However, in mice infected with vesicular stomatitis virus and immunosuppressed with CS, only a temporal state of antigen-specific unresponsiveness could be achieved. The two first mentioned types of virus cause a persistant productive infection, whereas the latter does not replicate to considerable titers in mice and viral antigens do not persist. These results concur with those from the cardiac allograft model in the rat (White and Lim, 1988) in that early treatment with CS for a limited period can induce immunological tolerance in rats and mice provided that the foreign antigen is available for sufficiently long periods. The intriguing question is how these findings might have a bearing on the use of CS as an inducer of antigenspecific tolerance in clinical transplantation? Yasunami et al. (1990) have successfully induced donor-specific unresponsiveness to each rat strain by a short-course of CS following intraportal grafting of mixed islets from two rat strains as the donor for single transplantation. However, it should be considered that islet cells do not express class I1 antigens. Indeed, it has been clearly established that different organs are rejected with different kinetics. Vriens et al. (1994) have unequivocally demonstrated that tissue-specific differences were critical not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts. They studied various combinations of simultaneous or sequential skin, lung, or heart allografts from PVG into DA rats using preoperatively four doses of rabbit antirat thymocyte globulin or postoperatively ten doses of CS. Subsequent transplants were performed at least 40 days later and without additional immunosuppression. While only cardiac allografts were accepted indefinitely, skin transplantation induced the rejection of subsequently transplanted organs from the same donor strain with accelerated kinetics. Interestingly, lung transplantation modified the recipient’s immune system resulting in long-term acceptance of subsequently placed allografts, regardless of the fate of the primary allograft. Lim and Li (1991) have confirmed that induction of tolerance to vascularized skin allografts, even in a low responder rat strain combination and despite an extended treatment protocol of excessive doses of CS, could not be achieved. The few animals with long-term skin survival over 200 days would eventually reject later; there was one exception. Various protocols have been investigated for their potential to induce donor-specific unresponsiveness in rodents; in particular the use of donor cells or peptides in combination with CS alone or additional immunosuppressive means. Induction of allograft unresponsiveness mediated by suppressor T cells has been demonstrated in heart grafted mice treated with donorspecific blood transfusion and CS (Mottram et al., 1990). Salom et al. (1992) have used a rat cardiac allograft model for tolerance induction involving perioperative injection of donor spleen cells and injection of CS (25 mg/kg

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i.m.) at Day 2 after transplant. They could show that this treatment protocol was associated with suppression of interleukin-2 and y-IFN production, and that the absence of these molecules may promote T-cell receptor occupancy by alloantigens which as a consequence induces a state of anergy in vivo. Jamshidi et al. (1991) have obtained allograft tolerance with neonatal skin in mouse recipients treated with various combinations of antilymphocyte serum, bone marrow cells, and a limited course of CS. This specific tolerance could not be abrogated by injection of normal donor spleen cells and, when the recipients were challenged much later, it was extended to adult donor tissue grafts, whereas third-party grafts were rejected promptly. Induction of donor-specific unresponsivenesshas been achieved in a rat renal allograft model with soluble or membrane-bound class I major histocompatibility complex antigens and a subtherapeutic dose of CS (Foster et al., 1992). Similarly, rat heart allografts survived indefinitely when transplanted into recipients treated with a synthetic class-I derived peptide in combination with a subtherapeutic dose of CS (Nisco et al., 1994).This treatment resulted in long-term donor-specificunresponsivenesswhich appeared to be mediated by anergic donor reactive cells and there was no evidence for the involvement of suppressor cells. Using the rather weak renal allograft model of LEW into DA rats, Simms et al. (1980) found that established allografts were not rejected when reimplanted into new, untreated allogeneic hosts. Drug persistence and clonal deletion could be ruled out because neither factor could apply to the secondary host. In contrast, when examining antigen modulation of vascularized osteochondral allografts in the strong DA into LEW rat stain combination, Sakai et al. (1993)observed that after reimplantation of established allografts from immunosuppressed recipients into naive, untreated recipients all transferred grafts were rapidly rejected. However, when host tolerance was examined by removal at various periods after cessation of CS and reimplantation of a fresh allograft into the same recipient, some of the second grafts survived for prolonged periods without immunosuppression, indicating that some degree of donor-specific unresponsiveness had developed. The cellular and molecular mechanisms that allow donor-specific unresponsivenessin rats with long-term cardiac allograft survival after treatment with a short course of CS have been analyzed by Pearce et al. (1993a,b). In the model of DA recipients of PVC heart allografts the CD4 T cells from unresponsive rats transfer suppression to irradiated DA rats with PVG grafts. However, these CD4 suppressor cells from CS-treated rats with longsurviving grafts respond normally in various test systems to PVG alloantigen (Hall et al., 1990). Moreover, these CD4 suppressor cells are short lived and their survival depends on contact with specific alloantigens and cytokines, one of which is interleukin-2. In the absence of these factors, the CD4 suppressor cells did regain the capacity to initiate graft rejection in irradiated

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rats (Pearce et al., 1993b). CD8 cells failed to transfer unresponsiveness to irradiated rats, but the host-derived CD8 cells are important in reestablishing unresponsiveness (Hall et al., 1990). It was further demonstrated by Pearce et al. (1993a) that CD4 cells from CS-treated rats retain their capacity to transfer specific unresponsiveness only if maintained in a cytokine-rich supernatant. In Vitro they exert normal alloreactivity in mixed leukocyte cultures and in a cell-mediated lysis assay to donor-specific and third-party alloantigens. The unexpected reemergence of normal reactivity may relate to the greater immunogenicity of donor strain cells in the mixed leukocyte reaction, compared with the relatively weaker stimulus of the donor heart. This discrepancy between in vitro and in vivo studies renders these conventional in vitro tests inappropriate for detecting whether a recipient has developed donor-specific unresponsiveness to an allograft. In contrast to the specific unresponsiveness described in the rat cardiac allograft model by Hall et al. (1990) and in which suppressor T cells play a key role, the tolerance state in the murine lymphocytic choriomeningitis virus model investigated by Stitz (1992) does not appear to depend on the action of suppressor cells, since adoptive transfer of spleen cells from CStreated, infected donors did not reveal evidence for the presence of operative virus-specific cytotoxic effector cells. In addition, in the Borna disease virus model in the rat, adoptive transfer of spleen cells from CS-treated, virusinfected donors into infected, cyclophosphamide-treated recipients did not result in pathological changes, whereas spleen cells from virus-infected, but CS-untreated animals, caused clinical symptoms (Stitz et al., 1989). This again demonstrates, like in the above murine model, that CS-treated, virusinfected rats do not respond immunologicallyto viral antigen, which strongly suggests that this tolerance is not affected by the emergence of suppressor cells. Wood and Monaco (1980)had shown that the survival of skin allografts in antilymphocyte serum-treated mice could be significantly prolonged by injection of donor bone marrow one week postgrafting. Moreover, spleen cells of such mice bearing long-term skin grafts were able to transfer unresponsiveness to secondary syngeneic recipients. The state of unresponsiveness was specific and was shown to be mediated by suppressor T cells. In further experiments CS was added to this protocol to explore whether it would act synergistically to induce a more durable unresponsiveness. When CS was started on Day 9 (after marrow on Day 7 and skin on Day 0) in antilymphocyte serum-treated mice injected with bone marrow, the skin allografts survived over 150 days in more than 50% of the recipients (Wood et al., 1988). The synergistic effect of CS in this trebble treatment was suggested to be due primarily to the ability of CS to promote the expansion of suppressor T cells, since adoptive transfer assays indicated that the spleens had a more potent and/or larger number of suppressor cells than in the spleens obtained with the previous protocol without CS.

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Tchervenkov et al. (1995) also explored the effect of CS on the induction of tolerance using a stringent rat cardiac allograft model. The longest survival rate was observed when donor-specific transfusions were given both periand postoperatively in combination with a prolonged posttransplant CS coverage of 60 days. The addition of an anti-T cell monoclonal antibody did further improve the results. A similar protocol was used in a canine renal allograft model to determine synergism of the three components in a higher species and to examine the in vitro (ex vivo) reactivity of lymphocytes from long-term survivors. Kidney allografts were sustained throughout the CS treatment period (20 mg/ kg/day for 60 days) without rejection in six out of eight recipients receiving antilymphocyte serum plus fractionated donor bone marrow and CS. Peripheral blood or bone marrow cells from long-term survivors proliferated normally in response to mitogens and were responsive to third-party cells in a mixed lymphocyte culture, but did not have reduced responsiveness to donor alloantigen in all cases. These results show that long-term renal allograft survival with donor-specific functional unresponsiveness can also be obtained in the dog with the combined treatment of antilymphocyte serum plus fractionated donor bone marrow and a limited course of CS (Hartner et al. 1991). These experiments in dogs were extended and included CS treatment for 120 days. Using divided donor-derived bone marrow cell dosing, it was clear that this treament led to improved graft function during CS therapy. Paradoxically, although additional posttransplantation immunosuppression with antilymphocyte serum to bracket the day + 1 of bone marrow infusion promoted graft function and prevented rejection during maintenance therapy with CS, it appeared to prevent developments that can promote long-term graft survival without immunosuppression (Hartner et al., 1995). Specifictolerance and the resulting immunocompetencewas investigated by Deeg et al. (1987) in bone marrow allografted dogs which were treated with methotrexate and CS. Only haploidentical (not completely allogeneic) chimeras became immunocompetent against third-party antigens. By one year after transplantation they still failed to generate cytotoxic cells against host cell targets. There was evidence that both clonal abortion and the presence of specific suppressor cells were associated with the state of tolerance observed in these canine chimeras. Further attempts to produce specific tolerance in adult animals have been performed in the porcine renal allograft model. Kamada et al. (1983) had demonstrated earlier that liver allografts in rats induce tolerance to themselves when transplanted accross complete major histocompatibility antigenic mismatches without any immunosuppression. Liver grafts are known to be less susceptible to immune injury. However, there was a substantial, but self-limiting allograft reaction in the liver that resolved spontaneously, leaving the recipient specifically tolerant to donor tissues. During

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the allogeneic engagement donor Kupffer cells were replaced by those of recipient origin (Gassel et al., 1987) and donor leukocytes migrating from the graft survived long term in the tolerant recipients (Sriwatanawongsa et al., 1993). Calne et al. (1994) have since attempted to reproduce the tolerizing liver effect by injecting donor spleen cells intravenously at the time of kidney grafting to pigs given, in addition, 7 days of CS, leaving a dose-free window of 48 h designed to allow a transient interaction between the graft (cells plus kidney) and the recipient, in order to favor the induction of a specifically tolerant state. This protocol was effective in securing long-term kidney graft survival resistant to challenge by donor skin grafting in about half of the recipient pigs. Donor skin was, however, rejected but more slowly than third-party skin. Sophisticated studies on the induction of specific unresponsiveness in kidney allograft transplantation in miniature swine have been performed by the group of D. H. Sachs. Two-haplotype class I disparate, class I1 matched donor-recipient pairs were used and the recipients were treated with CS (10 mg/kg/day i.v.) for 12 days only. This protocol led to the induction of tolerance in all recipients, whereas the nonimmunosuppressed controls had rejected their grafts within 2 weeks. Subsequent challenge with skin grafts showed prolonged donor-specific survival of the grafts compared with skin bearing third-party class I antigens. Tolerant recipients had markedly diminished or absent antidonor mixed lymphocyte as well as cellmediated lymphocytotoxicity responses, but maintained normal reactivity to third-party antigens. Some recipients had detectable levels of antidonor IgM antibodies, while none demonstrated the presence of antidonor IgG which was found in all rejecting controls (Rosengard et al., 1992). These results concur with the increasing evidence that CS appears more effective in suppressing antidonor class I responses to allografts when help is generated solely through the indirect pathway of alloactivation (see also Fukuzawa and Shearer, 1989). In another study, the same short course of immunosuppression by CS was used in renal allografts mismatched selectively for two haplotypes at class I1 (Fishbein et al., 1994). Long-term graft survival was observed in five out of seven miniature pigs and the five acceptors expressed specific tolerance by their response either to donor-matched skin grafts or to a second donor-matched kidney transplant without further immunosuppression. In conclusion, the results of these two studies suggest the existence of a common pathway for induction of specific transplantation tolerance to major histocompatibility complex antigens, when these antigens are recognized on vascular endothelium under conditions of altered cytokine production as effected by CS. The same short course of CS treatment failed consistently to produce specific unresponsiveness in pigs transplanted with allografts bearing class I plus class I1 disparities. However, the drug does prolong survival of fully

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mismatched grafts which are ultimately rejected at the latest when drug treatment is discontinued. Therefore, it was attempted to induce tolerance to a fully allogeneic kidney graft by injecting single-haplotype class IImismatched bone marrow before transplanting a kidney from a donor class 11-matchedto the bone marrow donor and treating the recipients with a 12day course of intravenous CS (10 mglkglday). All four pigs had normal renal function for over 200 days. They had specific hyporesponsiveness to both bone marrow and renal donor-type alloantigens in mixed lymphocyte culture and cell-mediated lympholysis assays. Significant donor-specificskin graft prolongation compared to third-party grafts was also observed (Smith et al., 1992). Gianello etal. (1995b)have further studied tolerance induction in donorrecipient combinations sharing one full haplotype, which mimics the clinically relevant transplant combination of parent to offspring.Whereas nonimmunosuppressed controls uniformely rejected their kidney allograft around 11 days, an intravenous course of CS (10-13 mgkglday) during the first 12 postoperative days induced longterm acceptance of the allograft in 4 out of 6 recipient miniature swines. Their results suggest the feasibility by CS of inducing specific tolerance across a single haplotype mismatch in the majority of the cases. The mechanisms allowing for induction and maintenance of donorspecific tolerance were investigated in this model. Rosengard et al. (1991a) tried to break tolerance to class I disparate renal allografts in miniature swine by placing multiple skin grafts bearing donor class I plus third-party class I1 antigens on tolerant recipients. Prior to the skin graft challenge these animals had no detectable antidonor activity in mixed lymphocyte reaction or cell-mediated lymphocytotoxicity assays, but after repeated skin grafts they developed marked antidonor immunity without a break in tolerance. When such animals were retransplanted with kidneys matched to their first allografts, the second graft was accepted permanently without further immunosuppression, indicating that graft adaptation was not necessary for the maintenance of tolerance (Rosengard et al., 1994). It was further demonstrated that the CTL generation was directed toward the identical donor class I antigens as expressed by the kidney donor and that detection by CTL of peptides expressed by skin but not by kidney could not explain the results, since skin grafts from the kidney donor were also prolonged. Under this special condition (limited to class I difference only), the helper pathway necessary for activation of precursor CTL appears to be suppressed in vivo in tolerant animals, possibly by differences in patterns of cytokine expression by graft infiltration cells (cf., Gianello et al., 1994). In contradistinction, the sensitization by skin grafts elicited in these long-term tolerant swines antibody production against class I1 and antithird-party class I antigens but not against antidonor class I antigen (Gianello et al., 1995a). These findings

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suggest that in this model tolerance is a stable, centrally mediated phenomenon that cannot be broken by donor antigen. Rosengard et al. (1991b)isolated graft infiltrating leukocytes from tolerated kidneys. These cells were lacking antidonor cell-mediated lymphocytotoxicity and were shown in coculture experiments to suppress antidonor but not antithird-party cell-mediated lymphocytotoxicity. Therefore, it was concluded that local specific suppression may be a contributing factor to maintain tolerance. Finally, there has been evidence for a T-cell help deficit at the time of the first exposure of the host’s immune system to alloantigens, resulting from a deficiency of interleukin-2 induced with the perioperative course of CS. To prove this assumption, Gianello et al. (1994)have administered intravenously exogenous recombinant interleukin-2 on the postoperative Days 8 to 10 to animals receiving the full CS tolerizing regimen. This led to acute rejection in all animals. Thus, limitation of T-cell help at the time of first antigen exposure seems to be required in this porcine model to prevent rejection during the time necessary for active tolerance to develop. However, maintenance of tolerance does not seem to require continuous limitation of T-cell help. Permanent lymphohematopoietic chimerism has also been induced in major histocompatibility complex-disparate miniature swine by bone marrow transplantation after lethal total body irradiation. Guzzetta et al. (1991) have shown that this procedure led to permanent tolerance to a bone marrow donor-matched vascularized allograft (kidney) without requirement for exogenous immunosuppression. Such animals, however, had regained ex vivo responsiveness to third-party alloantigens. Kawai et al. (1995) have used a nonmyeloablative preparative regimen to induce mixed chimerism and renal allograft tolerance between major histocompatibility complex-disparate cynomolgus monkeys. Their protocol included nonlethal, fractionated total body irradiation, local thymic irradiation, antithymocyte globulin to deplete mature T-cell subsets, donor bone marrow infusion, and the addition of CS for one month. All four monkeys receiving this regimen developed multilineage chimerism and accepted longterm renal allografts with no further treatment. They showed donor-specific nonreactivity as confirmed by mixed lymphocyte reaction and skin transplantation. Their experiments demonstrated that at least transient engraftment of the donor bone marrow inoculum appears to be essential for establishing chimerism as the prerequisite for inducing donor-specific tolerance in this preclinical nonhuman primate model. To make this protocol more clinically applicable, the morbidity of the preparative regimen should be reduced, possibly by the use of radiomimetic drugs rather than irradiation, and the timing of T-cell depletion with respect to the kidney transplant would have to start not more than 24 h prior to transplantation for cadaver donor transplants.

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Nowadays, many human recipients of organ transplants develop longterm functioning grafts, suggesting that they undergo induction and maintenance of donor-specific hypo- or unresponsiveness. Wramner et al. (1987) have used cell-mediated lympholysis to test the donor-specific response of peripheral blood mononuclear leukocytesfrom transplant patients with wellfunctioning, one haplotype-mismatched kidney grafts from related donors. These recipients were transfused prior to transplantation and treated with conventional immunosuppression, i.e., without CS. There was a donorspecific cellular unresponsiveness,as indicated by an abolished cell-mediated lympholysis reactivity against donor cells, whereas the antithird-party response remained normal. There was also evidence for the presence of a donor-specific cellular suppressor mechanism underlying the specific unresponsiveness in those long-term kidney allograft recipients. Recently, Wramner et al. (1993) studied the cell-mediated immune responses in renal transplant recipients treated with CS and prednisolone with and without azathioprine. The two CS protocols allowed the induction of donor-specific unresponsiveness in terms of cell-mediated lympholysis in nearly all recipients and in terms of mixed lymphocyte reaction in half of the patients with stable renal function after the first transplant year. The antithird-party reactivity was low during the first posttransplant year in recipients with triple therapy but not in those without azathioprine. There are more clinical data available in the literature supporting the concept that immunologic adaptation as expressed by hyporeactivity to donor antigens following transplantation does occur. However, the problem remains to assess with reliability the immune status and the graft outcome of patients who appear to have developed donor-specific hyporeactivity, as determined by in vitro assays in order to decide whether it is possible to withdraw or reduce maintenance immunosuppressive therapy. It is known that class I antigens serve as the primary target structures for cytotoxic T cells, while class I1 targets stimulate the majority of the proliferative response assayed in a mixed lymphocyte reaction (for review cf., Bach, 1985). Although a selective loss of functional antidonor cytolytic T-cell precursors following donor-specific blood tranfusions in long-term renal allograft recipients has been demonstrated (Hadley et al., 1992) and in many instances a diminished or abolished cytotoxic reactivity in the cell-mediated lympholysis assay has been observed, the correlation of cell-mediated lympholysis hyporeactivity with graft survival has only limited prognostic value (Goulmy et al., 1989). Given the important role that CD4 cells, presumably reactive with allogeneic class I1 antigens, play in graft rejection, Reinsmoen et al. (1990) have suggested that decreased proliferative response, reflecting primarily the CD4 cell response, may be a better indicator of immunologic adaptation to the graft than hyporeactivity of cytotoxic cells. Proliferation may correlate with the relative absence or nonreactivity of reactive cells that would result in graft rejection. Therefore, their alternative approach for

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determining donor-specific hyporeactivity or unresponsiveness has been to identify hyporeactivity to stimulation by homozygous typing cells defining the disparate donor human leukocyte class I1 antigens DR and DQ present on the donor organ. Development of donor-specifichyporeactivity was demonstrated in about one-third of the patients immunosuppressed with CS tested 1 year posttransplantation (Reinsmoen et a!., 1990, 1991). Very recently, there has been an exciting report by Ramos et al. (1995) about a prospective clinical trial in which 59 long-term liver transplant recipients had been selected for stepwise drug weaning. Complete weaning was accomplished in 16 patients, in 28 recipients drug weaning was still progressing, and in 15 patients this attempt had failed but without graft loss. In spite of the inability to accurately predict which patients can be successfully weaned, it became clear that a significant percentage of selected long-surviving liver recipients can unknowingly achieve drug-free graft acceptance. Two major points must be emphasized here. Firstly, it has become clear that tolerance induction and maintenance cannot be attributed to a single mechanism but to a combination of interacting pathways. Secondly, the assessment of the immune status of a stable transplant patient, as determined by his reactivity in various in vitro assays, reflects only the status quo under the prevailing conditions (Goulmy et al., 1989) and has no reliable predictive value for altered conditions, i.e., when immunosuppression is being modified or under the outbreak of even benign viral infections. Indeed, a virus can act as a most powerful immunostimulator which will promptly upset the established but fragile immune balance. Such changing conditions might explain sudden and unexpected rejection crises to occur in hitherto seemingly stable and donor-specifichyporeactive patients (e.g., Wijngaard et al., 1993). In view of these facts, it is evident that the suppressive spectrum of CS should be advantageously enlarged through its combination with other selective means and agents which might act in an additive or possibly synergistic fashion. This approach might obviate the clinical side effects observed with higher doses of CS alone. It could also markedly enhance the suppressive potency during the early sensitization phase and, thereafter, be more effective in inducing and sustaining donor-specific unresponsiveness or even tolerance. The optimal drug combinations may not necessarily be the same for supporting each of these different immunological steps. Basic considerations for establishing immunosuppressive protocols with CS were reviewed by Bore1 et al. (1989, pp. 298-307). However, with the advent of new chemical agents much experimental work has been achieved (Hollander et al., 1996; Kahan et al., 1993,1994; see also experimental papers in Transplant. Proc. 26, 3025-3061, 1994). Promising results have recently been obtained by combining moderate to low-dose CS with monoclonal antibodies, e.g., antiinterleukin-2 receptor MAb (Hancock et al., 1990; Ueda et al., 1990), antitumor necrosis factor-a MAb (Seu et al., 1991), anti-CD4 MAb (Lu

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and Borel, 1995) or with the soluble CD 28 receptor analogue (Bolling et al., 1994). These efforts may effectively further contribute to “the quiet revolution of immunosuppressive therapy” as it has been referred to by Bach (1994).

IV. Other Biological Effects Associated with the ImmunosuppressiveActivity As shown in Table 11, several biological effects other than immunosuppression exhibited by CS and some of its analogues are either linked or in some yet unknown manner associated with the immunosuppressive activity.

A. Chronic Inflammation Since the chronicity of an inflammatory reaction seems primarily caused and maintained by the direct participation of activated T lymphocytes, it seems evident that the potent anti-inflammatory activity of CS and some of its analogues are mediated through their suppressive effect on T cells. In particular, CS exerts a potent suppression in most arthritis models (Wong, 1993; Kovarik et af., 1994b). However, there exists intriguing differences between some derivatives which still remain unexplained. Thus, the derivative (Va12)dihydro-CS(as well as a few others) is not only effective in suppressing experimental allergic encephalomyelitis,but also sustains remission in the chronic relapsing encephalomyelitismodel in rats and prevents further relapses even after treatment has stopped which is in marked contrast to CS (Chabannes et af., 1992; Feurer et af., 1988). Whereas CS only delays the onset of the disease relapse until discontinuation of treatment, certain lysolecithin derivatives, especially SDZ MLS 337, also exerted in most animals a curative effect, although this compound lacks the activity spectrum typical for immunosuppressant drugs (Kovarik et af., 1995). SDZ MLS 337 is a cyclic ether analogue of the antitumour agent ET-18-OCH3 and the active enantiomer of SRI 62-834 (Chabannes et af., 1992). In addition, the latter compound showed efficacy in the treatment of experimental autoimmune diseases, such as systemic lupus erythematosus and Lyme arthritis in mice (P. Hiestand, Sandoz Pharma, Basel; manuscript in preparation).

B. Prolactin Antagonism Hypophysectomy or administration of prolactin inhibitors results in suppression of the immune response which is reversed by the administration of prolactin. Specific binding sites for prolactin have been demonstrated on human peripheral blood lymphocytes from which prolactin is displaced by CS, but not by a biologically inactive analogue (for review cf., Borel et al.,

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1989; page 306). CS acts also as a competitive antagonist to prolactin receptors on a breast tumor cell line and inhibits the growth of these mammary cancer cells (Bernard et al., 1991).Clevenger et al. (1991)have demonstrated the requirement of nuclear prolactin for the proliferation of cloned T lymphocytes in response to interleukin-2 stimulation. Using highly purified B cells from tonsil samples, Morikawa et al. (1993) were able to suppress in vitro the early stage of the proliferative response of resting and activated B cells with bromocriptine, a dopamine ergot alkaloid, which inhibits the release of pituitary prolactin. The same authors also demonstrated that bromocriptine suppresses T-cell proliferation by means of blocking interleukin-2 production by T cells as well as the mixed lymphocyte reaction in a dose-dependent manner (Morikawa et al., 1994). Wqra et al. (1995) reported that transcription of the human prolactin gene was specifically inhibited by CS and tacrolimus in vitro, whereas rapamycin exerted a more general effect on transcription and/or translation in pituitary cells. Chen and Johnson (1993) further showed that treatment with prolactin enhanced the respiratory burst and phagocytosis of peritoneal macrophages in mice. Moreover, Compton et al. (1994) have demonstrated in vivo that the use of bromocryptine alone was sufficient to significantly prolong both allogeneic and xenogeneic skin graft survival in a mouse model. Since the evidence that prolactin may be involved in the maintenance of T-cell immunocompetence is accruing (Russell, 1989), it was suggested by Hiestand et al. (1986a) that the immunosuppressive effect of CS might also be mediated by its competitive prolactin antagonism. Morikawa et al. (1994) have recently reported an additive effect for the suppression of Tcell proliferation and CD2S antigen expression in vitro when combining both bromocriptine, a dopamine type 2 agonist which prevents the secretion of pituitary prolactin, and CS or tacrolimus (FK 506). Hiestand et al. (1986b) used bromocriptine with low dose CS for preventing renal allograft rejection or localized graft-versus-host reaction in rats and they observed a superadditive inhibitory effect. Several other groups have confirmed the clear additive effect of this drug combination, e.g., in the cardiac allograft model in rats (Wilner et al., 1990; DiStefano et al., 1990), in the rat heart-lung en bloc allograft model (Eckes et al., 1988), the pancreas allograft model in rats (Ferrero et al., 1987), and also in the experimental autoimmune uveitis model in the rat (Palestine et al., 1987). However, it is essential to treat the animals with bromocriptine or a similar drug both before and after antigenic challenge in order that their prolactin level is well depressed at the time of immune reactivity. The opposite has also been shown, namely that restoration of prolactin levels to normal, either by adding prolactin in vitro to a cell line (Yu-Lee, 1988) or by treating animals with a prolactin-releasing compound (P. C. Hiestand, Sandoz Pharma, Basel; unpublished results) does effectively restore the depressed immune response in several test systems.

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Some clinical trials have also been performed to assess the effect of lowered prolactin levels in CS immunosuppressed patients. Thus, Carrier et al. (1990)have used bromocriptine as an adjuvant to CS in heart transplanted patients. Administration of bromocriptine (three daily doses) appeared to improve the immunosuppression by CS, at least during the early postoperative period when the risk of rejection and infection is higher. Three other similar clinical trials were done in autoimmune diseases. A small open trial in rheumatoid arthritis failed to show any significant difference in clinical and laboratory variables, possibly due to subtherapeutic bromocriptine dosage (Dougados et al., 1988). In a study including 14 corticosteroid-resistant patients with chronic sight-threatening uveitis, the combination of bromocriptine (three to four times daily) with low-dose CS led to significant improvements in vision or in inflammation in 10 out of 14 patients after 6 months of therapy (Palestine et al., 1988). Finally, an open pilot study was undertaken by Atkinson et al. (1990) to test the combined effects of bromocriptine (three daily doses) and CS in the treatment of newly diagnosed patients with insulin dependent diabetes mellitus. The results did not indicate a significant benefit from concurrent bromocriptine and CS therapy, except that there was a trend that bromocriptine might have some protective effect on preserving endogenous insulin secretory capacity. In conclusion, too little experimental and clinical attention has yet been given to explore the important neuroendocrine-immune connection and the promising avenue to successful hormonal manipulations of the immune process after organ transplantation or in autoimmune diseases. C. Possible Interactions with the Central Nervous System

Felten et al. (1985) have demonstrated that lymphoid organs are innervated by both the sympathetic and parasympathetic parts of the autonomic nervous system. Furthermore, lymphocytes have binding sites for numerous neurotransmitters and neuropeptides which are able to modulate cellular immune responses and natural killer cell activity (O’Dorisio et al., 1985; Smith et al., 1985). Therefore, a functional relationship between the immune system and the neural and neuroendocrine systems was postulated. The participation of the immune system in processes primarily considered to be central nervous system phenomena has been suggested by several studies demonstrating the ability of various immune modifiers to attenuate opiate withdrawal severity. CS (Dafny et al., 1985b), a-interferon (Dafny et al., 1985a), cyclophosphamide (Montgomery et al., 1985) or destruction of the immune system by y-irradiation (Dafny and Pellis, 1986; Dougherty et al., 1990) dramatically reduced the severity of naloxone-precipitated withdrawal in morphine-dependent rats. These findings suggest that opiate addiction may at least in part involve the immune system. In the case of a -

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interferon it has been speculated that this immunoenhancing compound may be involved with opiate receptors, since it has opiate-like properties in vitro and in vivo (Blalock and Smith 1981).In contrast, CS and y-irradiation exert potent immunosuppressive activities that have no demonstrable effects on opiate receptors. This enigmatic relationship between the immune system and morphine withdrawal remains controversial. Dantzer et al. (1987) were unable to observe after treatment with CS or a-interferon an attenuation of the reduction in body weight or of the behavioral suppression induced by naloxone in morphine-dependent rats trained to press a lever for food reinforcement on a fixed-ratio schedule. Unfortunately, these authors did not study the possible effect of CS and a-interferon on the spontaneous behavioral withdrawal syndrome. In spite of this, they suggest that the interaction demonstrated previously (Dafny et al., 1985a) may only be the consequence of disturbances in thermoregulation. In contradistinction, Berthold et al. (1989) have demonstrated that CS significantly suppressed the spontaneous stereotyped behavior characteristics for the naloxone-induced morphine withdrawal syndrome in mice. However, this effect was obtained at a dose of CS which is regarded as too low for being immunosuppressive in the mouse. Since this immunosuppressive agent was also effective in the nude mouse lacking an intact immune system or after selective ablation of the immune system by whole body irradiation, a direct effect of CS on the central nervous system structure was postulated. Nevertheless, specific cellular activitiesor factors derived from lymphoid cells are probably required for the expression of opiate withdrawal as demonstrated in the case of the behavioral prevention observed after irradiation: adoptive transfer of splenocytes to irradiated rats prior to chronic morphine treatment restored the severity of all withdrawal signs precipitated by naloxone. In contrast, adoptive transfer of fractionated splenocyte subpopulations only partially restored withdrawal severity and transfer of irradiated splenocytes, red blood cells, or diluted numbers of normal splenocytes did not have any noticeable restorative effect (Dougherty et al., 1987, 1990). Moreover, a potential role of the opiate alkaloid-selective(mu3)receptor cannot be excluded, since this receptor is proposed to be an important neuroimmune link (Makman, 1994).This very link is likely to play a significant role in a variety of responses involving the immune system, including the response of the organism to stress. Unfortunately, the affinity of CS for this type of receptors has not yet been investigated. Although the few papers published on this topic express controversial viewpoints, it appears reasonable to postulate a potential pharmacological role for CS, and possibly some of its derivatives, which may interact along the neuroimmune connection. [Studying further this connection, Gardier et al. (1994)presented data which suggest that the decrease in brain serotonin levels in rats, that occurs after antigen administration may reflect a specific short-lasting CS-dependent

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release of serotonin at frontocortical nerve terminals at a time when the splenic immune response is maximal.]

D. Major Side Effects Nephrotoxicity, hepatotoxicity, and hypertension are the major side effects of the immunosuppressive cyclosporins both in preclinical studies and in the clinic (Ryffel, 1982; Mihatsch et al., 1986; Feutren and Mihatsch, 1992; Mimran et al., 1995). As mentioned, we have not succeeded in achieving a wider therapeutic window of biological significance in the clinic with any CS derivatives. However, there were a number of quantitative differences in adverse reaction profiles of some of these compounds in experimental animals, especially (Nva12)-CS(cyclosporin G) and SDZ IMM 125. Because certain selected nonimmunosuppressive derivatives possessing other pharmacological activities show partly differing side effect patterns, this would indicate that some of these, in particular nephrotoxicity and hypertension, may be closely associated with immunosuppression. Therefore, adverse reaction profiles obtained in rat models were compared between CS, some other interesting immunosuppressive analogues and several selected cyclosporins lacking significant immunosuppression. The list of compounds is shown in Table V. The adverse reaction profiles of these cyclosporin derivatives have been investigated for the selection of least toxic compounds in Wistar, Sprague Dawley, or spontaneously hypertensive (Kyoto) rats. These animal models were used at an early developmental stage when only limited amounts of compounds were available. The experiments were always carried out in parallel with the reference compounds CS or (Nvalz)-CS (cyclosporin G) under equidosed conditions. The route of administration was oral by gavage or by subcutaneous injection. The compounds were dissolved in a maize or olive oil solution given orally at a volume of 5 mVkg or subcutaneously at a volume of 1 mVkg. With the exception of all immunosuppressive compounds and of SDZ PSC 833, which is presently in phase I1 development, all nonimmunosuppressive analogues were only administered for 2 weeks to assess their adverse reaction profiles. The parameters examined included clinical signs, body weight development and food consumption, clinical chemistry in serum and urine, creatinine clearance, macro- and microscopical pathology of major organs, such as liver, kidney, gastrointestinal tract and lymphatic tissues. In addition, an unspecific radioimmunoassay was used to measure plasma concentrations in order to gain an estimate of the systemic exposure of the animals to the compound. In some experiments designed for optimal dosage selection and for investigating the responsive level of clinical chemistry parameters, CS was orally or subcutaneously administered at 12.5, 25, and 50 mg/kg/day for 10 days. The results obtained in normotensive Wistar male rats and in spontaneously hypertensive male Kyoto rats showed in both strains that

TABLE V

List of Immunosuppressive and Nonimmunosuppressive Cyclosporins Analyzed for Toxicological Effects in the Rat Model

SDZ number Immunosuppressive cyclosporins SDZ 027-400 SDZ 037-325 SDZ 216-125 SDZ 034-271 Nonimmunosuppressive cyclosporins SDZ 037-049 SDZ 037-839 SDZ 211-811 SDZ 215-833 SDZ 205-120 SDZ 203-218

Chemical name

Other nomenclature

Cyclosporine (CS) (Nva)'-CS D-Ser(0-Zhydroxyethyl) *-CS ( Val)'dihydro-CS

Cyclosporin A Cyclosporin G

MeBmt(3-deshydroxy)'-CS (D-MeVal)"-CS (Melle)'-CS MeBmt(3-keto)'-Val'-CS MeBmt(8-OH) '-CS MeBmt(8-OH-8-oxo)'-CS

Cyclosporin F Cyclosporin H Cyclosporin 29 (3-keto)-CyclosporinD AM1; M17 AMlA

Dihydro-Cyclosporin D

References

(OL 27-400) (OG 37-325) (IMM 125) (OD 34-271)

(NIM 811) (PSC 833) (OL 17)

1,9,10 2 3 4 2 2 5 6 798 798

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magnesium, urea, and bilirubin were the most sensitive parameters (Table VI). They were affected at the lowest dose of 12.5 mg/kg/day, corresponding to an exposure in the range of 30 to 40 pg.h/ml and peak plasma concentrations in the range of 1.2 to 2 pglml after either subcutaneous or oral administration. As shown in Table VII, the effects were generally more marked in the Kyoto rat at similar exposure than in the Wistar rat. This also was true for histopathological findings, which mainly consisted of focal tubular regenerative changes, tubular vacuolizations, corticomedullary mineralization and atheropathies in the kidney, and single hepatocellular necrosis and bile duct hyperplasia in the liver (data not shown). The dosedependence of adverse reaction profiles and exposure values of the immunosuppressive cycloporins CS and SDZ IMM 125 are listed in Table VIII. These data clearly indicate marked quantitative differences of toxicity patterns between these pharmacologically equipotent immunosuppressive cyclosporins, especially if considering the respective exposure values. When the first toxicological profiles on the nonimmunosuppressive cyclosporins MeBmt (3-deshydroxy)'-CS (cyclosporin F) and (D-MeVal)"CS (cyclosporin H) were obtained, they showed clear differences from the immunosuppressive cyclosporins in several aspects (Table VIII). As was to be expected, they did not cause atrophy of the lymphatic tissues as it occurred at high dose levels with the immunosuppressive cyclosporins. An important finding was that the contrasting profiles of these two nonimmunosuppressive natural cyclosporins occurred at exposure values at which the reference compound CS responded (cf., Table VI). In addition, it was interesting to note that the effects on renal parameters, including also creatinine clearance, were absent compared with CS and other immunosuppressive cyclosporins. Liver specific parameters, in particular total bilirubin, were also much less affected. These markedly attenuated effects of the two nonimmunosuppressive cyclosporins were confirmed by the histopathological results. Similar results as for these two compounds were also obtained at a later stage with other nonimmunosuppressive cyclosporins. Another characteristic difference between immunosuppressive and nonimmunosuppressive cyclosporins was observed when serum magnesium was included in the toxicologic chemistry profile. This parameter had been shown to be affected in early human trials with CS. Similarly, administration of CS and several other immunosuppressive cyclosporins to rats caused a dose-dependent moderate to marked decrease of serum magnesium and magnesium wasting (Mason, 1989). It was of great interest also to see that the selected cyclosporin D derivative (Val)2dihydro-CS(SDZ 034-271) caused CNS effect in the form of reversible impairment of locomotion and excitatory behavior (not shown on Table VIII). To demonstrate similar CNS effects with CS, dose levels of 100 mg/kg/day were necessary. These adverse effects might at least partly be associated with the marked decreases in serum magnesium. No effects on serum magnesium were seen with nonimmunosuppressive cyclosporins

Dose Dependency of Clinical Chemistry Profiles in Wistar and Kyoto rats after 10 day Subcutaneous Administration. Changes Relative to Controls Which are Equal to 1.0

TABLE VI

Dose level mglkgld

12.5 25 50 12.5 25 50

Rat strain

Wistar

Kyoto

Mgt +

Urea

Creatinine

Cr-CI

Bilirubin

Cmax pglml

A UC pg.hlml

0.7 0.7 0.6 0.7 0.75 0.8

1.3 1.45 1.5 1.4 1.8 2.3

1.1 1.25 1.3 1.2 1.35 1.6

0.85 0.8 0.7 0.8 0.6 0.5

1.4 1.7 2.1 2.6 2.6 3.1

1.3 3.5 7.2 1.4 2.9 6.3

29 76 155 30 67 135

TABLE VII Adverse Reaction Profiles of Cyclosporins in Wistar Rats after I 0 day Administration. Changes Relative to Controls (n = 12) Which Are Equal to 1.0 Histopafhology" mean scores

Compound

cs SDZ 027-400

SDZ 216-125

a

Dose level mglkglday

N

Mg

Urea

Creatinine

Bilirubin

AST

Kidney AP

REGd

Liver VACd

Cmaxb pghl

AUC pg.h/ml

10 20 40 80 20 40 80 160

6 6 6 4' 6 6 6 6

0.7 0.64 0.6 0.58 0.8 0.75 0.6 0.65

1.1 1.3 1.3 1.0 1.1 1.1 1.4 1.3

1.o 1.2 1.25 1.1 1.o 1.0 1.05 1.05

1.25 1.45 2.1 3.0 1.05 1.05 1.2 1.3

1.o 0.9 0.95 1.7 1.1 1.o 0.95 1.2

0.3 1.0 2.5 3.0 0.3 0.2 0.5 0.8

0.1 0.1 1.0 2.3 0.0 0.3 0.8 0.8

0 0 0.3 2.2 0.0 0.2 0.5 0.8

2.9 6.9 16.2 23.1 0.2 0.62 4.9 4.0

37 107 284 418 2.7 9.2 65 78

Histology score: 0 = none, 1 = mild, 2 = moderate, 3 = severe. Cmax after 8 days of treatment. 216 animals died. AP, arteriolopathy; REG, regeneration; VAC, vacuolization.

TABLE Vlll Immunosuppressive and Nonimrnunosuppressive Cyclosporins at 50 mg/kg/day Relative to Controls (control = I .O), Including Histopathology and Plasma Exposure Values

PO.

Values of Major Clinical Chemistry Parameters

Histopatboloeyb Compound Immunosuppressive SDZ 027-400 SDZ 037-325 SDZ 216-125 SDZ 034-271 Nonimmunosuppressive SDZ 037-049 SDZ 037-839 SDZ 211-811 SDZ 215-833 SDZ 205-120' SDZ 203-218' Creatinine-clearance.

MgCt

Urea

Creatinine

Cr-CP

Bilirubin

Kidney

Liver

Cmax pglml

A UC pg.blml

0.5-0.7 0.7-0.9 0.7-0.8 nd

1.5-2.0 1.4-1.8 1.1-1.4 1.1-1.7

1.2-1.5 1.1-1.3 1.1-1.2 1.2-1.5

0.6-0.8 0.7-0.9 0.9-1.1 0.5-0.7

1.4-3.0 1.5-2.5 1.1-1.2 2.0-3.5

2-2.5 1.5-2.2 0.5-1.5 2-2.5

1-2 nd 0-1 nd

14-22 16-24 3-6 -16

190-280 180-290 40-80 -280

nd nd 0.9-1.1 1.0-1.1 0.8-0.9 0.9-1.0

1.0-1.2 1.0-1.2 0.9- 1.1 1.0-1.1 1.0-1.2 1.0-1.1

0.9-1.0 0.9- 1.1 1.0-1.1 1.0-1.1 1.1-1.2 1.l-1.2

1.0-1.1 1.0-1.1 0.8-0.9 1.1-1.3 0.9-1.1 0.9-1.1

1.5-1.8 1.3-1.6 1.1-1.2 1.5-2.0 1.0-1 * 1 1.3-1.6

* Scores (1 = mild, 2 = moderate, 3 = severe). Subcutaneous administration.

0-1 0 1-1.5 0 0-1 0-1

nd nd nd 1-2 0-1 0- 1

15 4.5 12 6 0.6 15

-225 75 195 110 10 168

-

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in rats. These compounds included SDZ NIM 811 and SDZ PSC 833 as well as the CS metabolites SDZ 205-120 and SDZ 203-218. Adverse events in human volunteers with SDZ PSC 833 were shown to be in accordance with those observed in the rat model. In addition, there is some evidence indicating that this might also pertain to metabolite 17 (SDZ 205-120). Even though this metabolite has never been administered to man, it attains markedly different exposure values relative to CS in kidney, liver, heart, and bone marrow transplant patients (Wang et al., 1988).Since the incidence of adverse effects in these patients bearing different organ allografts does not exhibit relevant differences, it is unlikely that this major metabolite 17 may play a significant role as a contributing factor. An important parameter in the comparison of the qualitative and quantitative adverse reaction profiles was the determination of exposure to the compounds, preferably at different dose levels. By considering the exposure values the importance of the induced effects on an organ function could be related with those of the reference compound. In conclusion, comparison of adverse profiles between immunosuppressive and nonimmunosuppressive cyclosporins in the rat indicated moderate to marked quantitative differences within the two classes of cycloporins as well as important qualitative differences between these classes. With respect to the latter it became evident that parameters associated with renal function were clearly affected in the immunosuppressive cyclosporins, whereas they were not or only marginally affected in the nonimmunosuppressive group. This finding was confirmed by histopathological examinations. Moreover, the adverse reactions on hepatic function were also less pronounced in the nonimmunosuppressive cyclosporins. An exception seems to be SDZ PSC 833, which due to an action on multidrug resistance glycoproteins in the canalicular system of the liver, might cause changes of bile acid andor bilirubin handling (Bohme et al., 1993). Consideration of the above results makes, therefore, the possibility of widening the therapeutic window by modifying the CS molecule in such a way as to save or even increase its immunosuppressive potency while significantly reducing or eliminating the toxicological effectsvery unlikely. It rather seems that some molecular mechanisms elicited by CS and other derivatives, which are essential for inducing immunosuppression, are concomitantly causing the side effects. It would, therefore, be of major importance for understanding the CS-induced toxic effects to learn, for instance, how cyclophilin-binding and alteration of calcineurin activity by the cyclosporins are affecting cellular functions in non-T cells. As recently reported by Ryffel et al. (1994),the demonstration of immunophilins and calcineurin in cytosolic extracts of the kidney together with the observation of specific immunophilin-drug-calcineurin complexes in the presence of CS or tacrolimus (FK 506),but not of sirolimus (rapamycin), suggest that CS and tacrolimus might use identical signaling pathways for the immunosuppressive and toxic effects, e.g., inhibition of the calcineurin

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phosphatase. Although the molecular mechanism leading to nephrotoxicity remains unknown, there is some evidence that the substrates dephosphorylated by calcineurin may be found in the pathways leading to renin and/or endothelin synthesis (see also Section VI1.C.).

V. Biological Effects Possibly Correlated with the Immunosuppressive Activity A. Development of Suppressor Cells

In contrast to other immunosuppressive agents, CS seems at least to allow, if not possibly to promote, the development of mononuclear cells expressing suppressor activity (cf., Section 1II.C.). Thus, it has been reported that T lymphocytes with donor-specific or nonspecific suppressor effects were found in vivo and ex vivo in allograft recipients, particularly in rodents, but the characteristics of these cells were variable, depending on the test model and animal species chosen (for review Section 1II.C and Bore1 et al., 1989). Both T cells of different phenotypes and macrophages have been shown to exert suppressor function. Moreover, some mononuclear cells were demonstrated to secrete soluble suppressor factors. In summary, it is generally agreed that T-suppressor cell induction does not depend on T-helper function, but the persistence of antigens, such as the presence of an allograft or donor cells, is necessary (cf. Section 1II.D.). Suppressor T cells are claimed to be interleukin-2 dependent, short-lived, and cyclophosphamide sensitive. In conclusion, the clinical relevance of the many but often contradictory results with suppressor cells in animal experiments is still uncertain. It is unlikely, however, but not proved, that nonimmunosuppressive CS congeners would allow or induce the development of cells with suppressor characteristics. B. Interference in the Regulation of Tolerance to Self and Nonself

Discrimination of self antigens from nonself antigens is achieved by negative and positive selection of T cells in the thymus (von Boehmer and Kisielow, 1990).In normal mice (Kappler etal., 1987)as well as in transgenic mice (Kisielow et al., 1988) tolerance to self is due to clonal elimination rather than suppression. Moreover, tolerance induction may occur in the thymus at the time immature thymocytes are selected to move into the mature thymocyte pool (Kappler et al., 1987) and it appears that CD4 and CD8 accessory molecules are involved in the deletion process (Kisielow et al., 1988). In addition, there exists in the developing thymus a dynamic interplay of cytokines that control the passage of precursor cells through

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different stages of developmentin this organ and many cell surface molecules seem responsible for mediating in a temporal manner the interactions between stromal cells and thymocytes prior to acquisition of the T-cell receptor (reviewed by Carding et al., 1991). Early studies revealed that after a single oral administration to mice CS caused a reduction in thymus weight with signs of increased cell death and regeneration (Ryffel et al., 1981; Ryffel, 1982). The salient feature was a cellular depletion of the thymus cortex reminiscent of glucocorticosteroid effect. However, it was experimentally shown that the immunosuppressive activity of CS in vivo did not depend on the adrenals (Ryffel et al., 1981). Systemic treatment over a few weeks also induces involution of the thymus with a normally reversible loss of the medullary epithelium (Beschorner et al., 1988a; Schuurman et al., 1990; Fabien et al., 1992). The destruction of the medullary microenvironment is accompanied by an atrophied or depleted major histocompatibility class I1 positivity (Brayman et al., 1986; Schuurman et al., 1990). In contrast, the cortex of CS-treated rodents (Beschorner et al., 1987; Hirarnine et al., 1988) as well as chickens (Wick et al., 1986) is relatively intact. Hiramine et al. (1989) observed that prolonged treatment of mice with CS affected both thymus and spleen cells in particular the CD4 helper subset. However, lymph node cells seemed to be relatively spared from the in vivo effect of CS. Histological analysis of thymic architecture as well as surface staining and quantification of thymocytes at different developmental stages have been performed and several possible mechanisms have been discussed to explain the effects of CS treatment. The further investigation of the profound thymic changes described in the literature may reveal how CS interferes with the regulation of tolerance to self and nonself, because CS is not only able to induce unresponsiveness to nonself, but it can, under defined conditions, also break tolerance to self (cf. review by Bore1 et al., 1989, pp. 309-318). The disrupting effects of CS both in vitro and in vivo on T-cell maturation, clonal deletion, and induction of autoimmunity are well documented (Hollander et al., 1994a; Prud’homme et al., 1991; Fischer et al., 1991a; Kosaka et al., 1990). CS, tacrolimus (FK 506), and a few other drugs have been found to induce an autoimmune-like graft-versus-host disease in lethally irradiated rats following syngeneic or autologous bone marrow reconstitution (Hess et al. 1995). First reported by Glazier et al. (1983), it occurred in young adult rats treated for 40 days with CS followingirradiation and bone marrow transplantation. Clinical symptoms and histologic lesions of graft-versus-host disease were observed after discontinuation of CS (Beschorner et al., 1988b). This phenomenon has been confirmed in rats (Sorokin et al., 1986; Bos et al., 1988) and in a few mouse strains (Cheney and Sprent, 1985; Bryson et al., 1991b; Prud’homme et al., 1991) but not in others (Chow et al., 1988; Kosugi et al., 1989; Prud’homme et al., 1991).

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CS-induced graft-versus-host disease after autologous bone marrow transplantation has also been observed in patients treated for acute myeloid leukemia (Yeager et al., 1993). In all cases, the presence of the thymus is essential and experimentally the disease can be adoptively transferred to irradiated secondary recipients injected with spleen cells from symptomatic donor rats. Implantation of a normal syngeneic thymus or shielding of the thymus or cotransfer of an excess of normal lympoid cells blocked the disease in the secondary recipients (Tutschka, 1987; Sorokin et al., 1986). More recently, Beijleveld et al. (1995b)concluded that the role of the thymus in CS-induced autoimmunity was the production of T cells having undergone aberrant selection under influence of CS and that X-irradiation of the thymus was not required. Provided the peripheral autoregulatory circuits were eliminated, these autoreactive, thymus-derived T-cells were able to cause autoimmune phenomena. In addition, there is unpublished evidence that syngeneic graft-versus-host disease can only be induced in “dirty” animals kept under conventional conditions, but not in recipients kept under specific pathogenfree conditions. Long-lasting investigations by Hess and co-workers have disclosed that after discontinuation of CS the syngeneic graft-versus-host reaction is primarily acute with epithelial infiltrates of CD8 T cells and lamina propria infiltrates that include immature double-positive thymocytes. Thereafter follows a rapid transition to chronic syngeneic graft-versus-host disease in which the residual mucosal infiltrate is now dominated by double-positive lymphocytes while the lamina propria infiltrate has more mature helperphenotype T cells (Beschorner et al., 1988b). Fischer and Hess (1990) have demonstrated that age-related factors played an important role in the CSinduced development of syngeneic graft-versus-host disease. Not only does the incidence rapidly decrease in recipients which are over 4 weeks old, but the age of the marrow donor is even more critical, since marrow from over 3 months old donors was virtually incapable of eliciting the disease. The dendritic cells represent the major antigen-presenting cells of the thymus. While the medullary dendritic cells normally recover promptly after CS treatment, rats receiving mediastinal irradiation demonstrated minimal recovery after CS discontinuation (Beschorner and Armas, 1991; de Waal et al., 1992). Therefore, the prolonged deficiency of medullary dendritic cells could represent an essential step for the generation of autoaggressive cells resulting in the loss of self-tolerance and the development of syngeneic graft-versus-host disease. However, Damoiseaux et al. (1994) have recently reported that a substantial number of dendritic cells could always be isolated from CS-treated rats and that these very cells exhibited an identical phenotype and function as those isolated from control animals. This finding strongly suggests that the partial deficiency of dendritic cells cannot be held as essential for loss of tolerance.

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Exploring further the role of aberrant major histocompatibility complex antigen expression in the pathogenesis of CS-induced autoimmunity, Parfrey and Prud’homme (1990) concluded that class I1 expression alone was insufficient to lead to cell injury in this model. However, results from adoptive transfer studies by Hess et a2. (1993) provided evidence that the major histocompatibility class I1 antigens are recognized in vivo in this autoaggression syndrome; in particular, administration of monoclonal antibodies to the recipients indicated that RT1 .B( I-A) class I1 determinants were preferentially recognized. In contrast, monoclonal antibodies to class I determinants were ineffective. They had previously shown that large numbers of CD8 autoreactive T cells could transfer acute syngeneic graft-versus-host disease that resolved within 2 weeks. However, for transfer with low numbers of CD8 T cells, CD4 T helper cells were required and these animals exhibited a much more pronounced reaction with the transition to chronic disease (Hess et al., 1990). In an earlier study, Bos et al. (1988)had demonstrated that in the syngeneic graft-versus-host disease CS treatment selectively suppressed T-helper cell repopulation, as monitored in peripheral blood, and that this effect persisted as long as CS was given since these cells completelyrecovered only after withdrawal of the drug. Their reappearance in the peripheral circulation coincided with the development of disease symptoms, suggesting a role of the Thelper cells in initiating or causing the disease. In a comparison of the effect of CS on thymocytic and peripheral T-cell populations, Prud’homme et al. (1991)observed that CS-induced alterations were similar both in rats and in a disease-prone mouse strain, and that CS-treated syngeneic bone marrow chimeras had transiently increased numbers of peripheral double-positive T cells. However, examination of T-cell receptor Vp expression in CS-treated mice revealed a normal pattern of clonal deletion in all strains suggesting that CS may induce autoimmunity without blocking intrathymic clonal deletion. To test the hypothesis that the autoreactive T cells that develop as a consequence of CS treatment may result from inhibition of clonal deletion and could be responsible for the development of this disease, Bryson et al. (1991a)have analyzed T-cell receptor expression as a measure of tolerance induction in a series of syngeneic radiation chimeras with and without CS treatment. In essence, the development of CS-induced autoreactive T cells as assessed by Vp T-cell receptor expression showed strain variations in mice that did not correlate with the induction of syngeneic graft-versus-host disease and suggested that other mechanismsmay be involved in the development of this autoimmune phenomenon. However, it has to be emphasized that the induction of this autoimmune disease requires two essential components, one being the emergence of autoreactive lymphocytes from the thymus and the other being the elimination of a T cell-dependent peripheral autoregulatory mechanism. Studies using this rat model to characterize the regulatory system that modifies the autoimmune potential of autoreactive effector cells revealed that although

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CS did not interfere with the effector function of the established autoregulatory T cells, it prevented the reconstitution of the regulatory system after syngeneic bone marrow transplantation. Additional studies indicated that effective autoregulation was a dynamic process which required a specific interaction of the T-cell receptor-dp on the autoregulatory cells with the major histocompatibility complex class I1 determinants on the autoreactive lymphocytes (Hess et al., 1994). Recent studies in rodents have disclosed that elimination of a particular T-cell subset from the peripheral T-cell pool leads to spontaneous development of various organ-specific autoimmune diseases (reviewed by Sakaguchi and Sakaguchi, 1992). It appears that an important condition required for CS to induce autoimmunity is its interaction either with the developing immune system early in life or during reconstitution of the immune system following irradiation and bone marrow transplantation. It is, therefore, when new T cells are developing in the thymus and, in addition, the regulatory system is not yet or still insufficiently functional that animals may be most vulnerable to adequate exposure to certain environmental and chemical insults which may result more easily in expansion of self-reactive T cells. The presence and accumulation of immature, unselected, self-reacting, or otherwise aberrant T cells both in the thymus and in the periphery under various but well defined in vivo conditions has been documented by many authors (Babcock et al., 1990; Kosugi and Shearer, 1991; Chen-Woan and Goldschneider, 1991; Classen and Shevach, 1991; Urdahl etal., 1992; Zadeh and Goldschneider, 1993; Sai et al., 1994; Beijleveld et al., 1995a, Huby et al., 1995). Similarly, the effects of CS on T-cell development have also been confirmed and analyzed in thymic organ cultures (Kosaka et al., 1990; Siege1 et al., 1990; Takeuchi et al., 1990). However, the fact should be stressed that, despite the presence of self-reactive T cells that have escaped clonal selection, CS treatment of adult animals possessing a mature immune system is not sufficient to induce overt autoimmune symptoms. This implies that a radiation-sensitive, autoregulatory peripheral mechanism of tolerance must first be eliminated to allow the onset of autoimmune disease (Urdahl et al.,1992; Severino et al., 1993). In summary, CS allows autoreactive T cells to escape negative thymic selection and hinders positive selection of regulatory T cells essential for persistent peripheral self-tolerance ( Wang et al., 1995). Continued immunosuppressive treatment inhibits T-cell activation and would thus prevent the appearance of autoimmune symptoms. However, on withdrawal of drug treatment, activation of circulating selfreactive T cells would result in an immune response directed against self (Hollander et al., 1994a). There is agreement that CS treatment during hematopoietic recovery inhibits positive selection of T-cell receptor double positive thymocytes to single positive T cells without apparently affecting earlier stages of Tcell ontogeny, in particular the development of y/S T cells (Gao et al., 1988;

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Jenkins et al., 1988; Fischer et al., 1991a; Bader et al., 1991). Meanwhile, Wells et al. (1993) have demonstrated that T cells expressing y/S antigen receptors are subject to positive selection during development and that CS, which blocks positive selection of the Culp T cells, also inhibited $6 T-cell development. Phenotypically mature, single positive T cells were reduced to about 10% of their normal frequency during CS administration and, with the use of V p l l (Gao et al., 1988) or VP17 (Jenkins et al., 1988) as markers for thymic negative selection, it has been shown that deletion was prevented in this small maturing subset, since these T-cell receptor phenotypes were still generated. Because the aJp T-cell receptor-mediated positive selection of CD4 single positive thymocytes is calcineurin dependent, the action of CS could be to inhibit this calcineurin-dependent activation, possibly by changing not only the quantity but also the character of signal transduction on T-cell receptor-mediated stimulation of thymocytes (Hollander et al., 1994a,b). In support of this assumption is the work by Curnow and SchmittVerhulst ( 1994) which provides direct evidence that presentation of antigen to thymocytes can result in deletion or activation depending on not only the differentiation status of the cell but also on the parameters of T-cell receptor-antigen interaction, and that these events are affected by CS. Nakayama and Nakauchi (1993) have further demonstrated that CS may block the transition from double to single positive thymocytes by inhibiting the CD4/CD8 downregulation induced by protein kinase C activation, possibly at the level of transcriptional regulation factors. In addition to the above-mentioned mechanisms, CS has been shown to interfere directly with apoptosis or programmed cell death (Shi et al., 1989; Liu and Janeway, 1990; Prud’homme et al., 1991; Yasutomi et al., 1992; Little and Flores, 1992; Saiagh et al., 1994a and 1994b), which is considered to constitute the ultimate mechanism whereby useless or potentially harmful, self-reactive developing thymocytes are deleted intrathymically. Moreover, activation-induced peripheral T-cell death provides also an important mechanism for clonal deletion to ensure the homeostasis of the peripheral immune system (Kabelitz et al., 1993). During programmed cell death DNA undergoes fragmentation within the affected cells, but in the presence of CS both in vitro and in vivo loss of cell viability and DNA fragmentation were prevented (Shi et al., 1989; Yasutomi et al., 1992). It has been suggested that CS interferes in this calcium-dependent process by blocking activation of a gene or biochemical pathway directly involved in apoptosis following activation via the T-cell receptor. It would also appear that calcineurin is required in the induction of apoptosis in thymocytes which might explain why CS and tacrolimus but not rapamycin are inhibitory (Bierer etal., 1993; Fruman et al., 1994a).The recent work of Genestier et al. (1994) confirms that in the murine WEHI-231 B lymphoma cell line, in which apoptosis can be triggered by ionomycin, CS selectively inhibits calcineurin without impairing the rise of intracellular calcium and protects

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these cells against apoptosis. Liu and Janeway (1990) have used an in vitro model in which ligation of the T-cell receptor of a T helper-cell clone in the absence of accessory cells rapidly leads to cell death. This process was inhibited by CS and the CS effect could be overrided by the addition of exogenous y-interferon. Ucker et al. (1992)have demonstrated that alternative cellular responses of cell death or cell proliferation in nontransformed T cells was triggered solely as a function of quantitative differences in the doses of identical stimuli and that either response was sensitive to CS. Interestingly, CS blocked in vivo regression of the anuran tadpole tail, which occurs through hormone-induced programmed cell death, but the mechanism by which the blockade is effected remains unresolved (Little and Flores, 1992). Whereas only the suppressor effects of CS on apoptosis have until now been discussed, we have also to consider a number of papers in which an enhancing effect of the drug has been reported. Saiagh et al. (1994b) hypothesize that thymocytes receive in vivo an inducing signal of apoptosis, but that these thymocytes die in vivo outside the thymus, as they do after 24 h in ex vivo cultures (demonstrated by DNA electrophoresis and DNA labeling), and that CS accelerates thymocyte apoptosis. Their observations based on short-term drug administration support the concept that the majority of cells undergoing an induced apoptotic process by CS die outside the thymus and also explain the marked decrease in thymus weight, the complete disappearance of single positive cells, and the decrease in the absolute number of double positive and double negative cells. Therefore, it seems probable that CS interferes with the positive selection process inducing apoptosis of double positive cells surviving the negative selection and normally differentiating into single positive cells. Viciana and Ruiz (1994) found that chronic CS treatment in rats caused a perturbation of normal T-cell maturation pathways in the thymus and the spleen which resulted in a selective elimination of thymocytes bearing high-density alp T-cell receptor and CD2. They also observed the absence of thymic ED-1 positive epithelial and macrophage cell populations following CS administration, which coincided with a change in CD2 expression, but they did not indicate how the drug might affect normal lymphocyte maturation. McCarthy et al. (1992) have used an in vitro model of murine thymocyte clonal deletion to analyze the effects of intracellular stimuli and immunosuppressants on immature thymic T cells. They concluded that CS, tacrolimus, and sirolimus (rapamycin) affected both mature peripheral T cells and developing immature thymic T cells by similar mechanisms. They have identified a mechanism in double positive thymocytes which spares these cells from apoptosis and which is inducible by TcWCD3-mediated signals. However, activation of this protective mechanism is blocked by CS which also blocks TcWCD3-induced activation pathways in mature T cells. This implies that apoptosis now can proceed in the presence of CS.

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CS and tacrolimus preferentially inhibit B-cell activation caused by stimuli which induce a rise of intracellular calcium. Inhibition of antiimmunoglobulin-activated B cells with these agents leads to cell death (Wicker et al., 1990). Gottschalk et al. (1994) have demonstrated that these two compounds themselves induced apoptosis in WEHI-231 B cells, but only in sublines susceptible to anti-immunoglobulin-mediatedprogrammed cell death. In the other B-cell lines resistant to anti-immunoglobulin- and immunosuppressant-induced apoptosis, CS and tacrolimus caused growth arrest. In cunclusion, it is evident that CS interferes with apoptosis of both mature and immature T and B cells. The reason for the controversial findings reported above may be twofold. Firstly, CS apparently does not affect T and B cells by the same mechanisms as reported by Wicker et al. (1990) and Gottschalk et al. (1994). Secondly, it seems that several different inducer pathways exist which are capable of triggering physiological cell death both in vivo and in vitro (cf., Hueber et al., 1994). In addition, the work of Vanier and Prud’homme (1992) investigating the opposing effects of CS on superantigen-induced peripheral T-cell deletion and anergy induction clearly demonstrated that the degree of the responses were highly dependent on the dose and the schedule of CS administration as well as on the number of superantigen injections. In view of their results, the extent of tolerance in CS- and superantigen-treated mice depends on the balance between opposing effects, i.e., enhancement of peripheral deletion versus abrogation of anergy. These observations taken together suggest that various cell activation pathways may have different sensitivities to CS and that the effects of the drug are mainly dependent on the prevailing experimental conditions. Finally, it also would seem that the mechanism(s) responsible for the effect of CS on apoptosis is closely linked with the immunosuppressive activity. C. Therapeutic Effects in Psoriasis

An increasingly important indication for CS has been its therapeutic use in dermatology (Kauvar and Stiller, 1994), especially in psoriasis (Fry, 1991). Both the experimental and clinical approaches have been reviewed by Borel et al. (1989).More recent and larger clinical studies have further substantiated the marked efficacy of the drug, particularly in plaque-type psoriasis (Ellisetal., 1991; Christophers etal., 1992)and in atopic dermatitis (van Joost et al., 1992, 1994). Studies directed at the detection of biochemical defects in psoriatic skin have revealed a number of abnormalities such as an increased number of several different cells of the immune system, including lymphokine-secreting activated T cells, activated antigen-presenting cells, polymorphonuclear leukocytes, and hyperproliferating keratinocytes. The effector function of the skin immune system is realized by a unique combination of proinflammatory,

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upregulating keratinocytes, paralleled by a simultaneous increase in the migratory activity of antigen-trapping and antigen-presenting cells which induce expansion of specific lymphocytes in the skin-draining lymph nodes. These lymphocytes can enter the skin as a result of expression of skin-specific adhesion molecules that interact with their upregulated counterreceptors on the endothelial cells of the dermal perivascular units (cf., review by Bos and Kapsenberg, 1993). The immunosuppressive activity of CS exerts a potent antipsoriatic effect and this observation has been advanced as an argument in favor of an immunopathologic origin of the disease. Furthermore, the successful therapy of severe psoriasis with anti-CD4 monoclonal antibody provides strong evidence for a critical role of CD4 lymphocytes in this disease (Morel et al., 1992). However, serious consideration should also be given to direct effects of CS on epidermal cells like Langerhans cells and keratinocytes. Gerritsen et al. (1994) have investigated the influence of systemic CS on proliferation and differentiation in the tape-stripped uninvolved skin of psoriatic patients using immunohistochemical methods. This is a model which provides the opportunity for studying epidermal regeneration in the absence of a significant accumulation of T lymphocytes. However, in the dermis total CD4 and CD8 T-cell numbers were approximately halved, and both activated CD4 and CD8 T cells were substantially decreased. It was concluded that epidermal hyperproliferation and abnormal keratinization were not modulated directly by CS at therapeutic doses in vivo, but that the antipsoriatic effect of CS appeared mediated by the immune system. Baker et al. (1988) had shown that there was a preferential recruitment of CD4 T cells into psoriatic lesions. In chronic psoriatic plaques there were increased numbers of dendritic (Langerhans) cells associated with approximately equal numbers of activated CD4 and CD8 T cells in the epidermis. They postulated that the abnormal epidermal proliferation in psoriasis was mediated by factors released by interacting CD4 T lymphocytes and Langerhans cells. In the epidermis of patients treated with CS total CD4 and CD8 T-cell numbers were substantially decreased and these reductions correlated with a decrease in the disease (PASI)scores. While the epidermal activated CD8 T cells were also markedly decreased, the number of epidermal activated CD4 T cells was, in striking contrast, little affected. However, in the dermis total CD4 and CD8 T-cell numbers were approximately halved, and both activated CD4 and CD8 T cells were substantially decreased. It was speculated that CS exerted its therapeutic effect mainly by inhibiting the release of lymphokines by CD4 T cells into the epidermis, since the rapid relapse following drug discontinuation supported the concept that activated CD4 T cells had been reversibly inactivated by CS. Van Joost et al. (1992) have studied the modulation in the expression of immunologic markers in lesional skin in atopic dermatitis patients under CS treatment. A statistically significant reduction in the number of activated T cells and in that of cells

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expressing the interleukin-2 receptor (CD25) paralleled a marked improvement in the disease and supported the view that atopic dermatitis is based on a T cell-mediated immune inflammation. Petzelbauer et al. (1991) and Petzelbauer and Wolff (1992) have reported that CS inhibits the expression of intercellular adhesion molecule-1 by papillary endothelium in healing psoriatic plaques. It could not be decided, however, whether the observed suppression occurs directly at the level of the gene responsible for the adhesion molecule synthesis or by CS interfering primarily with the secretion of cytokines that regulate the expression of this molecule by the papillary endothelial cells. While it is clear that in addition to inhibition of T cells CS can also affect the function of Langerhans cells and keratinocytes in vitro (seebelow), it remains uncertain to what extend these effects contribute to the antipsoriatic activity of CS in vivo. The investigations performed by Khandke et al. (1991) suggest that the effects of CS on psoriatic skin are unrelated to direct effects on autocrine growth regulation of keratinocytes via transforming growth factor a production or of epidermal growth factor receptor modulation. In addition, Arnold et al. (1993)failed to show inhibition of epidermal cell proliferation during systemic treatment with CS. Whether CS treatment also works via antigen-presenting cell inhibition depends on whether or not the lesion is primarily maintained by signals from macrophages, which are resistant to CS concentrations achieved in vivo, or by Langerhans cells, which are sensitive to levels of CS achieved during psoriasis therapy (Cooper et al., 1990a,b). It is generally recognized that the inhibitory effects of CS on keratinocytes and Langerhans cells occur at much higher concentrations than those required to inhibit T-cell function (Furue et al., 1988; Furue and Katz, 1988). However, Fisher et al. (1988) have demonstrated that human epidermis contains a high concentration of CS after oral administration, and that concentrations of CS within the range found in vivo can inhibit growth of cultured keratinocytes. This controversial issue has been debated, since not all authors agree whether during daily oral CS therapy the drug levels in blood and skin achieved in patients are high enough to account for a substantial inhibitory effect of CS on antigenpresenting Langerhans cells and/or keratinocytes (see: Letters to the editor, J. Invest. Dermatol. 98, 259-261, 1991). In conclusion, the mechanism of action of CS in the treatment of psoriasis has been pertinently summarized by Wong et al. (1993). CS inhibits Tcell production of interleukin-2 and, consequently, the interleukin-2 driven proliferation of activated T cells as well as the secretion of other T-cell cytokines important in the immunological functions of keratinocytes, nonkeratinocyte antigen-presenting cells, and polymorphonuclear leukocytes in psoriatic skin. In addition, CS has immunomodulatory effects on keratinocytes and antigen-presenting cells, key members of the immunological network in the psoriatic plaque. Although this latter activity is apparently exerted at higher concentrations than those needed to affect T cells, these

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cumulative effects may result in disruption of the self-propating mechanisms of inflammation in psoriasis. (See also Section V1.A.)

D. Therapeutic Effects in Asthma? Asthma is a chronic inflammatory condition of the airways which are hyperreactive and constrict easily in response to diverse stimuli, giving rise to episodic, reversible airway obstruction. The inflammatory cell infiltrate is characterized by a predominance of eosinophils, but increased numbers of lymphocytes, mast cells, and neutrophils have also been reported (Djukanovic et al., 1990). In addition, plasma exudation, edema of the airway mucosa, smooth muscle hyperplasia and hypertrophy, mucous plugging, and epithelial damage are evident (Djukanovic et al., 1990). The effects of CS on this characteristic type of pulmonary inflammation in asthmatics has not yet been reported. However, decreased disease exacerbation, reduced symptoms, and an improvement in lung function, in addition to a reduction in concomitant therapy, have been reported in patients with chronic, severe, corticosteroid-dependent asthma during treatment with CS (Szczekliket al., 1991; Alexander et al., 1992). Consequently, the mechanisms whereby this substance exerts beneficial effects in asthma are presently under experimental investigation. Although CS is not a bronchodilator (Chapman et al., 1993) and has not been reported to influence airway hyperreactivity in experimental animals (Morley, 1992; Elwood et al., 1992), several studies have examined the effects of CS on the pulmonary accumulation andlor activation of leukocytes and other proinflammatory cells considered to contribute to inflammatory pathology in the asthmatic lung. I . Eosinophils

Eosinophilic infiltrates are present in the conducting airways of asthmatics and increased numbers of eosinophils in peripheral blood (Durham et al., 1989), sputum (Bouquet et al., 1990), bronchoalveolar lavage fluid (Diaz et al., 1989), and bronchial tissue (Djukanovic et al., 1990) have been reported. Indeed, the presence of toxic proteins derived from activated eosinophils in these fluids and tissues together with evidence that drugs exhibiting clinical benefit in asthma reduce eosinophil numbers in blood, sputum, or lungs of patients has led to the hypothesis that the eosinophil is a major effector cell (Wardlaw and Kay, 1987) or even a causative cell (Venge and Hakansson, 1991) in the chronic inflammatory process that underlies bronchial asthma. In animal models of lung inflammation the accumulation of eosinophils as well as other leukocytes in bronchial alveolar lavage fluid recovered from actively sensitized guinea pigs (Norris et al., 1992; Lagente et al., 1994), rats (Elwood et al., 1992) or mice (Nogami et al., 1990) following exposure to allergen was inhibited by prior administration of CS. Furthermore, La-

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gente et al. (1994)demonstrated inhibition by CS of pulmonary eosinophilia induced by platelet activating factor and leukotriene B4; effects that were attributed to a reduction of the chemotactic activity of these agents on eosinophils. In addition to influencing inflammatory cell accumulation, direct inhibitory effects of CS on eosinophil activation, in particular the release of cytokines and granular proteins, may serve to prevent both local tissue damage and shorten cell survival in inflamed asthmatic lung tissue as well as averting further cell recruitment. Indeed, therapeutic concentrations of CS abolished ionomycin-stimulated granulocyte/macrophage-colonystimulating factor and interleukin-3 release from human isolated peripheral blood eosinophils (Kita et al., 1991) and could potentially influence the release of interleukin-5 from activated eosinophils (Broide et al., 1992). In contrast, CS did not inhibit the major basic protein-stimulated production of IL-8 by eosinophils (Kita et al., 1995).

2. Lymphocytes Increased numbers of activated CD4 helper T cells are present in bronchoalveolar lavage fluid from atopic asthmatics (Corrigan et al., 1988) and have been associated with increased serum concentrations of interleukin-5 (Corrigan et al., 1993); a cytokine that together with interleukin-3 and granulocyte/macrophage-colonystimulating factor is critical for the development and maturation of the eosinophil granulocyte (Sanderson, 1989). Recent experimental evidence implicates activated T helper-2 cells in orchestrating eosinophilicpulmonary inflammation in animals. For example, treatment of actively sensitized guinea pigs with a monoclonal antibody to interleukin5 (a lymphokine selectively secreted by T helper-2 cells) attenuated lung eosinophilia following allergen exposure (Gulbenkian et al., 1992). Additionally, prior exposure to aerosolized interferon-y, a T helper-1 lymphokine suppressing T helper-2 function, prevented antigen-induced eosinophilia in actively sensitized mice (Nakajima et al., 1993).Furthermore, deletion of the gene expressing interleukin-4, which is a cytokine implicated in committing T cells to the T helper-2 phenotye, inhibited blood and pulmonary eosinophilia in parasite-infected mice (Anderson et al., 1993).Together with the observation that CS inhibited interleukin-5 release from isolated human mononuclear cells (Anderson et al., 1992; Mori et al., 1994), it is tempting to speculate that selective inhibition of lymphokine release from T helper-2 cells by CS prevents pulmonary eosinophil recruitment. 3. Mast Cells

Increased numbers of mast cells have been observed in bronchoalveolar lavage fluid (Tomioka et al., 1984), bronchial brushings (Gibson et al., 1993), and lung biopsies (Pesci et al., 1993) from asthmatic patients and the contribution of the mast cell to the pathology of asthma has been extensively documented (Schulman, 1993; Page and Minshall, 1993).Results

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from several studies have indicated that therapeutic concentrations of CS (300 to 800 nM) inhibited the release of preformed mediators such as histamine as well as de novo synthetised mediators such as prostaglandin D2 and leukotriene C4 following IgE-dependent and IgE-independent activation of human lung mast cells and basophils in vitro (Triggiani et af., 1989; Cirilo et al., 1990) and basophils ex vivo from volunteers (Marone et al., 1993).However, while these observations have led to speculation that inhibition of mediator release following mast cell activation accounts for the therapeutic action of CS in vivo, such conjecture is not supported by whole animal experiments or clinical studies. Hence, treatment with CS neither impaired the expression of acute passive cutaneous anaphylaxis in rats (Chapman et al., 1993) or mice (Geba et al., 1991) nor acute allergic bronchospasm in guinea pigs (Chapman et al., 1993)-both manifestations of mast cell activation. Moreover, while marked clinical benefit was observed during prolonged treatment with CS, cutaneous responses to house dust mite were either unaltered or enhanced in patients with atopic dermatitis (Munro et al., 1991b). The discrepancy between these in vivo observations and reported effects of CS on isolated human mast cells in vitro and ex vivo remains unexplained, but whole animal and clinical data indicate that CS does not suppress acute manifestations of mast cell activation in vivo. Mast cells have the capacity to synthesize and release cytokines including interleukin-3, interleukin-5, granulocyte/macrophage-colonystimulating factor and interleukin-4 (Gordon et af., 1990). It is not known whether CS can differentially inhibit the synthesis or release of mast cell cytokines without influencing release of inflammatory mediators such as histamine or leukotriene in vivo. However, the precise role of mast cells in invoking inflammatory events in the asthmatic lung is contentious and the finding that actively sensitized mast cell-deficient mice developed marked airway eosinophilia in response to transnasal ascaris extract (Nogami et al., 1990) or aerosolized ovalbumin (Brusselle et af., 1994) indicates a noncritical role for mast cells in the induction of eosinophilic inflammation. Tissue mast cells and circulating basophils can initiate many IgEdependent immunologic reactions. Neutrophil infiltration associated with IgE-dependent cutaneous inflammation in mice is mast cell-dependent and tumor necrosis factor-a contributes significantly to this response. Both the in vitro and in vivo findings in mice by Wershil et al. (1995) indicate that CS can have at least three actions that interfere with the pathogenesis of IgE, mast cell- and cytokine-dependent inflammatory reactions: (1)suppression of the IgE-dependent increase in tumor necrosis factor-a mRNA by mast cells, (2)inhibition of the IgE-dependent production of tumor necrosis factor-a protein by mast cells, and (3) diminution of the responsiveness of target cells to this cytokine. These findings in mice raise the possibility that similar drug actions in humans may account for some clinical efficacy of CS in allergic diseases.

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4. Neotrophils

While the predominant granulocyte in asthmatic lung is the eosinophil, increased numbers of neutrophils have been reported following exposure of patients to diisocyanates (Fabbri et al., 1987), adenosine (Driver et al., 1991)or exercise (Vengeet al., 1990) and, more recently, increased numbers of neutrophils have been associated with sudden-onset fatal asthma (Sur et al., 1993). Effects of CS on the pulmonary accumulation of neutrophils have not been documented, but inhibition of formylpeptide-induced human neutrophil activation by CS, and interestingly by the nonimmunosuppressive (D-MeVal'l)-CS analogue has been reported ( Wenzel-Seifert et al., 1991). 5. Endothelial and Epithelial Cells

Direct or indirect inhibitory effects of CS on adhesion molecule expression by endothelial cells or leukocytes could limit cell recruitment into inflamed lung tissue and, while it has been reported that CS impairs bovine aortic endothelial cell function in vitro (Benigniet al., 1992),it is not known whether CS prevents adherence or diapedesis of inflammatory leukocytes by influencing the function of pulmonary vascular endothelium. Increased expression of intercellular adhesion molecule-1, which was associated with increased leukocyte numbers following bronchial allergen challenge in asthmatics (Montefort et al., 1993), and inhibition of intercellular adhesion molecule-1 expression on papillary endothelial cells isolated from CS treated psoriasis patients (Petzelbaueret al., 1991) may indicate an additional mechanism by which CS modulates leukocyte recruitment. However, expression of intercellular adhesion molecule-1 in mucosal biopsies from asthmatic lungs was not different from volunteers and was not reduced despite decreased mucosal eosinophil numbers following prolonged treatment with inhaled steroids (Montefort et al., 1992). Effects of CS on the expression of other adhesion molecules remain to be reported. There is increasing evidence that airway epithelial cells may play an important modulatory role in inflammation via generation of eicosanoids, expression of adhesion molecules, and release of proinflammatory cytokines such as granulocytdmacrophage-colony stimulating factor (Devalia and Davies, 1993) and RANTES (a chemokine regulated on activation in normal T cells expressed and secreted), a member of the interleukin-8 family with specific chemoattraaant activity for eosinophils (Alam et al., 1993). While these characteristics make airway epithelium an interesting target, inhibition of the release or expression of such agents by CS from these cells has not been reported. In summary, while suppression of lymphocyte function has been suggested to account for inhibition of pulmonary leukocyte recruitment in experimental animals (Norris et al., 1992; Elwood et al., 1992; Nogami et al., 1990; Lagente et al., 1994), inhibition by CS of granulocyte activation,

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adhesion molecule expression by endothelial cells, and possibly cytokine release by airway epithelial cells implies that alternative or additional mechanisms may contribute to its actions. Taken together, these clinical and experimental observations indicate that by impairing inflammatory events in asthmatic airways CS may have utility as a novel therapy in asthma. E. Effects on Hair Follicles

Stimulation of hair growth or hypertrichosis is a common side effect that has been observed in patients receiving CS (Wysocki and Daley, 1987) and which also occurs in most other species, in particular in the congenitally athymic nude (ndnu) mouse and in the normal rat (Pendry and Alexander, 1982; Hozumi et al., 1994; Gilhar et al., 1991). In contrast, there seems to be no report showing that tacrolimus induced hypertrichosis, even though this immunosuppressive drug possesses a mode of action very similar to that of CS. Sainsbury et al. (1991)have compared the differential effects of CS and tacrolimus on hair regrowth in Dundee experimental bold rats, a model for alopecia areata. Systemic administration of CS was effective in establishing complete hair regrowth and strongly downregulated the cutaneous inflammatory infiltrate around hair follicles, whereas systemic tacrolimus was less effective in clearing the cellular infiltrates and induced only marginal hair growth. However, Yamamoto and Kato (1994) have found that topical application of tacrolimus to the skin of mice, rats, and hamsters markedly stimulated hair growth, but not following oral administration, even at a dose causing profound immunosuppression. In vitro studies revealed that tacrolimus directly stimulated hair follicles, suggesting that this activity may apparently be unrelated to its immunosuppressive effect. To investigate whether the hair growth stimulating effect of CS was linked with its immunosuppressive activity, congenitally athymic B6 nude mice were orally treated with 3 nonimmunosuppressive CS derivatives: (DMeVal)"-CS, MeBmt (3-keto)'-VaP-CS, and (MeIle)4-CS(see also Table V). After 25 days of treatment it became evident that none of the immunologically neutral analogues were able to induce hair growth; thereby demonstrating that this stimulatory activity on the hair follicles was very likely to be associated with the immunosuppressivemechanism. The effect was transient since in the weeks following drug discontinuation all animals had lost all their hair (L. Kuntz and J. F. Borel, Sandoz Pharma, Basel; unpublished results). Moreover, Iwabuchi et al. (1995) have studied the effects of CS, tacrolimus, ascomycin, and rapamycin (all inhibitors of the peptidyl-prolyl cis-trans isomerase)on hair growth initiation in the mouse by topical application. Their results suggest that anagen hair induction was independent of isomerase inhibition and that a single application of adequate quantity of CS or tacrolimus was sufficient to initiate hair growth.

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VI. Biological Effects Appearing Independently of ImmunosuppressiveActivity

Besides its selective and powerful suppression of many lymphocyte functions as described in this review, CS itself and several congeners have also been noted for exerting other pharmacological activities which do not correlate with immunosuppressive properties as shown in Table IV (Borel, 1989). These observations entail that CS may also affect nonlymphoid cells (Thornson, 1992). Moreover, the assumption that a biological effect is not linked with immunosuppression can be proved by using a nonimmunosuppressive derivative. This has been performed for many of the effects listed in Table IVYbut, due to various reasons, this has not been feasible for all of them. Therefore, some of these effects are still assumed to be independent of the immunosuppressive activity, as for instance the insecticidal effect and the inhibition of exocytosis. The arguments supporting our assumptions will be given below. A. Effects on Nonlymphoid Cells 1. The Efeas of CS on the Antigen-Presenting Cells

The effects on CS on monocytes and macrophage functions have been reviewed by Borel et al. 1989 (pp. 345,377 to 382).Although several papers have reported a direct effect of CS on antigen-presenting cells (Little et al., 1990; Benson and Ziegler, 1989; Furue and Katz, 1988; Manca et al., 1988), the results should be interpreted with caution. The reason for this controversy could be due to the fact that 2 to 10 ng/ml of CS is sufficient to modulate the functions of lymphocytes while a hundred- to thousandfold higher concentration is necessary to interfere with macrophage functions. Even if after preincubation with drug the macrophages are washed extensively, the possibility that they may subsequently release low amounts of previously ingested drug cannot be excluded. These low concentrations could, however, still be adequate for affecting lymphocyte functions. Such results are, therefore, biased by drug carryover and may mimic a drug effect on antigen presentation. In order to obviate this effect, a limited intermediate incubation period of one to several hours in fresh culture medium followed by a second washing procedure should be included. Muller et al. (1988) have thus avoided this pitfall and concluded that CS had no influence on antigen presentation to lysozyme-specific T-cell hybridomas. However, Furue and Katz (1988), who in a careful in vitro study also avoided drug carryover, demonstrated that CS directly inhibited accessory cell functions of epidermal Langerhans cells. Using an extensive washing procedure and subsequent verification that all noncellular CS was removed from the incubated cells for the duration of the assay, Little et al. (1990) also concluded that there was an inhibitory effect of CS on class 11-positive cells in their

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ability to stimulate an allogeneic response in T cells. In contradiction, Granelli-Piperno et al. (1988),who analyzed separately the effects of CS on T cells, dendritic cells, and monocytes from human blood, concluded that the inhibitory effects of CS primarily involve the lymphocyte rather than the accessory cell (see Table I1 in the above reference). Since Manca et al. (1988)have suggested, depending on whether the antigen was taken up by constitutive or by receptor-mediated endocytosis, that accessory cells could be functionally defined as resistant or sensitive to CS, it appears that the cell source, time of drug addition, and test system used are of major importance. Thus, using a very different murine delayed hypersensitivity model in vivo and cell transfer experiments, Knight et al. (1988)presented convincing evidence that CS prevented acquisition and presentation of antigen by dendritic cells. (For review, cf., Roberts and Knight, 1994.) Using the homogeneous THP-1 cell line as source of pure monocytes, CS was identified as a potent inhibitor of tumor necrosis factor a with effective in vitro concentrations in the 10 to 20 nM range. By contrast, the compound was about ten times weaker on interleukin-lp synthesis and release and clearly much less active on interleukin-6 release (effectivevalues at 1 pM) (P. H. Cooper, unpublished results, Sandoz Pharma Ltd.). Reisman et al. (1991), using the same THP-1 cell line and a high CS concentration of 1 pglml, observed a suppressive effect on IL-1 cytokines. The results reported by Nguyen et al. (1990)confirm that CS blocks lipopolysaccharideinduced production of tumor necrosis factor from macrophages both in vitro and in vivo without decreasing tumor necrosis factor mRNA. In contrast to the former group, Goldfeld et al. (1993)have investigated the ability of CS to block in vitro the induction of tumor necrosis factor-a mRNA in an untransformed murine T-cell clone that responds to stimulation with antigen and T-cell receptor ligands. They identified a tumor necrosis factor-a promoter element, ~ 3 as, a CS-sensitive regulatory element required for this gene transcription in activated T cells. Activation of this nuclear ~3 binding factor, which resembles the preexisting component of nuclear factor of activated T cells (NF-AT), appears to require posttranslational modification and/or translocation to the nucleus via a calcium-dependent, CS-sensitive pathway. If this were the case, this mechanism of inhibition would appear linked or identical with that effecting immunosuppression. Indeed, it seems reasonable to anticipate that most cellular pathways, which are CS-sensitive in lymphoid cells, should also be affected by CS when they are present in other cell types and, for instance, used for cytokine production. This assumption seems validated by the results from Pigatto et al. (1990) who found that the unstimulated psoriatic blood monocytes have enhanced activity and produce factors that increase neutrophilic functions, but that unstimulated normal monocytes do not secrete. When CS was added to such monocytes cultured in vitro, they demonstrated a significant dose-dependent downregulation of all neutrophilic functions, such as chemotaxis and super-

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oxide radical production, in the supernatants from psoriatic monocytes, which also contained very low levels of tumor necrosis factor (inhibition greater than 90% at 200 ng/ml CS). Evidence supporting an effect of CS on macrophages also in vivo stems from the proliferative immune complex glomerulonephritis of chronic serum sickness in the rat, where the early immunopathogenetic events do not require T-cell activation. When CS treatment started before (but not after) the onset of proteinuria, the course of the disease was altered significantly, because progression from the moderate to the severe stage of nephritis did not occur. The early protective effect of CS was associated with the failure of glomerular macrophages to express abnormal phenotype markers (ED3 antigen) and may be the consequence of a direct influence of the responses of the macrophages and mesangial cells (Ren et al., 1993).Jephthah-Ochola et al. (1988) have studied the regulation of the major histocompatibility complex expression in the mouse in vivo and shown that bacterial lipopolysaccharide induces class I and I1 antigen products in the murine tissues by a T cell-independent but CS-sensitive mechanism. Similar experiments in mice performed by Halloran et al. (1988) and Cockfield et al. (1993) also indicated that non-T cells producing interferon-y following stimulation with lipopolysaccharide in vivo were inhibitable by oral treatment with high doses of CS. Burke et al. (1994a)have observed a significant fall in peripheral blood levels of tumor necrosis factor a after the first month of treatment with CS of newly diagnosed insulin dependent diabetes mellitus subjects. In Vivo kinetic studies in mice by Nguyen et al. (1990) revealed that CS in a very similar way as did an antimurine tumor necrosis factor antibody, inhibited both local and systemic lipopolysaccharide-inducedtumor necrosis factor production, but without decreasing mRNA or cell-associated tumor necrosis factor. CS also prevented the neutrophilia and lymphopenia that developed after a lipopolysaccharide challenge, but it did not block the lung neutrophilic infiltrate. Geratz et al. (1995) have demonstrated that subcutaneous injections of CS in rats receiving intravenous injections of particulate glucan prevented the rise in chemoattractant activity and suppressed the intra- and extravascular monocyte/macrophage accumulation. Finally, CS has further been demonstrated to inhibit zymosan-induced production of interleukin-10 in the mouse tissue chamber model, where macrophages have been shown to be the major cells responsible for its secretion (Dawson et al., 1993). This may suggest a direct action of CS on macrophages. A repeat of this experiment using athymic mice also demonstrated inhibition of interleukin-10 production, strongly indicating that T cells are not involved in this process (J. Dawson and U. Hurtenbach, unpublished observations, Sandoz Pharma Ltd.). Yamaguchi etal. (1993)have reported that administration of CS dramatically affected the distribution of several macrophage subpopulations in the rat hepatic allograft. However, it is not clear whether this reduction in graft

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infiltrating macrophages, which are involved in the rejection process, is exerted directly on these cells or only indirectly via other cells. 2. Epidermal Langerhans Cells

These cells are very effective accessory and antigen-presenting cells and within the normal epidermis are the only dendritic cell population expressing class I1 antigens. Furue and Katz (1988) have, as previously mentioned, unequivocally demonstrated that high, but physiologically relevant skin concentrations of CS (5 to 10 puglml), were able to directly inhibit in uitro the accessory cell functions of Langerhans cells without affecting their class I1 antigen expression. Reconstitution experiments using recombinant interleukin-1 failed to restore these functions. Teunissen et af. (1991) have confirmed these results and shown that CS concentrations up to 5 pg/ml did not alter the production of interleukin-1 or prostaglandin by Langerhans cells. Similarly, Dupuy et al. (1991) have demonstrated that CS (1 pg/ml) inhibited the antigen-presenting function of freshly isolated human Langerhans cells in uitro, but did not significantly modify interleukin-1 and prostaglandin E2 amounts nor the expression of several major histocompatibility antigens. Demidem et al. (1991) reported that CS concentrations of 1 and 10 pg/ml inhibited the accessory cell functions of peripheral blood monocytes, fresh and cultured human Langerhans cells. However, the expression of class I1 molecules on B cells and macrophages was unchanged by exposure to CS. To determine whether the proliferative capacity of Langerhans cells is affected by CS treatment, Haftek et af. (1990/91)used the model of normal human skin grafts on athymic “nude” mice. Their results indicate that Langerhans cell DNA synthesis was impaired by therapeutic levels of CS. In most of the above series of in uitro experiments, relevant controls and reversibility assays have shown that the effective CS concentrations were not cytotoxic for the epidermal Langerhans cells or macrophages. There is, however, a controversy about CS affecting directly or indirectly via drug carryover the antigen-presenting cell function of Langerhans cells. The direct inhibition of CS on these cells as presented above by Dupuy et af. (1991) and Demidem et af. (1991) have been challenged by PkguetNavarro et af. (1991)who performed very similar experiments. They claimed that CS-treated Langerhans cell-enriched epidermal cells kept in culture for several days still released enough drug into the supernatant to account for the inhibition measured in vitro in the mixed epidermal cell lymphocyte reaction. (The debate is represented in 1. Invest. Derrnatof. 97, 953-954, 1991.) 3. Keratinoqe Proliferation

It is controversial as to whether the therapeutic effect of CS in dermatology can also be explained by a direct inhibition of keratinocyte proliferation (cf., review by Wong et al., 1993). Besides negative evidence, several authors

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have claimed that CS can in vitro inhibit keratinocyte proliferation in a dose-related manner but this effect appears influenced by the culture conditions used and requires rather high drug concentrations. Duncan (1994)has studied the differential inhibition of epidermal cell proliferation from guinea pigs by CS, tacrolimus (FK 506),and sirolimus (rapamycin). Her findings showed that tacrolimus failed to have any effect on keratinocyte growth, whereas CS and sirolimus were very effective in inhibiting their proliferation. Furue et al. (1988) have obtained positive results using 3 to 10 pg/ml CS with human and murine cell lines in serum-free cultures. Sharpe and Fisher (1990) and Nickoloff et al. (1988) have performed similar experiment but using primary cultured keratinocytes. It is of interest to note that the cytostatic effect of CS is reversible which indicates that this action is not cytocidal and also may explain the relapse after discontinuation of therapy (Nickoloff et al., 1988). Furthermore, the same investigators demonstrated a marked inhibitory effect at 5 to 10 pg/ml of (D-MeVal")-CS (cyclosporin H), a nonimmunosuppressive derivative, to inhibit reversibly keratinocyte proliferation but not T-cell activity. This analogue may provide an important pharmacological tool for future clinical investigations of the mechanism of action of CS in dermatological diseases. If (D-MeVal")-CS would improve psoriasis, this would suggest that the cellular target of CS in psoriatic skin is the keratinocyte rather than the lymphocyte. The results with this CS derivative have been confirmed by Ramirez-Bosca etal. (1989)and Amsellem et al. (1992) in similar experiments. This may imply that the mechanism causing the arrest in keratinocyte proliferation is quite different from the one underlying the effect of CS on lymphocytes as suggested by the work of Richter et al. (1995). They have determined an ICso of 2.5 p M CS to inhibit the proliferation of transformed keratinocytes (A431 cells). The ICso for blocking growth factor-induced mitogenesis of fibroblasts was approximately 1.5 pM.When using pyrethroids as potent calcineurin inhibitors, they found that the blocking effect of CS was unlikely to result from inhibition of calcineurin alone. There is now concurrent evidence that CS may act on a pathway common to a number of growth factors and downstream of receptor phosphorylation. In this context, the in vivo results of Ramirez-Bosca et al. (1990)using human epidermal keratinocytes xenografted onto congenitally athymic mice are of interest because they show that CS exerted a cytostatic effect by decreasing the rate of DNA-synthesising epidermal cells also under conditions devoid of T cell-mediated immunity. In another in vivo assay in mice, Kietzmann et al. (1990)have assessed the influence of CS given subcutaneously on epidermal hyperproliferation induced by abrasion of superficial epidermal layers of normal skin. They found a dose-dependent inhibition of epidermal proliferation as measured by two different parameters. At variance are the results from Arnold et al. (1993)who investigated the effect of oral doses of CS in psoriatic patients on epidermal proliferation following

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standardized skin injury. They concluded that systemic treatment with CS doses sufficient to produce a pronounced clinical effect did not inhibit epidermal proliferation in vivo. Keratinocytes produce multiple cytokines to a variety of stimuli but it is not clear whether CS therapy occurs through modulation or cytokine production by keratinocytes. Won et al. (1994) have confirmed that CS inhibits the growth of human and murine keratinocytes in culture. They have further shown that concentrations of 5 to 10 pg/ml of CS selectively inhibited the expression of specific cytokine genes, such as for interleukin1 and -8 and tumor necrosis factor a,which are chemotactic for leukocytes, in both human and murine keratinocyte cultures in a dose-dependent fashion. Elder et al. (1993) and Kojima et al. (1993) have also produced data supporting an in vivo effect of CS in psoriasis to reduce the production by keratinocytes of several cytokines, including interleukin-lp, -8 and the groa peptide belonging to the intercrine-a family. However, since CS pretreatment had no inhibitory effect on cytokine expression in cytokine-stimulated cultured keratinocytes, these findings strongly suggest that CS does not exert its antipsoriatic effects directly on the keratinocyte but rather by inhibiting activated T cells to produce signals leading to increased keratinocyte cytokine expression. Previously, Gallo et al. (1992)had also demonstrated that shortterm CS exposure did not inhibit the expression of granulocyte/macrophage colony-stimulating factor in murine keratinocytes in vitro. Finally, Gilhar et al. (1991) have reported that systemically applied CS was able to directly inhibit normal mouse serum-induced class I1 antigen expression on skin keratinocytes in nude mice. 4. Other Cell Types Affected by CS

Yard et al. (1993) have investigated the inhibiting effect of CS on interleukin-la enhanced tumor necrosis factor a production by cultured human proximal tubular epithelial cells and found a dose-dependent effect with a maximal effect at 250 ng/ml corresponding to 90 percent inhibition. It has been shown that CS may interfere in vitro with the secretory function of some cell types, especially the mast cell and the basophil as reported above (cf. Section V.D.) Martin and Bedoya (1990) have tried to characterize the short-term effects of CS on secretagogue-induced insulin release by isolated rat islets. Their data indicate that a concentration of 0.5 pg/ml CS was able to block glucose-induced release in isolated islets. This effect was not reversed by using substances known to activate the protein kinase C or the calcium-dependent branches of the stimulus-secretion coupling system in p-cells, indicating that the site of action of CS might be located in distal steps of the stimulus-secretion coupling of glucose-induced insulin release. Despite the extremely controversial reports in the literature, which have been reviewed by Bore1 et al. (1989, p. 289) and more recently by Jindal(1994),CS in general does not create serious concerns as a diabeto-

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genic drug. In particular, CS did not impair glucose homeostatis in a clinical study examining intravenous glucose tolerance and pancreatic islet P-cell function in a group of 22 nondiabetic multiple sclerosis patients before and during a 2-year course of CS or placebo therapy (Robertson et al., 1989). Several studies have demonstrated that CS increases the regenerative response after partial hepatectomy (Kim et al., 1988; Kahn et al., 1988) or orthotopic reduced-size hepatic transplantation in the rat (Kikuchi et al., 1993). The hepatocyte regeneration during liver allograft rejection in the rat was also positively influenced by CS (Takata et al., 1993), but the intracellular mechanisms affected by the hepatotropic effect of CS remain yet unknown. Results by Masuhara etal. (1993)confirmed that CS stimulates rat liver proliferation in vivo and that this effect did not entail changes in the production of hepatocyte growth factor or transforming growth factors a and 01. Hudnall (1991) has reported that CS renders some human target cells resistant to immune cytolysis, possibly by blocking some target cell biochemical pathway important in the suicidal cytolytic process which is linked to some early cell cycle events. His in vitro results would indicate that the increased risk of Epstein-Barr virus-associated lymphoproliferative disease in human organ transplant recipients may be augmented by CS-induced resistance of such virus-transformed B lymphocytes to immune cytolysis, this in addition to the suggested blocking effect of CS on T cell-dependent responses to EBV-transformed B cells. Sharma et al. (1993) have recently published a clinical study in which they observed that CS increased muscular force generation in Duchenne muscular dystrophy, when it was administered during 8 weeks at the moderate dose of 5 mg/kg/day. The increment in isometric force became apparent within 2 weeks and continued until therapy was stopped. In another clinical study in juvenile dermatomyositis, CS treatment led also to recovery of muscle strength and function and to resolution of complications (Heckmatt et al., 1989). One can only speculate on the mechanism underlying this therapeutic action. As will be reported in Section VII, CS does not seem to have a direct modulatory effect on smooth muscle cell proliferation in vitro. However, contraction of smooth muscle in isolated rat aorta was induced by a high concentration of CS and reversed by the calcium channel blocker verapamil (Xue et al., 1987).Furthermore, the results from Meyer-Lehnert and Schrier (1989), who studied the effect of CS on calcium kinetics and contraction in primary cultures of rat aortic smooth muscle cells, suggest that the drug stimulates transmembrane calcium-influx, thereby increasing arginine vasopressin-sensitive intracellular calcium pools in these cells. If CS did increase calcium available for release from the sarcoplasmic reticulum of skeletal muscle in patients with Duchenne muscular dystrophy, this could account for the increased force generation observed in the study of Sharma et al.

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(1993). Interestingly, Goldberg et al. (1989) found a dissociation between the immunosuppressive activity of CS derivatives and their effects on intracellular calcium signaling in cultured rat mesangial cells, because (D-MeVa1")CS (CsH), which is nonimmunosuppressive, caused a large increase in peak vasopressin-stimulated intracellular free calcium release, similar to that of CS. However, two major CS metabolites OL-17 and OL-1, which have a much reduced immunosuppressive activity compared to CS itself, had no effect on the peak intracellular free calcium response. 6. Effects on Various Cellular Functions I . Reversal of Mukidrug Resistance of Cancer Cells

One major cause of therapeutic failure in cancer is the presence of intrinsically anticancer drug resistant cells and/or the emergence of resistant clones after repeated courses of chemotherapy. In order to avoid development of drug-specific resistance, chemotherapy treatment protocols frequently use several drugs with different structures and modes of action. This problem is further exacerbated by the observation that tumors may often be cross-resistant to other chemotherapeutic agents, even though these drugs were not used in the initial treatment, belong to unrelated structural classes and have different mechanisms of action. This phenomenon is widely known as multidrug resistance (Morrow and Cowan, 1990). The most common and better characterized mechanism by which tumor cells acquire multidrug resistance is the overexpression of a particular class of transmembrane glycoprotein encoded by a small family of mdr genes and called the P-glycoprotein (Gros and Buschman, 1993). P-glycoprotein is a 170 to 180 kDa integral plasmamembrane glycoprotein expressed in a variety of human tissues and tumors. P-glycoprotein molecules seem to confer resistance by acting as an energy-dependent efflux pump with a capacity to recognize several antineoplastic drugs. By exporting anticancer drugs from the cell, the P-glycoprotein lowers their effective intracellular concentration below their active (cytostatic) threshold. With cell lines, the level of Pglycoprotein expression correlates with decreased drug accumulation in the cells and increased cell resistance to several drugs. Several reports have indicated the resistance-modulating activity of CS (for review see Bore1 et al., 1989; Loor, 1994; Bohme et al., 1994; Colombo et al., 1994). By studying a variety of CS-derivatives, the known immunosuppressive activity could be clearly separated from the resistance-modulating effect (Gavkriaux et al., 1989). The in vitro resistance-modulating activity of the nonimmunosuppressive CS analogue SDZ PSC 833, which is (3'-KetoMeBmt-(1)-Val2)-CS,was found to be much higher than those of classical resistance-modulating agents, especially its capacity to restore the anticancer drug-sensitivity of the in vitro growth of multidrug resistant tumor cells (Gavkriaux et al., 1991; Boesch et al., 1991b; Jachez et al., 1993) and the

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intracellular retention of daunomycin, an anticancer drug (Boesch et al., 1 9 9 1 ~Boesch ; and Loor, 1994), as well as rhodamine 123, a nonanticancer drug P-glycoprotein probe (Pourtier-Manzanedo et al., 1992). The cyclosporin D derivative SDZ PSC 833 behaves like actual P-glycoprotein blockers (Didier and Loor, 1995; Jiang et al., 1995) and does not bind to cyclophilins A, B, and C (V. Quesniaux, personal communication, Sandoz Pharma Ltd., Basel). Evidence for the in vivo activity of SDZ PSC 833 in a murine multidrug resistant tumor graft model was obtained (Boesch et al., 1991a). A therapeutic window for multidrug resistance cancer chemotherapy could be restored by a combined treatment of doxorubicin and SDZ PSC 833 with acceptable myelotoxicity (Froidevaux and Loor, 1994). Keller et al. (1992b) have also tested in vivo the potency of CS and SDZ PSC 833 to overcome P-glycoprotein-mediated multidrug resistance of the murine L1210 leukemia. In Vivo the drug-resistant cell line was completely unresponsive to intravenous monotherapy with adriamycin at its maximum tolerated dose. SDZ PSC 833 enhanced the activity and toxicity of adriamycin which was about three times lower than when given alone. On this basis, the maximum tolerated dose of intravenous adriamycin in combination with oral SDZ PSC 833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred. A striking increase in antineoplastic activity and toxicity of the cytostatic agent etoposide was also found in combination with SDZ PSC 833 in vivo in rats (Keller et al., 1992a).This effect was paralleled by marked changes in the pharmacokinetic parameters of etoposide in vivo, but the underlying mechanisms of the reversal of multidrug resistance by the cyclosporins are not yet understood. Watanabe et al. (1995) have in essence confirmed the above findings using similar in vitro and in vivo models. In particular, SDZ PSC 833 significantly enhanced the increase in life span by more than 80 percent in doxorubicin-resistant P388-bearing mice and also showed significant potency in the doxorubicin-resistant colon adenocarcinoma 26-bearing mouse model. Unfortunately, similar effects have so far not been reproduced in two clinical trials when using CS (Osieka et al., 1988; Verweij et al., 1991). In a third phase I trial of etoposide with CS as a modulator of multidrug resistance, Yahanda et al. (1992) have reported tumor regression in 4 out of 57 patients. The study indicated that highdose, 3-day continuous infusions of CS in combination with reduced doses of etoposide could be administered with acceptable toxicity (Lum et al., 1992). 2. Seleaive Formylpeptide Receptor Antagonism

Human neutrophils possess several classes of receptors with different yet overlapping functions. Among those are the chemoattractant receptors and, in particular, that for the chemotactic formylpeptide (Lew and Krause,

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1993). BocPLPLP is one of the most potent formylpeptide receptor antagonists, which inhibit formylpeptide-stimulated azurophilic granule release and high affinity GTP hydrolysis in rabbit neutrophils. In contrast to CS, (D-MeVal")-CS, which lacks immunosuppressive activity and does not bind to cyclophilin, has been described as a potent inhibitor of formylpeptideinduced superoxide anion formation in human neutrophils and also to prevent formylpeptide binding in HL-60 membranes ( Wenzel-Seifert et al., 1991). Recently, the same group has demonstrated that this natural CS congener acts as a significantly more potent and selective formylpeptide receptor antagonist than BocPLPLP ( Wenzel-Seifert and Seifert, 1993). (D-MeVal")-CS may become a valuable compound to elucidate the still poorly defined role of endogenous and bacterial formylpeptides in the pathogenesis of inflammatory processes in vivo. C. Antibiotic Effects The modulation by CS of host defense mechanisms against infections and its various effects against microorganisms have been reviewed by Bore1 et al. (1989; pp. 339-351) and High (1994). The present discussion will be limited to some newer aspects stemming from recent work. I . Inhibition of HIV- l Replication

The evaluation of the antihuman immunodeficiency virus type 1(HIV-1) activity in vitro of more than 200 CS derivatives showed that the antiviral activity did not correlate with the immunosuppressive potential. However, the structure-activity relationship revealed a strong correlation of the antiviral activity with the cyclophilin-binding capacity of the compounds (Thali et al., 1994). Similar findings were reported by Bartz et al. (1995). The most effective CS analogue found was (Me-Ile4)-CS(SDZ NIM 811) which is devoid of immunosuppressiveactivity but exhibits a high cyclophilin binding capacity and a potent anti-HIV-1 activity (Rosenwirth et al., 1995). It selectively inhibits HIV-1 replication in CD4 T-lymphocyte cell lines, in a monocytic cell line, and in HeLa CD4 T cells. Furthermore, its antiviral activity was demonstrated against laboratory strains and against clinical isolates from geographically distinct regions in primary CD4 T lymphocytes and in primary monocytes. SDZ NIM 8 11does not inhibit proviral gene expression or virus-specific enzyme functions, neither free nor bound to cyclophilin, and it does not influence CD4 expression or inhibit fusion between virusinfected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells (Rosenwirth et al., 1994). The mechanism of the antiviral activity of CS and its effective derivatives appears closely linked with their potent cyclophilin binding capacity. The retroviral Gag protein is capable of directing the assembly of virion particles independent of other retroviral elements and plays an impor-

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tant role early in the infection of a cell. Luban et al. (1993) have identified two host proteins, cyclophilins A and B, which interact specifically with the HIV-1 Gag polyprotein. The results by Franke et al. (1994) indicate that the interaction of Gag with cyclophilin A is necessary for the formation of infectious HIV-1 virions. In addition, Thall et al. (1994) have demonstrated that drug-induced reduction in virion-associated cyclophilin A levels was accompanied by reductions in virion infectivity indicating that the association is functionally relevant. Moreover, SDZ NIM 811 inhibited the replication of HIV-1 but was inactive against SIVMc, a primate immunodeficiency virus closely related to HIV-1 which does not incorporate cyclophilin A. It has previously been suggested that immunosuppression by CS may have a beneficial effect in HIV disease since it would block CD4 T-cell activation, which is required for HIV replication, inhibit different steps of the autoimmune process leading to the killing of the CD4 lymphocytes, and possibly counteract HIV-induced apoptotic death of these T cells (Klatzmann and Gluckman, 1986). However, the few clinical trials undertaken have not indicated any beneficial effect of CS in HIV disease (Andrieu et al., 1988; Phillips et al., 1989). Perhaps the most revealing clinical evaluation is the survey on patients with HIV-transmission by transplantation performed by Schwarz et al. (1993). They evaluated the case reports of 53 patients with HIV-infection by an infected transplant or by blood transfusion during or shortly after transplantation. The cumulative incidence of AIDS was significantly lower in 40 transplant patients with an immunosuppressive regimen including CS than in 13 patients receiving no CS. The 5-year cumulative risk of AIDS was 31 percent versus 90 percent. It could be concluded that even if administered at the time of infection as in transplant patients CS cannot totally prevent the progression of HIV-1 infection but it does seem to have a moderating effect. However, the efficacy of the novel compound SDZ NIM 811, which acts synergistically with azidothymidine, has not yet been assessed under clinical conditions. 2. Antifingal Properties of Cyclosporins

Newer investigations by Dreyfuss (Sandoz Pharma Ltd., Basel; unpublished results) on a possible correlation between the antifungal and immunosuppressive activities and cyclophilin-binding capacity of a series of selected cyclosporins have yielded negative results. His results clearly show that the antifungal activity as measured in the ramification test with Neurospora crassa and in the agar diffusion assay with Aspergillus niger does not correlate with immunosuppression nor with cyclophilin binding. Borel et al. (1989, p. 345) have previously reviewed the impact of CS on phagocyte-mediated host defense against infections. Recently, Roilides et al. (1994) have assessed the effects of CS on the activity of circulating phagocytes and macrophages against hyphae and conidia of Aspergillus fumigatus both in vitro and ex vivo. Therapeutic drug concentrations up

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to 250 ng/ml did not alter the superoxide anion production of stimulated polymorphonuclear leukocytes, but concentrations of 500 ng/ml and above significantlysuppressed it. However, incubation of monocytes with 100 ng/ ml of CS inhibited their antihyphal activity. No essential change in phagocytic activity of monocyte-derived macrophages was detected after 2 to 4 days of incubation over the range of 10 to 1000 ng/ml CS. When rabbits were treated with very high CS doses of 20 mg/kg/day intravenously for 7 days, the superoxide anion production and hyphal damage caused by polymorphs or monocytes against hyphae were not affected and the phagocytosis of conidia by pulmonary alveolar macrophages was also not significantly suppressed. 3. The Antiparasitic Efecrs of the Cyclosporins

In parasitology, CS and some few derivativeshave been employed almost exclusively as a research tool. It seems unlikely that CS itself could ever be used clinically to control parasite infection in man or animals; however, the more immediate interest of the cyclosporins is in their use as probes with which to explore the immunobiologic interactions of the host-parasite relationship (for review, cf., Chappell and Wastling, 1992; Bore1 et al., 1989, pp. 349-351). Similar studies to those of Dreyfuss (see above C.2.) were performed by Bell et al. (1994) who analyzed the roles of peptidyl-prolyl cis-trans isomerase (cyclophilin) and calcineurin in the mechanism of antimalarial action of CS, tacrolimus (FK 506), and sirolimus (rapamycin).The peptidylprolyl cis-trans isomerase activity was detected in extracts from Plasmodium falciparum and was completely inhibited by concentrations of CS above 0.1 p M but not by FK 506 or rapamycin. Comparison of the antimalarial and antiisomerase activities of a series of selected CS derivatives failed to reveal a correlation between the two properties. FK 506 and rapamycin were also active antimalarials although at higher concentrations than the cyclosporins but in the absence of a detectable macrophilin isomerase (FKBP) activity in P. falciparmm. Therefore, the mechanism of action by which these compounds act as antimalarials remains unknown. Grau et al. (198713)have investigated the influence of CS in a murine model of cerebral malaria and shown that an extremely low dose of CS (1 mg/kg/day orally on Days 4 to 8), which has no effect on parasitaemia and is not immunosuppressive in mice, displayed a potent protective effect on neurovascular complications. A similar protective effect was also observed with two almost nonimmunosuppressive CS derivatives, SDZ 205634 and SDZ 207-594, which are an ester derivative of (Threonin2)dihydroCS and [(6’E)-8’-Nor-7’-pheny11]-CS, respectively. Higher doses of all these compounds were parasiticidal but, paradoxically, they did not afford protection against neurological manifestations. Grau et al. (1987a) have convincingly demonstrated that tumor necrosis factor was an essential mediator in

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murine cerebral malaria. Later work which included other experimental models of inflammation has substantiated the evidence for CS possessing an exquisite inhibitory action on the expression and/or release of the tumor necrosis factor (G. Grau, WHO and University of Geneva, Geneva; personal communication). While the direct in vitro antiparasitic effect of the active cyclosporins against P. falciparum occurs in the range of 0.05 to 0.3 p M (Bell et al., 1994) and mostly affects the asexual blood stages of P . berghei in infected mice (Murphy et al., 1988), it appeared that CS exerts parasiticidal effects in vitro against Leishmania major promastigotes only in extremely high concentrations of 5 pg/ml according to Bogdan et al. (1989) or 25 pg/ml (Behforouz et al., 1986). Interestingly, Behforouz et al. (1986) have demonstrated that the CS congener 1-(8’methoxy)-dihydro-CS (SDZ 205-549), which possesses only marginal immunosuppressive activity, was enhancing resistance in L. major infected mice like CS when given prophylactically. Bogdan et al. (1989)reported that CS profoundly enhanced the degradation by macrophages of both intracellular L. major promastigotes and amastigotes but another very similarly structured CS derivative (desoxy-C,’)-CS, which has antifungal but lacks immunosuppressive activity, was ineffective. Sypek and Wyler (1990)have identified two different mechanisms for the activation of macrophages to exert their antileishmanial defense in vitro (ulcerative leishmaniasis). One is lymphokine dependent and CS sensitive, whereas the second might involve the interaction of a lymphocyte membrane-associated macrophage-activating factor with its receptor on the macrophage and is fully CS resistant. It has been suggested that CS inhibits the lymphokinedriven ability of T cells to recruit macrophages, which are required by L. major for multiplication (cf. Chappell and Wastling, 1992); however, this explanation is not satisfactory in view of the above facts. Murray et al. (1992) have analyzed and discussed the complexity of the tissue immune response in initial versus established immunity to visceral leishmaniasis in mice; CS was used as an experimental probe. The antihelmintic activity of CS has recently been investigated in depth by Chappell and Wastling (1992) who have shown the drug to be a potent schistosomicide with unusual and potentially exploitable properties. All the reports have clearly demonstrated that the action of CS on schistosomes in mice was dose- and time-dependent and also that the route of drug administration was important (Chappell and Wastling, 1992).Thus, CS has been unequivocally shown to be prophylactic when administered subcutaneously (but not orally) at least up to 100 days prior to infection. In addition, CS markedly reduced the worm burden when given at or after infection (Munro et al., 1991a). There is clear evidence to suggest that CS exerts its schistosomicidal effects by mechanisms distinct from its immunomodulatory properties (Munro and McLaren, 1990a,b). Weakly or nonimmunosuppressive analogues such as 1-(8’methoxy)-dihydro-CS (SDZ 205-549) are com-

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parably active against schistosomes (Chappell and Thomson, 1988) and these compounds retain their antischistosomal activity in athymic and other immunodeficient mice which is also unaffected by concomitant administration of SDZ ADA 202-718, a cytokine inducer (Brannan et al., 1989). The doses of CS necessary to protect mice from schistosome infection are rather low, i.e., in a suboptimal range for achieving immunosuppression. Moreover, transfer of spleen cells from CS-treated mice do not confer resistance on infected but untreated mice (Chappell and Thomson, 1988). There is, however, controversy about the killing capacity of CS in vitro and, similarly with some other parasites, the antiprotozoal activity may be due to some yet unresolved host-mediated mechanism. Chappell et al. (1989) have further demonstrated a moderate shortlived and reversible action of CS and of its 1-(8’methoxy)-dihydro-CSderivative (SDZ 205-549) against cestodes like Hymenolepis microstoma in mice. In contrast, the closely related helminth H. diminuta is resistant to CS and parasite growth was enhanced when treating mice with the drug immediately following infection (Wastling et al., 1990). These contrasting effects on tapeworms reveal that CS is antiparasitic against some species, while it may be immunosuppressive for other infections. The direct effects of CS on the morphology of H. microstoma correlate with the antihelminthic activity, since CS treatment dramatically reduces worm growth, retards migration into the bile duct, and limits parasite survival (Wastling et al., 1992).Recent data by Wastling and Chappell (1994) have confirmed that CS treatment in vivo disrupts the functional integrity of the worm tegument, one facet of which is impaired acquisition of glucose. The data from Yoshimura et al. (1993), who have studied the effect of CS on Angiostrongylus cantonensis infection in mice, would also suggest a direct damaging activity of CS against certain developmental stage(s) of the parasite. In their review, Chappell and Wastling (1992) presented several instances in which CS acts in two distinct modes against a single parasitic infection behaving in part as an immunosuppressant and in part as an antiparasitic compound. Thus, Trichinella spiralis in the mouse model responds in two apparently opposing ways, i.e., that expulsion of adult worm from the mouse gut is significantly delayed, whereas the muscle larvae count is markedly depleted in CS-treated mice compared with controls (BolasFernandez et al., 1988; Boulos et al., 1992a,b). These conflicting results clearly reflect, on the one hand, a drug-diminished immune response to the adult parasite and, on the other, an antihelmintic effect directed at reproduction of adult females. In contrast, CS seems to act solely in an antiparasitic mode against T.pseudospiralis (Cabaj, 1990). In conclusion, there is no doubt that CS as well as some of its nonimmunosuppressive but parasiticidal derivatives still have an important role to play as biological probes for studying various immunobiologic aspects of the host-parasite relationship.

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VII. Chronic Allograft Rejection A. Clinical Situation

After a difficult and prolonged learning process in which the efficacy and the side effects of the drug were explored, CS is now being used as the mainstay in clinical immunosuppression. The question concerning the therapeutic window in either induction or maintenance therapy is whether one can get adequate immunosuppression without irretrievable and irreversible damage to the kidney (Burke et al., 1994; Lorber, 1991; Stiller and Opelz, 1991; Koote et al., 1988). The benefits of CS in comparison with alternative immunosuppressive protocols seen in the first 3 months to 2 years are sustained (Opelz, 1992; Thorogood et al., 1992; Kriett and Kaye, 1991; Grattan et al., 1990; Sutherland et al., 1989).Consequently, the major impact of CS is to prevent acute, i.e., cell-mediated, early rejection (Fischer et al., 1991b; Gilki et al., 1990; Terasaki et al., 1989). However, as mentioned in many single center and collaborative studies, the research emphasis is shifting from early posttransplant events to late events that influence longterm outcome (Offermann et al., 1993; Dunn and Kahan, 1992; Opelz, 1992; Almond et al., 1992; Klare et al., 1991; Montagnino et al., 1991; Gruber et al., 1991; Amend et al., 1990; Canafax et al., 1990; Ettenger et al., 1990; Lewis et al., 1990; Monaco et al., 1990; Moreno et al., 1990). Next to drug toxicity and posttransplant malignancies as a complication of immunosuppression, it is now chronic graft rejection which moves into the focus of attention (Shaikewitz and Chan, 1994; Ferguson, 1994; van der Woude et al., 1994). CS, though effective at reducing graft loss due to acute rejection, has had little impact on the incidence of chronic rejection which is a prime barrier to long-term organ allograft survival (Almond et al., 1993; Hong et al., 1992; Modena et al., 1991; Salomon, 1991; Tilney et al., 1991; Monaco et al., 1990).Many studies have demonstrated that renal transplant recipients receiving CS have a clear trend of slowly declining allograft function which was comparable with control patients not treated with CS and which is consistent with the effect of chronic rejection (Varenterghem and Peeters, 1994; Isoniemi et al., 1994; Matas, 1994; Almond et al., 1993; Bergmann et al., 1993; Hong et al., 1992; Fischer et al., 1991b; Tilney et al., 1991; Lewis et al., 1990). The major causes of graft loss beyond the first year posttransplantation are chronic rejection, followed by death with a functioning graft, patient noncompliance (Didlake et al., 1988), and sepsis. Similar results have also been reported for cardiac transplant patients by Grattan et al. (1990), though the term graft atherosclerosis or graft coronary artery disease is used instead of chronic rejection as for renal recipients (see also Berry et al., 1993; Gao et al., 1993; Pfau and Bender, 1993; Schoen and Liby, 1991). The primary etiology of late acute rejection

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may have included subtherapeutic levels of CS (Almond et al., 1993; Burke et al., 1994; Salomon, 1991; Wrenshall et al., 1990). Acute rejection of a kidney graft diagnosed by standard clinical criteria with histologic confirmation can be treated with pulse methylprednisolone and/or antilymphocyte globulin preparations and is different from chronic rejection which is defined as the presence of a slow and relentless deterioration of renal function in conjunction with proteinuria and hypertension and documented histologically by the presence of interstitial fibrosis, tubular atrophy, and vascular wall thickening involving the intima only (Billingham,1994; Shaikewitz and Chan, 1994; Sumrani eta!., 1993; Olson, 1986).Although the pathogenesis of chronic rejection remains unknown, with both immunologic and nonimmunologic factors implicated, early acute rejection episodes have been associated with an increased allograft loss from chronic rejection (Matas et al., 1994; Yokoyama et al., 1994; Sumrani et al., 1993; Tesi et al., 1993; Gulanikar et al., 1992). Odland and Kasiske (1993) have recently reported that kidneys from female donors were at an increased risk for chronic allograft rejection, while Grattan et al. (1990) found that male cardiac recipients died of graft atherosclerosis significantly more often than did female recipients. It is evident today that the major unresolved clinical problems in transplantation concerns the long-term outcome of organ allografts. It appears that CS may not have significantly improved the relentless attrition rate in long-term graft survival. Interestingly, however, Rosenblum et al. (1988) and Rowe et al. (1988) have each used CS to treat chronic renal allograft rejection in two small-sized patient groups and concluded that CS induced a slowing of the rate of loss in graft function but it could not reverse chronic rejection. Addonizio et al. (1993)have demonstrated a significant decreasing incidence of coronary artery disease in pediatric cardiac transplant recipients using increased immunosuppression, particularly high CS doses. Tullius and Tilney (1995) have presented an excellent updated overview on the alloantigen-dependent and -independent factors influencing chronic allograft rejection.

B. Experimental Approaches Chronic rejection has a variety of histopathological manifestations depending on the type of the transplanted organ allografts, but common to all of them is obliterative arteriopathy associated with an ongoing inflammatory response and affecting the intraorgan muscular arteries (cf., Gouldesbrough and Axelsen, 1994). Since chronic rejection has now emerged as the major obstacle of long-term graft survival, it is imperative to investigate how these interactions are regulated. Only recently have experimental models in vivo been elaborated which allow study of the development of accelerated allograft arteriosclerosis. Mennander et al. (1991a) have developed an animal

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model for chronic rejection in which aortic allografts were exchanged between histoincompatible rat strains. They further reported that allografted rats immunosuppressed with CS (5 mg/kg/day p.os) exerted an early inflammatory lesion in the subendothelial space (endothelialitis) which was followed by an influx of proliferating smooth muscle cells into the intima and resulting in intima thickening and accelerated arteriosclerosis (Mennander et al., 1991b). The same group has also demonstrated that cytomegalovirus infection was enhancing allograft arteriosclerosisin the rat model (Lemstrom et al., 1993). They furthermore found that triple drug immunosuppression (CS: 10 to 20 mg/kg/day) had a protective effect on vascular wall histology in this rat cytomegalovirus-enhanced allograft arteriosclerosis model (Lemstrom et al., 1994). CS provides effective immunosuppression for preventing arterial and venous allograft rejection and failure. Schmitz-Rixen et al. (1988) have performed aortic allograft studies in the rat and examined the grafts at different time intervals for patency, aneurysmal dilation, gross structural changes, inflammatory responses, and infiltration of lymphocytes. Aneurysmal dilation was reduced or prevented by CS correlating with medial smooth muscle preservation, and both cellular infiltration and intimal thickening in the graft were delayed. The degree of improvement was clearly dependent on the dose and the length of CS treatment. Augelli et al. (1991) have transplanted fresh or cryopreserved vein allografts in dogs and assessed vein patency under CS treatment. Their results demonstrate that long-term patency of saphenous vein allografts was achieved only with the combination of cryopreservation and immunosuppression with continued CS. Miller et al. (1993)have also shown that cryopreserved venous allografts in the dog remain patent with antiplatelet and immunosuppressive therapy in spite of loss of functional smooth muscle. Both Belitsky et al. (1993) and Steele et al. (1993) were not able to demonstrate a significantadvantage of CS in modifying the vascular changes of chronic rejection in the rat aortic allograft model. In their excellent review, Hayry et al. (1993; Table XVIII) tested different immunosuppressive drugs in the rat aortic allograft model and demonstrated that CS at 5 mg/kg/day orally had a significant beneficial effect on adventitial inflammation but a negative one on intimal thickness. However, in the adult male ACI donor and Lewis recipient combination in the same rat model with a dose of 10 mg/kg of CS given per 0s daily, Stoltenberg et al. (1994) have observed a clear preventive effect on the development of graft arteriosclerosis. We have been able to confirm such an effect with oral doses of 10 to 15 mg/ kg/day of CS for 2 months, but in a different strain combination: DA into WF rats (Lu and Borel, Sandoz Pharma AG, Basel; unpublished results). Continuous high oral dose treatment (10 mg/kg/day or more) with CS seems crucial for achieving successful prevention of chronic rejection, although a lower intraperitoneal dose of 6 mg/kg/day was also reported to be fully

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effective in preventing graft-vessel disease in rats after cardiac transplantation (Meiser et al., 1991). Hisatomi et al. (1995) have also been successful with 6 mg/kg/day of CS injected intramuscularly for 90 days, but the lower dose of 2 mg/kg had no protective effect. From preliminary experiments using the above rat aorta allograft model, Lu and Bore1 (unpublished results) have concluded that the prevention of graft vascular disease may be linked with immunosuppression or/and anti-inflammatory effects. Thus, the CS analogue 1-(8'methoxy)-dihydro-CS (SDZ 205-549), which possesses marginal immunosuppressive but marked anti-inflammatory activity, was moderately effective at 15 mg/kg/day and was as active as CS at the double dose. In contrast, the nonimmunosuppressive analogues (D-Me-Val)"-CS (cyclosporinH), MeBmt (3-keto)'-Val2-CS(PSC 833), and (MeIle)4-CS(NIM 811),of which only the last congener binds to cyclophilin, were all found to be ineffective at the same dose as used for CS. Furthermore, Andersen et al. (1994) have used the aorta-allografted, cholesterol-fed rabbit model, in which plasma cholesterol is clamped at human levels, to investigate the effect of CS on the development of allograft arteriosclerosis. Their results suggest that human therapeutic levels of CS substantially attenuated the severity of transplant arteriosclerosis and that this effect was at least partially mediated by a large decrease in aortic lipoprotein permeability. Yilmaz et al. (1992a,b) and Diamond et al. (1992) have convincingly demonstrated that histoincompatible renal allografts in the rat after a short course of CS, which is sufficient to prevent acute rejection, resulted in chronic progressive rejection for 3 to 6 months, though the grafted kidneys were not acutely rejected nor was chemical evidence of chronic renal insufficiency observed. However, assessment of parameters of renal function and histological differentiation clearly reflected chronic glomerular abnormality beginning with albuminuria and progressively increasing to decline in renal blood flow and glomerular filtration rates, changes associated with glomerulosclerosis. The sequence of the histopathological lesions in this rat allograft model was an early persistent interstitial and perivascular inflammation with a strong pyroninophilic component in the inflammatory infiltrate, followed by glomerular sclerosis and a very strong, occlusive intimal response in the allograft vascular tree. These striking features were not observed in the syngeneic controls. When analyzing further the multifaceted etiology of this process in which there is evidence of the involvement of both antibodyand cell-mediated immune activity (Tilney et al., 1993), it was found that neutrophils and macrophages played a prominent role in the pathophysiology (Heemann et al., 1993) and a novel subpopulation of infiltrating leukocytes in long-term surviving allografts was detected (Stein-Oakley et al., 1993). Tullius et al. (1993) have shown that changes of chronic renal allograft rejection were reversible after retransplantation into the original donor strain up to 12 weeks posttransplantation. After Week 12, intense cellular infiltration and progressive fibrosis made the process irreversible despite

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retransplantation. In addition, Yilmaz and Hayry (1993)have observed that acute renal allograft rejection in the rat model carries a highly significant correlation with the development of chronic rejection, as it is the case in the clinical situation (see above). Therefore, the early stage of chronic rejection appears to be driven by immunologic factors and is reversible up to a critical point after which nonimmunologic factors may become important in causing irreversibility of the process (see also Hansson, 1993). Braun et al. (1993) have recently discovered that the T cells mediating early acute kidney allograft rejection in the rat were CD4 T cells induced by direct pathway and were different from those responsible for chronic rejection, possibly selfrestricted CD4 T cells sensitized by the indirect pathway. Forbes et al. (1994) have provided good evidence that CD8 T cells are not involved in the development of chronic vascular rejection in a rat allograft model since recipient CD8 T-cell depletion does not influence its initiation nor its course. Another experimental model for studying graft arteriosclerosis has been described by Cramer et al. (1989), Fellstrom et al. (1990) and Adams et al. (1992). Basically, the model consists of exchanging heterotopic cardiac allografts between donor and recipient pairs that differ for MHC class I antigens or for minor, non-MHC antigens only. Fellstrom et al. (1990) used in addition a short-course treatment with CS to prevent acute rejection. The results obtained by Cramer et al. (1989) demonstrated that in strain combinations with mild and prolonged allograft reaction, donor hearts exhibited diffuse, interstitial myocardial and perivascular fibrosis and intimal proliferation in arteries of the myocardium. Fellstrom et al. (1990) also observed after 2 to 3 months excessive proliferative changes of the vascular intima and endocardium along with fibrosis and fibrin deposition. Adams et al. (1992)reported that the majority of arteries showed significant intimal disease and that histological lesions in long-term surviving allografts demonstrated fibrous intimal thickening. All these authors observed pathological changes which are consistent with those of graft arteriosclerosis seen clinically (for review see Ip et al., 1990). Both Cramer et al. (1990) and Adams et al. (1992) found that the limited course of CS was associated with a modest or even substantial reduction of arteriosclerotic changes. Handa et al. (1993) have convincingly demonstrated that late CS treatment did significantly ameliorate established coronary graft disease in rat allografts. However, this effect was not permanent and the progressive graft disease recurred after CS therapy was discontinued. Recently, Guttmann et al. (1994) have studied the effect of CS on the prevention and reversal of the so-called transplant arteriosclerosis in a rat cardiac allograft model using a major histocompatibility complex identical system. They clearly showed that a CS dose of 15 mg/kg/day is effective in both preventing and reversing this type of vasculopathy. Paul et al. (1994a) have confirmed these results in the same rat strain combination, but only when they used the renal allograft model. Nagamine et al. (1994) have also used the heterotopic

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cardiac allograft model in a weak genetic rat strain combination (Fischer 344 into Lewis) in which only chronic rejection occurs. Comparing the effects of CS and 15-deoxyspergualin, they found that both drugs were effective, but the latter drug was superior to CS in preventing graft coronary arteriosclerosis. In marked contrast, Paul et al. (1994b)reported that continuous subcutaneous treatment with 15 mg/kg/qod of CS increased the extent of atherosclerosis in both syngeneic and allogeneic, heterotopic cardiac transplants in various rat strains at 100 days posttransplantation. However, the combination of CS with the angiotensin-converting enzyme inhibitor cilazapril had no effect on graft atherosclerosis itself, but it decreased the degree of luminal narrowing significantly. Rowan and Billingham (1990) have undertaken a morphologic study of myocardial hypertrophy, vascularity, and fibrosis in long-term human heart recipients and they concluded that CS treatment was not responsible for significant hypertrophy or fibrosis in most transplants. The effect of fish oil combined with long-term, low-dose CS on accelerated graft coronary arteriosclerosis was assessed in Brown Norway to Lewis rat heterotopic cardiac allografts. The data demonstrated that fish oil supplementation inhibited progression of arteriosclerosis (Sarris et al., 1989), while in the reverse combination of Lewis to Brown Norway strain the same treatment did not exert any beneficial effect (Yun et al., 1991). When comparing the effect of FK 506 with CS on coronary graft disease of rat cardiac allografts, Arai et al. (1992) observed that FK 506, unlike CS, showed severe graft coronary disease. When investigating the effect of CS on progressive vascular rejection in MHC identical, indefinitely surviving rat heart graft model, Forbes et al. (1993) found that only long-term maintenance of CS immunosuppression was effectively preventing rejection. Aziz et al. (1993) used a similar rat intra-abdominal heterotopic model of heart transplantation. In comparison with controls or animals treated with low dose CS alone, the addition of low molecular-weight heparin significantly improved allograft survival and reduced both the frequency and the severity of accelerated graft coronary disease and the extent of parenchymal rejection. Additional studies in this rat model by Atkinson et al. (1993) suggest a role for calcium channel blockers in the prevention of graft coronary artery disease. Allogeneic small bowel transplantation in the rat is also subject to chronic rejection and leads to profound morphologic changes and functional impairments according to Heeckt et al. (1993). Changes in smooth muscle structure and function evolve before the clinical signs of graft rejection. C. Factors Involved in Chronic Allograft Rejection

The arterial response to a denuding injury (e.g., induced by balloon catheter) is dominated by proliferation of smooth muscle cells, which is under growth factor control, and infiltration of leukocytes in the vascular

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intima. Jonasson et al. (1988)observed that CS treatment at surgery caused a persistent inhibition of the intimal proliferative lesion and concluded therefore that intimal cell proliferation appears to be regulated by the immune system. These findings, namely that CS administration to rats inhibits smooth muscle hyperplasia, were essentially confirmed by Wengrowitz et al. (1990) and Saenz et al. (1991). Reidy (1991) has reviewed in an editorial the effect of CS on vascular smooth muscle cells. The finding of relatively large numbers of T lymphocytes in atherosclerotic lesions suggested that they were not just passive bystanders and that their presence was somehow actively contributing to disease progression. Recently, Blotnick et al. (1994) have reported that cultured T lymphocytes isolated from normal human peripheral blood synthesize and secrete both heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor. These growth factors act as potent mitogens for fibroblasts and smooth muscle cells suggesting that T cells may play a key role in mediating smooth muscle hyperplasia. The results from Hansson et al. (1991)are contradictive in as far as they clearly show that rats devoid of T cells developed larger proliferative arterial lesions which indicates that T lymphocytes modulate, possibly in an immunologically nonspecific manner, proliferation of smooth muscle cells during vascular repair. Emeson and Shen (1993) have also reported that mice given an atherogenic diet plus CS injections to suppress their T cells displayed even larger atherosclerotic lesions at all three times observed than the hyperlipidemic controls receiving placebo injections. The effect of CS on the stimulation of rat cardiac smooth muscle cells was also investigated in vitro. Thyberg and Hansson (1991)obtained similar results with CS on rat aortic smooth muscle cells and dermal fibroblasts. The drug inhibited the induction of DNA synthesis by peptide mitogens, i.e., several growth factors. Based on their results, Leszczynski et al. (1993) concluded that CS did not exert a direct modulatory effect on smooth muscle cell proliferation in vitro, but might inhibit this proliferation indirectly via yet unidentified endothelial cell-derived factors which abolish the mitogenic effect of endothelin. In an ex vivo study, Iwai et al. (1993)have demonstrated an increased gene expression of angiotensin type 1A receptor in aortic smooth muscle cells of CS-induced hypertensive rats. In tissue culture, endothelin induces smooth muscle proliferation and contraction by influx of extracellular calcium. CS has been shown by Bunchman and Brookshire (1991)to stimulate human endothelial cells in vitro to synthesize endothelin (but not cultured human renal cortical epithelial cells as reported by Ong et al. 1993) which in turn causes smooth muscle cell proliferation. This action was inhibited by the coincubation of a specific antibody to endothelin or a calcium channel antagonist. Atrial natriuretic peptide has also been reported by Bokemeyer et al. (1994) to inhibit the CS-induced endothelin production and calcium rise in rat vascular smooth muscle cells. Similarly, diltiazem seems to exert a protective role on renal

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arterioles in CS-treated human renal allograft recipients (Choy et al., 1994). Takeda et al. (1992) produced evidence that CS might act as a potential inducer of endothelin release from the mesenteric artery in rats. Recently, they suggested that the CS-induced synthesis of endothelin-1 by vascular smooth muscle cells may participate in the pathogenesis of CS-induced vasoconstriction and vasculopathy via an autocrine regulatory mechanism (Takeda et al., 1993).The results from Phillips et al. (1994)also support a role for CS-induced vascular endothelin synthesis rather than changes in endothelin receptor characteristics or mesenteric vascular responsiveness in the pathogenesis of CS-induced hypertension in rats. Endothelin has, of course, been implicated in CS-induced acute nephrotoxicity and hypertension (Grieff et al., 1993; Iwasaki et al., 1994; Hunley et al., 1995). When infused in animals, endothelin causes a decreased renal blood flow and glomerular filtration rate and an increased mean arterial pressure. It has further been demonstrated in patients with solid-organ transplants that a transient increased urinary endothelin secretion rate (Perico et al., 1992) and a transient elevation in circulating endothelin-1 followed each oral administration of CS. This elevation in endothelin levels might contribute to CS-associated nephrotoxicity and hypertension, particularly during long-term immunosuppressive therapy. Diminished intrarenal endothelin immunostaining, which is associated with renal endothelial damage, occurs during vascular kidney allograft rejection and is aggravated by chronic CS toxicity as reported by Watschinger et al. (1994).They suggested that disintegration of the renal endothelium may play an important pathophysiological role in the release of endothelin and in the exposure of the vascular smooth muscle cells directly to this potent vasoconstrictor peptide. It has also been shown that local and systemic pretreatment with antiendothelin antibodies (Perico et al., 1990) or with a specific endothelin receptor antagonist (Fogo et al., 1992) largely prevented CS-induced glomerular hypofiltration and hypoperfusion. Carrier et al. (1991)demonstrated a dosedependent effect of CS in renal arterial resistance in dogs. Renal vasoconstriction was induced by a local effect of CS at the arterial wall which appears to be at least partly mediated by endothelin release in the renal vessels and was prevented remarkably by calcium channel blockers. Clinical trials have confirmed that the prophylactic use of a calcium channel blocker such as isradipine after kidney transplantation ameliorates CS-induced nephropathy and appears to protect against early postoperative vascular complications (van den Dorpel et al., 1994). In this context, the in-depth review by Textor et al. (1994) on the clinical aspects of CS-induced hypertension after transplantation should be mentioned. Bloom et al. (1993)concluded that in rats the acute renal vasoconstriction induced by CS was mediated by endothelin1, since the vasoconstriction was entirely prevented by infusion of either the endothelin antiserum or the receptor antagonist. The clinical findings by Yamada et al. (1994) that the changes in the levels of plasma endothelin

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and plasma thrombomodulin run parallel with the changes in serum creatinine just after renal transplantation suggest a possibility that endogenous endothelin might at least in part be involved in the change in posttransplant renal functions and the development of acute tubular necrosis. However, a positive correlation between plasma endothelin levels and serum CS trough levels was not observed in those patients. Since the correlation between CS trough level and actual drug bioavailability is very low when using S a n d i m m d , their data may be inadequate for proving this point (cf. Section VII1.B.). It has further been suggested that CS-induced hypertension may damage vascular endothelium possibly by directly causing vasoconstriction. Richards et al. (1990) have proposed that the inhibition of endothelium-dependent relaxation by CS was mediated by an effect on vasodilatory prostanoids. Balligand and Godfraind (1991) showed that CS did not affect the synthesis of endothelium-derived relaxing factor by vascular wall cells but rather inhibited the endothelial cell mechanism responsible for this stimulated vasodilator release that follows muscarinic receptor activation and intracellular rise in calcium. The primary injury to the transplanted organ is caused by ischemia and reperfusion and there is convincing evidence that this early damage may negatively predispose the graft to chronic rejection. Kubes et al. (1991)have subjected isolated segments of cat small intestine to ischemia followed by reperfusion and observed that pretreatment with CS significantly attenuated neutrophil infiltration due to this injury. Suzuki etal. (1993)obtained similar findings in a rat liver model. CS inhibited the infiltration of neutrophils in the hepatic tissues as a result of ischemia and reperfusion injury. Kurokawa et al. (1992) have reported a potent protective effect of CS pretreatment on postischemic liver injury in rats. Although CS did not affect ischemia-induced mitochondrial dysfunction, it did accelerate the recovery of mitochondrial functions and of tissue adenosine triphosphate concentrations; in addition, it also mitigated leakage of several liver enzymes and adenine nucleotides after reperfusion. Hayashi et al. (1991) have also demonstrated a beneficial effect of CS pretreatment in canine liver ischemia. Their results suggested that the drug may induce the stabilization of lysosomal membranes. Finally, Kim et al. (1991) have reported that CS pretreatment alleviated warm ischemia and perfusion injury to the hepatic sinusoids in pig livers, particularly by ameliorating concurrent intravascular coagulation rather than by exerting a protective effect on parenchymal hepatocytes. The detrimental role of oxygen free radicals in ischemia/reperfusioninduced organ injury has been studied extensively in various experimental models (Takahashi et al., 1993a,b). It has been shown by Land etal. (1994) that the perioperative use in the clinic of the highly specific scavenger of superoxide ions, superoxide dismutase, resulted in a significant long-term improvement of the actual 4-year graft survival rate in such treated renal

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recipients compared with placebo-treated patients (74 versus 52%, respectively). Slakey et al. (1993) investigated the effects of the antioxidants ascorbic acid and a-tocopherol on cardiac allograft rejection in rats treated with low dose CS. Since a significant improvement in cardiac allograft rejection resulted from combined CS and antioxidant therapy, it was suggested that scavenging of oxygen derived free radicals is of importance for delaying graft destruction. Wang and Salahudeen (1994)have found, though in a different context, that an antioxidant inhibitor of lipid peroxidation (lazaroid) limits CS-induced renal toxicity in vitro and in vivo which suggested a pathogenic role for reactive oxygen species-mediated lipid peroxidation in CS-induced renal toxicity in the rat model. Studies by Kawano et al. (1993) on the prevention of warm ischemia reperfusion injury to the rat liver by CS suggested that one of the responsible mechanisms might be the diminished lipid peroxidative damage, indicating a superoxide scavenging activity for CS. The interesting work by Chiara and Sobrino (1991) has demonstrated that there was a cooperative effect between CS and glucocorticoids in vitro resulting in the modulation of the inhibition of the respiratory burst in peritoneal macrophages. They further provided evidence for the inhibitory effect of in vivo administration of CS on phorbol myristate acetatedependent superoxide anion production in mouse macrophages; however, this inhibition was abolished when macrophages were in the activated state. The adherence of recirculating lymphocytes to vascular endothelium is regulated by several cytokines. Renkonen et al. (1991) have shown that different mediators of inflammation causing enhanced lymphocyte binding to endothelial cells may operate via completely different intracellular signal transduction pathways. Asako et al. (1992)studied whether CS could modify the adhesion and migration of leukocytes in postcapillary venules from the rat mesentery exposed to inflammatory mediators. Their results indicated that CS prevented all of the adhesive and hemodynamic alterations induced by the platelet activating factor but not by leukotriene B4. Adhesion molecules participate in many stages of an immune response (reviewed by Arnaout, 1993)and it has been shown that treatment with monoclonal antibodies directed against the intercellular adhesion molecule-1 and the leukocyte function-associated antigen-1 may induce specific acceptance of cardiac allografts between fully incompatible mice strains (Isobe et al., 1992). Horrocks et a;. (1991) have examined psoriatic skin lesions for the effect of CS on adhesion molecule expression. CS treatment caused a loss of the intercellular adhesion molecule-1 on keratinocytes, but the expression of this adhesion molecule persisted on vascular endothelium, despite resolution of the skin lesions. CS had no effect on several other adhesion molecules, including the leukocyte function-associated antigen-1. Petzelbauer et al. (1991) have also confirmed that in CS-treated psoriatic patients there was a dramatic reduction in the intercellular adhesion molecule-1expression by papillary endothe-

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lial cells, but density, pattern, and phenotype of infiltrating inflammatory cells remained essentially unchanged. Platelet-derived growth factor is an important mitogen for vessel wall cells in vitro and it has been implicated as a key mediator for atherosclerosis and tissue remodeling in chronic inflammatory processes, including chronic renal allograft rejection (Fellstrom et al., 1989). Higgy et al. (1991) have reported an increase in platelet-derived growth factor receptor expression in chronic rejection of cardiac and renal grafts in the rat. However, the relevance of this receptor for the tissue remodeling of chronic rejection remains to be established. Prolonged renal and cardiac allograft survival in sensitized rats has been observed when they were treated with plateletactivating factor antagonists (Freiche et al., 1990; Makowka et al., 1990). Human coronary transplantation-associated arteriosclerosis is currently regarded as an ongoing immune-inflammatory reaction in the vessel wall with activation of endothelial cells expressing major histocompatibility I1 antigens that stimulate lymphocyte proliferation and sustain release of cytokines (Schoen and Libby, 1991). Clause11 et al. (1993) have studied the early features of the development of the postcardiac transplant coronary arteriopathy in piglets. They found an increase in interleukin-10 which upregulates fibronectin synthesis. Fibronectin could mediate adherence, transendothelial migration and trapping of inflammatory cells, and smooth muscle cell migration into the subendothelium. However, the effect of CS in this model has not been investigated. In conclusion, chronic rejection appears as being essentially allograft arteriosclerosis. In Vitro and in vivo studies of cytokine effects on vascular cells revealed that these signal substances of the immune system were able to modulate vascular tissue responses and thus were involved in the pathogenesis of atherosclerosis. In addition, there is clear evidence for an inflammatory process occurring in the vessel wall, heavily involving endothelial and smooth muscle cells. However, the multiple effects of the cytokines, which may be originating from various cells, mean that it is unclear how the immune involvement affects the various stages of the disease. Nevertheless, it seems possible that pharmacological interference with the cytokine network could be used to prevent or reverse pathological reactions in the vessel wall. Since animal models have recently been developed, the search for interesting candidates such as immunosuppressive compounds alone or in combination with anticoagulants, calcium channel antagonists, or other drugs should now be feasible (Hansson, 1993; Hayry et al., 1993). VIII. Impact of Galenic Formulation on Pharmacokinetics -

A. Clinical Pharmacokinetics of CS (Sandimmun) CS is a lipophilic molecule with a partition coefficient of about 4000 as measured in a bufferfliposome system (Fahr, 1993). The oil components

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of the oral CS formulation, CS and bile form micelles, are absorbed from the gastrointestinal tract. Bioavailability of CS (mean value approx. 30%, range 8 to 60%) is dependent on food, bile and other interacting factors (Fahr, 1993). CS is extensively metabolized in the liver and to some extent in the intestinal endothelium by the cytochrome P-450dependent monooxygenase (CYP3A) system, whose activity has a considerable interindividual variation (Kronbach et al., 1988). The free fraction of CS in plasma is very low (1 to 4%).Distribution of CS in the body (see Fig. 6 ) depends not only on its physicochemical characteristics, but also on biological carriers such as lipoproteins and erythrocytes in blood. Cyclophilin, a binding protein for CS, influences distribution of CS in the body. The pharmacokinetically determined volume of distribution has a range of 1.8 to 13.8 liter/kg body weight. Despite its lipophilicity, CS does not appear in the brain. The body distribution of metabolites, which are less hydrophobic than CS, can be different from that of CS itself. CS is a drug with a low to intermediate extraction ratio which does not undergo excessive first pass metabolism; the extraction of CS does not exceed 50% of the dose (Kahan, 1985). The clearance value ranges from 0.15 to tissue

FIGURE 6 Schematic overview of the distribution and the fate of ciclosporin (CS) and its metabolites in the body (From Fahr, 1993). Abbreviations: CP, cyclophilin; RBC, red blood cell; Cyt P450II1,cytochrome P-450dependent mono-oxygenase (CYP3A) system; GIT, gastrointestinal tract. CS is either given orally and absorbed from the GIT or injected intravenously. Distribution of CS is thought to be by passive diffusion over the biological barriers. CS binds avidly to cyclophilin and lipophilic binding material. Body elimination of CS is mainly by metabolism and excretion into bile.

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0.7 liter/h/kg body weight. The concentration-time profile in the systemic circulation (blood, plasma) is usually described by two exponential terms having half-lives of 0.1 to 1.7 h for the a-phase and 2.9 to 15.8 h for the @-phase. Elimination of the drug is mainly via bile (>90%) as metabolites of the parent drug; other routes, like urinary excretion, are of minor importance. As described above, pharmacokinetic parameters of CS vary greatly. These parameters depend on many partly interacting factors such as age, the state of the patient, the type of organ transplant, and comedication. Pediatric patients have a higher clearance than adult patients (Yee et al., 1986b, 1987). This must be balanced by higher dosages and/or more frequent administration. Hepatic function in liver allograft recipients, for example, can be poor after transplantation with a bile flow being reduced by up to 70% (Busuttil et al., 1986). The decreased absorption of CS caused by the reduced bile flow is partly compensated by the diminished capacity (70% of normal clearance) of the liver to clear CS by metabolization and excretion. Comedication of drugs like ketoconazole, erythromycin, and verapamil inhibits CS metabolism. CS dosage has, therefore, to be reduced in such cases (Yee and McGuire, 1990). Ketoconazole, for example, inhibits elimination of CS to such a degree that some authors even recommend ketoconazole for lowering CS dosage (First et al., 1989). Inhibition of CS metabolism in the gut wall seems also to be caused by grapefruit juice and increases thereby the blood concentration of CS by a mean value of 32% (Ducharme et al., 1993). Drugs like rifampicin, phenytoin, or barbiturates induce the CSmetabolizing cytochrome P-450system which must be compensated by higher dosages of CS (in some instances up to four times higher). Another important factor in the reported variability of CS pharmacokinetic parameters is the different analytical methods and conditions used (Fahr, 1993). Following the recommendations of recent consensus documents on how to monitor blood concentrations, this source of variability may diminish in the future. Several metabolites are reported to possess immunosuppressive activity, but this activity is less than that of the parent drug (Fahr et al., 1990). Metabolites with renal side effects have been reported (Yee et al., 1986a; Rosano et al., 1988). The findings in the literature are not conclusive, presumably because of the highly variable activity of the CS-metabolizing liver enzymes and the paucity of data available on metabolite pharmacokinetics. In summary, the therapeutic range and dosage of CS are highly dependent on many individual parameters in the patients. Immunosuppression and renal side effects of CS are dose related. CS dosages of up to 5 mg/kg/

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day, however, rarely cause renal side effects while maintaining adequate immunosuppression. This calls for further studies on CS and metabolites in clinical pharmacokinetics with the aim to correlate metabolite pharmacokinetics, activities, and adverse effects.

B. New Galenical Formulation of CS (SANDIMMUN NEORAL) Sandimmun is a potent immunosuppressive drug widely used in organ transplantation and autoimmune diseases (Feutren, 1992; Feutren and Mihatsch, 1992; Tugwell, 1992). However, the therapeutic window of the drug is narrow with respect to immunosuppressive effect and adverse events. In addition, the pharmacokinetic profile of CS after treatment with the current market form Sandimmun is variable and influenced by many factors such as bile flow, coadministration with food, or gastrointestinal motility (Kahan, 1985; Burckart et al., 1986; Lemaire et al., 1990; Grevel, 1992).This requires careful drug monitoring which is usually done by trough concentration measurements (Lindholm, 1991; Inoue et al., 1994). Since it is well known that the main source of the variability in the pharmacokinetics of CS is its erratic absorption, a CS formulation with a more predictable drug absorption process providing reliably consistent blood concentrations of CS would be desirable. With Sandimmun Neoral a new galenical formulation for the oral administration of CS has been developed. This formulation is based on the microemulsion technology (Meinzer and Vonderscher, 1993) and should avoid the problems associated with the variable absorption and provide more accurate prediction of the systemic exposure of patients to CS. It could be shown that Sandimmun Neoral provides improved dose linearity in CS exposure [area under the curve (AUC)](Mueller et al., 1994d), a more consistent absorption profile (Kovarik et al., 1994e) and less dependence from concomitant food intake (Mueller et al., 1994a)and from diurnal . combined properties yield a lower rhythm (Kovarik et al., 1 9 9 4 ~ )These within-patient variability in pharmacokinetics of CS (Kovarik et al., 1994d) and a stronger correlation between trough concentration (C,) and total . advantages will allow the exposure (Fig. 7 ) (Mueller et al., 1 9 9 4 ~ )These patient a more deliberate dietary time schedule with respect to the drug administration times. In addition, CS seems to be absorbed independently from bile when given as Sandimmun Neoral (Trull et al., 1993). This might be of special importance for patients with reduced bile flow (e.g., liver transplant patients or patients with cystic fibrosis). The pharmacokinetic advantages of Sandimmun Neoral do clinically translate into greater facility in reaching the desired target blood concentration range and a more rapid stabilization of CS therapy with fewer maintenance-dose adjustments (Taesch and Niese, 1994; Mueller et al., 1994b).

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i0

Cmin [ng/&l Correlation between ciclosporin C,,,," (trough level) and area under the curve (AUC) during steady-state oral treatment with Sandimmun (left panel) and Sandimmun Neoral (right panel) in stable renal transplant patients.

FIGURE 7

Moreover, in the long run, a reduction in the frequency of CS blood concentration monitoring, necessary in routine transplant patients management, as well as in less creatinine monitoring in patients with autoimmune diseases is expected. The improvement in the relationship between trough blood concentration and total exposure to CS by administration of Sandimmun Neoral should reduce the risk of over- (increased side effects, especially nephrotoxicity) or underdosing (risk of rejection) patients which is critical for drugs with a relatively narrow therapeutic window. In newly transplanted kidney recipients, renal functions improved to a greater degree and faster under Sandimmun Neoral therapy as compared with Sandimmun (Taesch and Niese 1994; Mueller et al., 1994b). In addition, less patients experienced a rejection episode and the time period free of rejection was longer compared with Sandimmun, indicating an improved maintenance immunosuppression during CS therapy with Sandimmun Neoral. References Adams, D. H., Tilney, N. L., Collins, J. J., and Karnovsky, M. J. (1992). Experimental graft arteriosclerosis. I. The Lewis-to-F-344 allograft model. Transplantation 53, 1115-1 119. Addonizio, L. J., Hsu, D. T., Douglas, J. F., Kichuk, M. R., Michler, R. E., Quaegebeur, J. M., Smith, C. R., and Rose, E. A. (1993). Decreasing incidence of coronary disease in pediatric cardiac transplant recipients using increased immunosuppression. Circulation 88, 11-224-11-229. Alam, R., Stafford, S., Forsythe, P., Harrison, R., Faubion, D., Lett-Brown, M. A., and Grant, J. A. (1993). RANTES is a chemotactic and activating factor for human eosinophils. 1. Immunol. 150,3442-3447.

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Wenzel-Seifert, K., Grilnbaum, L., and Seifert, R. (1991). Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation. /. Immunol. 147,1940-1946. Wenzel-Seifert, K., and Seifert, R. (1993). Cyclosporin H is a potent and selective formyl peptide receptor antagonist. Comparison with N-t-butoxycarbonyl-L-phenylalany1-Lleucyl-L-phenylalanyl-L-leucyl-L-phenylalanine and cyclosporins A, B, C, D and El. J. Immunol. 150,4591-4599. Wera, S., Belayew, A., and Martial, J. A. (1995).Rapamycin, FKSO6, and cyclosporin A inhibit human prolactin gene expression. FEBS Letters 358, 158-160. Wershil, B. K., Furuta, G. T., Lavigne, J. A., Choudhury, A. R., Wang, Z., and Galli, S. J. (1995).Dexamethasone or cyclosporin A suppress mast cell-leukocyte cytokine cascades. Multiple mechanisms of inhibition of IgE- and mast cell-dependent cutaneous inflammation in the mouse. /. Immunol. 154, 1391-1398. White, D. J. G., and Lim, S. M. L. (1988). The induction of tolerance by cyclosporine. Transplantation 46, 118S-121S. Whitham, R. H., Vandenbark, A. A., Bourdette, D. N. Chou, Y. K., and Offner, H. (1990). Suppressor cell regulation of encephalitogenic T cell lines: Generation of suppressor macrophages with cyclosporin A and myelin basic protein. Cell. Immunol. 126,290-303. Wick, G., HBla, K., Wolf, H., Ziemiecki, A., Sundick, R. S., Stoffler-Meilicke, M., and DeBaets, M. (1986).The role of genetically-determined primary alterations of the target organ in the development of spontaneous autoimmune thyroiditis in obese strain chickens. Immunol. Rev. 94, 113-136. Wicker, L. S., Boltz, R. C., Matt, V., Nichols, E. A., Peterson, L. B., and Sigal, N. H. (1990). Suppression of B cell activation by cyclosporin A, FK 506 and rapamycin. Eur J. Immunol. 20,2277-2283. Wijngaard, P. L. J., Schuurman, H., Gmelig Meyling, F. H. J., Jambroes, G., and Borleffs, J. C. C. (1993).Breaking of transplantation tolerance after reduction of immunosuppression. J. Thorac. Cardiouasc. Surg. 105, 183-184. Williamson, M. S., Miller, E. K., Plemons, J., Rees, T., and Iacopino, A. M. (1994).Cyclosporine A upregulates interleukin-6 gene expression in human gingiva: Possible mechanism for gingival overgrowth. J. Periodontol. 65, 895-903. Wilner, M. L., Ettenger, R. B., Koyle, M. A., and Rosenthal, J. T. (1990). The effect of hypoprolactinemia alone and in combination with cyclosporine on allograft rejection. Transplantation 49, 264-267. Wolf, G., Thaiss, F., and Stahl, R. A. K. (1995). Cyclosporine stimulates expression of transforming growth factor-p in renal cells. Transplantation 60, 237-241. Won, Y., Sauder, D. N., and McKenzie, R. C. (1994). Cyclosporin A inhibits keratinocyte cytokine gene expression. Br. J. Dermatol. 130, 312-319. Wong, R. L. (1993). Mechanism of action of cyclosporin A in animal models of rheumatoid arthritis. Inflammopbarmacology 2, 177-195. Wong, R. L., Winslow, C. M., and Cooper, K. D. (1993). The mechanisms of action of cyclosporin A in the treatment of psoriasis. Immunol. Today 14, 69-74. Wood, M. L., Gottschalk, R., and Monaco, A. P. (1988). The effect of cyclosporine on the induction of unresponsiveness in antilymphocyte serum-treated, marrow-injected mice. Transplantation 46, 449-451. Wood, M. L., and Monaco, A. P. (1980).Suppressor cells in specific unresponsiveness to skin allografts in ALS-treated, marrow-injected mice. Transplantation 29, 196-200. Wramner, L., Mjornstedt, L., Tydberg, L., and Olausson, M. (1993). Cell-mediated immune responses in renal transplant recipients treated with cyclosporin and prednisolone with or without azathioprine. Scand. 1. Immunol. 37, 656-660. Wramner, L., Olausson, M., Saderstcorn,T., Lindholm, L., Rydberg, L., and Brynger, H. (1987). Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients. Transplantation 44,390-395.

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Wrenshall, L. E., Matas, A. J., Canafax, D. M., Min, D. I., Sibley, R. J., Dunn, D. L., Payne, W. D., Sutherland, D. E. R., and Najarian, J. S. (1990). An increased incidence of late acute rejection episodes in cadaver renal allograft recipients given azathioprine, cyclosporine, and prednisone. Transplantation 50, 233-237. Wysocki, G. P., and Daley, T. D. (1987). Hypertrichosis in patients receiving cyclosporin therapy. Clin. Exp. Dennatol. 12, 191-196. Xue, H., Bukoski, R. D., McCarron, D. A., and Bennett, W. M. (1987).Induction of contraction in isolated rat aorta by cyclosporine. Transplantation 43, 715-718. Xue, B., Dersarkissian, R. M., Baer, R. L., Thorbecke, G. J., and Belsito, D. V. (1986). Reversal by lymphokines of the effect of cyclosporin A on contact sensitivity and antibody production in mice. J. Zmmunol. 136, 4128-4133. Yahanda, A., Adler, K. M., Fisher, G. A., Brophy, N. A., Halsey, J., Hardy, R. I., Gosland, M. P., Lum, B. L., and Siric, B. I. (1992).Phase I trial of etoposide with cyclosporine as modulator of multidrug resistance. J. Clin. Oncol. 10, 1624-1634. Yahata, H., Fukada, Y., Hayamizu, K., Okimoto, T., Ishikawa, T., Asahara, T., Ono, E., and Dohi, K. (1994). Mechanism of suppression of cloned human suppressor T cells. Transplant. Int. 7, Suppl. 1, S59O-SS95. Yamada, K., Gunji, Y., Hishikawa, E., Kashiwabara, H., Sakamoto, K., Arita, S., and Yokoyama, T. (1994). Possible involvement of endothelin in posttransplant acute tubular necrosis. I. Studies in renal transplant patients. Transplantation 57, 1137-1 139. Yamaguchi, Y., Goto, M., Makino, Y., Takata, N., Kikuchi, N., Hamaguchi, H., Hisama, N., Otsuka, Y., Mori, K., and Ogawa, M. (1993).Effect of cyclosporine on the distribution of macrophage phenotypes in the rat hepatic allograft. Transplant. Proc. 25,1796-1798. Yamamoto, S., and Kato, R. (1994). Hair growth-stimulating effects of cyclosporin A and FK506, potent immunosuppressants. J. Dermatol. Sci. 7 , Suppl., S47454. Yard, B. A., Pancham, R. R., Paape, M. E., Daha, M. R., van Es, L. A., and van der Woude, F. J. (1993). CsA, FK 506, corticosteroids and rapamycin inhibit TNFa production by cultured PTEC. Kidney Znt. 44, 352-358. Yasunami, Y., Kamei, T., Ryu, S., Terasaka, R., and Konomi, K. (1990).Use of mixed islets from two strains as the donor for single transplantation. Transplantation 49,1179-1 181. Yasutomi, D., Odaka, C., Saito, S., Niizeki, H., Kizaki, H., andTadakuma,T. (1992).Inhibition of programmed cell death by cyclosporin A: Preferential blocking of cell death induced by signals via TCWCD3 complex and its mode of action. Immunology 77, 68-74. Yeager, A. M., Vogelsang, G. B., Jones, R. J. Farmer, E. R., Hess, A. D., and Santos, G. W. (1993). Cyclosporine-induced graft-versus-host disease after autologous bone marrow transplantation for acute myeloid leukemia. Leukemia Lymphoma 11, 215-220. Yee, G. C., Kennedy, M. S., Self, S. G., Storb, R., and Deeg, H. J. (1986a). Pharmacodynamics of cyclosporine in patients undergoing bone marrow transplantation. Transplant. Proc. 18, 774-776. Yee, G. C., Lennon, T. P., Gmur, D. J., Kennedy, M. S., and Deeg, H. J. (1986b).Age-dependent cyclosporine: pharmacokinetics in marrow transplant recipients. Clin. Pharmacol. Ther. 40,438-443. Yee, G. C., Lennon, T. P., Gmur, D. J., Kennedy, M. S., and Deeg, H. J. (1987). Effect of age on cyclosporine pharmacokinetics in marrow transplant recipients. Transplant. Proc. 19, 1704-1705. Yee, G. C., and McGuire, T. R. (1990).Pharmacokinetic drug interactions with cyclosporin. Clin. Pharmacokinet. 19, 319-332; 400-415. Yilmaz, S., and Hayry, P. (1993).The impact of acute episodes of rejection on the generation of chronic rejection in rat renal allografts. Transplantation 56, 1153-1 156. Yilmaz, S., Taskinen, E., Paavonen, T., Mennander, A., and Hayry, P. (1992a). Chronic rejection of rat renal allograft. I. Histological differentiation between chronic rejection and cyclosporin nephrotoxicity. Transplant. Int. 5, 85-95.

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Yilmaz, S., Paavonen, T., and Hiiyry, P. (1992b). Chronic rejection of rat renal allografts. 11. The impact of prolonged ischemia time on transplantation histology. Transplantation 53, 823-827. Yokoyama, I., Uchida, K., Kobayashi, T., Tominaga, Y., Orihara, A., and Tadagi, H. (1994). Effect of prolonged delayed graft function on long-term graft outcome in cadaveric kidney transplantation. Clin. Transplant. 8, 101-106. Yoshimura, N., and Kahan, B. D. (1986).The requirement for the renal transplant to induce allograft unresponsiveness by the combination of extracted histocompatibility antigen and cyclosporine. Transplantation 42, 642-646. Yoshimura, N., Matsui, S., Hamashima, T., Kita, M., and Oka, T. (1988).The in vivo immunosuppressive action of suppressor cells from alloantigen-cyclosporine-treatedmice and the capacity of spleen cells to release interleukins and gamma-interferon. Transplantation 45, 157-162. Yoshimura, N., Oka, T., Amagai, T., Horii, Y., and Imanishi, J., (1991). Interleukin-2receptor gene expression in kidney transplant recipients treated with cyclosporin A. Clin. Exp. Immunol. 85, 326-330. Yoshimura, K., Sugaya, H., Ishida, K., Khan, W. I., Abe, T., and Unno, K. (1993).The effect of cyclosporin A on Angiostrongylus cantonensis infection and eosinophilia in mice. Int. J. Parasitol. 23, 997-1003. Yu-Lee, L. Y. (1988).Prolactin: Role in T-cell proliferation.Ann. N. Y.Acad. Sci. 546,245-247. Yun, K. L., Michie, S. A., Fann, J. I., Billingham, M. E., and Miller, D. C. (1991). Effects of fish oil on graft arteriosclerosisand MHC Class I1 antigen expression in rat heterotopic cardiac allografts. J. Heart Lung Transplant. 10, 1004-1011. Zadeh, H. H., and Goldschneider, I. (1993).Demonstration of large-scalemigration of cortical thymocytes to peripheral lymphoid tissues in cyclosporin A-treated rats. J. Exp. Med. 178,285-293.

Ian J. Okazaki' Joel Moss Pulmonary-Critical Care Medicine Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892

Mono-ADP-ribosylation: A Reversible Posttranslational Modification of Proteins

1. Introduction Mono-ADP-ribosylation involves the transfer of the ADP-ribose moiety of NAD to an acceptor protein or amino acid and is catalyzed by NAD :(amino acid) protein ADP-ribosyltransferases(Williamson and Moss, 1990), of which several bacterial toxin ADP-ribosyltransferases have been well characterized (for review see "ADP-ribosylatingToxins and G Proteins: Insights into Signal Transduction" (J. Moss, and M. Vaughan, Eds.), 1990. American Society for Microbiology, Washingtion, D.C.). Cholera toxin and the related E. coli heat-labile enterotoxin ADP-ribosylate an arginine residue in Gsa7the stimulatory, a-subunit of the heterotrimeric GTP-binding (G) protein, resulting in the activation of adenylyl cyclase which increases intra-

' To whom correspondenceshould be addressed. Advances in Pharmacology, Volume 35

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cellular CAMP levels leading to abnormalities in intestinal cell fluid and electrolyte flux (Moss and Vaughan, 1988).Pertussis toxin modifies a specific cysteine in the a-subunit of Gi, Go,and G, which results in uncoupling of the G-protein from its receptor (Ui, 1990).Diphtheria toxin and Pseudomonas aeruginosa exotoxin A ADP-ribosylate diphthamide, a modified histidine residue, in elongation factor-2, inhibit protein synthesis and cause cell death (Wick and Iglewski, 1990; Collier, 1990). Modification of arginine 177 of actin by a group of clostridial ADP-ribosylating toxins causes inhibition of actin polymerization and breakdown of the microfilament network (Aktories, 1994). NAD :arginine ADP-ribosyltransferase activity has been detected in numerous animal tissues including turkey erythrocytes (Moss et al., 1980), rat liver (Moss and Stanley, 1981b), rabbit skeletal muscle (Soman et af., 1984), Xenopus tissues (Godeau etal., 1984),chicken heterophils (Tanigawa et al., 1984), and several murine cell lines (Soman et af., 1991). Most of the transferases demonstrated the ability to modify arginine or other simple guanidino derivatives. The turkey erythrocyte (Moss et af., 1980; Yost and Moss, 1983), chicken heterophil (Tanigawa et al., 1984; Mishima et al., 1991), and rabbit skeletal muscle (Peterson et al., 1990; Zolkiewska et af., 1992) transferases have been purified and characterized. Likewise, the transferase coding region cDNAs have been cloned from rabbit (Zolkiewska et al., 1992) and human (Okazaki et al., 1994) skeletal muscle, and chicken heterophils (Tsuchiya et al., 1994).Unlike the poly(ADP-ribose)polymerase which catalyzes the formation of branched ADP-ribose polymers during DNA synthesis and repair (Alvarez-Gonzaleset al., 1994), the mono-ADPribosyltransferases modify a diverse group of endogenous substrates including G,a (Duman et af., 1991), Ply-actin (Matsuyama and Tsuyama, 1991), p33 (Mishima et al., 1991), and integrin a7 (Zolkiewska and Moss, 1993). The modification of arginine residues by ADP-ribosyltransferases can be reversed by ADP-ribosylarginine hydrolases which remove the ADPribose moiety and regenerate free arginine (Williamson and Moss, 1990). Hydrolase activity was detected in the soluble fraction of turkey erythrocytes (Moss et al., 1985),cultured mouse cells (Smith et al., 1985),and rat skeletal muscle (Chang et af., 1986);the rat brain hydrolase cDNA was subsequently cloned (Moss et al., 1992). A eukaryotic ADP-ribosylation cycle (Fig. 1)was proposed based on the reversible modification of proteins catalyzed by the transferases and hydrolases (Williamson and Moss, 1990). This regulatory cycle is similar to one established in the photosynthetic bacterium Rhodospirilfurn rubrum (reviewed by Ludden, 1994). Dinitrogenase reductase, which is part of a nitrogen-reducing enzyme complex of R. rubrum, is inactivated by ADP-ribosylation of arginine 101 by dinitrogenase reductase ADPribosyltransferase. The modified dinitrogenase reductase is activated on removal of the ADP-ribose group by dinitrogenase reductase ADP-ribose gly-

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ADP-ribosyltransferase

\ yd *

I

Arginine

ADP-ribosylarginine

Ribosylarginina

ADP-ribosylarginine Hydrolase

FIGURE I

Proposed ADP-ribosylation cycle in eukaryotic cells. ADP-ribosyltransferases catalyze the forward reaction while the cytosolic ADP-ribosylarginine hydrolases catalyze the removal of the ADP-ribose moiety, regenerating free arginine. In the case of the GPI-anchored skeletal muscle transferase, extracellular phosphodiesterases and phosphatases process the modified substrate.

cohydrolase regenerating the free guanidino. moiety. Recently, glutamine synthetase I11 from the symbiotic nitrogen-fixing bacteria Rhizobium meliloti was shown to be ADP-ribosylated at an arginine in uivo resulting in inhibition of glutarnine synthetase activity (Liu and Kahn, 1995). The activity of the modified enzyme was restored by treatment with the turkey erythrocyte ADP-ribosylarginine hydrolase (Liu and Kahn, 1995). Endogenous ADP-ribosylation of cysteine was reported in human erythrocytes (Tanuma et al., 1987) and a 27-kDa, NAD :cysteine ADPribosyltransferase that modified Gia in erythrocyte and platelet membranes was purified (Tanuma et al., 1988). A human erythrocyte ADPribosylcysteine hydrolase that catalyzed the reverse reaction was also identified (Tanuma and Endo, 1990). These data are consistent with the presence of an ADP-ribosylation cycle involving cysteine residues on proteins analogous to the cycle utilizing arginine described above. The nonenzymatic formation of an ADP-ribosylthiazolidine from the reaction of L-cysteine, Dcysteine, cysteamine, and L-cysteine methyl ester, but not dithiothreitol, P-mercaptoethanol, glutathione or N-acetyl-L-cysteine, with ADP-ribose was, however, recently reported (McDonald et al., 1992). The thiazolidine linkage was sensitive to N H 2 0 H and HgC12 whereas the thioglycoside ADP-ribosylcysteine was hydroxylamine resistant. It was not definitively established that ADP-ribosylcysteine methylester, generated by incubation of the erythrocyte transferase with cysteine and NAD (Tanurna etal., 1988),

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was synthesized enzymatically. A two-step mechanism whereby ADP-ribose generated by an NAD glycohydrolase reacted with free cysteine to yield a thiazolidine product needs to be excluded (McDonald and Moss, 1994). It appears, however, that free ADP-ribose reacts with the cysteine in proteins to yield a product with the HgC12 and hydroxylamine sensitivity of ADPribosylated transducin formed by pertussis toxin. Thus, with proteins, chemical sensitivity alone cannot distinguish enzymatic and nonenzymatic ADPribosylation (McDonald and Moss, 1993a). It should be remembered, however, that proteins ADP-ribosylated on cysteine have been identified in tissue extracts (Jacobson et al., 1990); whether ADP-ribose addition is enzymatic or nonenzymatic remains to be determined. It was previously reported that nitric oxide stimulated the ADP-ribosylation of a cytosolic protein, subsequently identified as glyceraldehyde 3phosphate dehydrogenase. Based on the HgC12 sensitivity of the linkage, it was hypothesized that a cysteine residue was involved. Further analysis of the reaction product established that the entire NAD molecule, not just ADP-ribose, was involved (McDonald, and Moss, 1993b). Thus, in this case as well, cysteine appears not to be ADP-ribosylated. It should be noted that in brain extracts nitric oxide-dependent ADP-ribosyltransferases were observed which modified both cysteine and arginine linked to solid supports (Schuman et al., 1994). The mechanism of this reaction is uncertain pending purification of the products. This chapter will focus on the characterization of NAD :arginine ADPribosyltransferases and ADP-ribosylarginine hydrolases which have been purified and cloned from vertebrate species. Recent evidence will be presented to demonstrate that the vertebrate transferases, like many of the bacterial toxins, possess regions of sequence similarity at the active site, suggesting a common mechanism for catalyzing mono-ADP-ribosylation reactions.

II. Mono-ADP-ribosyltransferases

A. Avian ADP-ribosyltransferases 1. Turkey Erythrocyte ADP-ribosyttransferases

Several different ADP-ribosyltransferaseshave been identified in turkey erythrocytes and two have been purified extensively. Transferases A (Moss etal., 1980)and B (Yost and Moss, 1983)were isolated from the erythrocyte cytosol, transferase C from the plasma membrane, and transferase A' was from the nucleus (West and Moss, 1986). Transferase A was identified in turkey erythrocytes based on the similarity of its reaction to that of cholera toxin (Mossandvaughan, 1978). It was subsequently purified over 500,000fold by sequential chromatography on phenyl-Sepharose, carboxymethyl

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cellulose, NAD-agarose, and concanavalin A-agarose (Moss et al., 1980). The 28-kDa transferase had a specific activity of 350 pmol.min-'*mg-' using arginine methyl ester as the ADP-ribose acceptor. This activity was several orders of magnitude greater than that observed with cholera toxin. The transferase-catalyzed reaction, like that of cholera toxin, is stereospecific; the a-anomeric ADP-ribosylarginine being generated from P-NAD (Oppenheimer, 1978; Moss et al., 1979). In the absence of NaCI, the transferase existed as a relatively inactive oligomer (Mosset al., 1981).With the addition of up to 200 mM NaCl, the oligomer dissociated and enzyme activity increased more than 10-fold. Histones (20 pg/ml) also converted the transferase to an active monomer (Moss et al., 1982). At low concentrations of ADP-ribose acceptor, transferase activity was enhanced by chaotropic salts Positively charged groups near the gua(SCN- > Br- > C1- > F- > nidino moiety of the substrate also affected transferase activity, with arginine methylester > agmatine 2 arginine > guanidinopropionate 2 guanidine (Moss et al., 1981). The Km for NAD at optimal concentrations of histone and NaCl was 15 p M (Moss et al., 1982); the K, for arginine methyl ester in the presence of NaCl was 1.3mM (Moss et al., 1981).NaCl and guanidino compounds also stimulated the arginine-independent NAD glycohydrolase activity of the transferase. Kinetic studies of the turkey transferase demonstrated a random rapid equilibrium model in which both NAD and the ADP-ribose acceptor, agmatine, bind randomly to the transferase with binding of one substrate having a negative effect on the subsequent binding of the other (Osborne et al., 1985). The kinetic mechanism of the transferase was identical to that of cholera toxin (Osborne et al., 1985). Histones and ovalbumin stabilized the monomeric transferase, and served as ADP-ribose acceptors in vitro (Moss and Stanley, 1981a). In the presence of agmatine, however, formation of ADP-ribosylagmatine by the transferase was favored over the formation of ADP-ribosylhistone.Additionally, there was >lO-fold activation of the transferase at histone concentrations of 10 to 20 pg/ml which were much lower than those required for histones to serve as ADP-ribose acceptors. In the absence of salt, ADPribosylagmatine formation was > l o times greater in the presence of histone than it was in the presence of ovalbumin. At high salt concentrations (300 mM NaCl), enzyme activity was maximal whether histone or ovalbumin was present (Moss et al., 1982), i.e., the effects of protein and salt were not additive. Transferase A activity was enhanced -6-fold by lysophosphatidylcholine derivatives (Moss et al., 1984a). Lysophosphatidylcholine was not as effective as NaCl or histones in stimulating enzyme activity. Unlike NaCl, however, lysophosphatidylcholine stabilized the enzyme against thermal denaturation. Both fatty acid (C16> c18 > CI4> C12> Clo = c8)and choline moieties were critical for activity, whereas lysophosphatidylglycerol, lyso-

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phosphatidylserine, lysophosphatidylethanolamine, and lysophosphatidic acid did not increase transferase activity. Although the apparent Km for NAD was unchanged in the presence of lysophosphatidylcholine, the V,,, in NaCl was -1.7-fold times that in lysophosphatidylcholine. In addition, the apparent Km for agmatine was not different in NaCI, lysophosphatidylcholine, or NaCl plus lysophosphatidylcholineconsistent with the hypothesis that lysophosphatidylcholine interacts directly with the transferase to stabilize the enzyme in an active conformation. The zwitterionic detergent, 3[(cholamidopropyl)dimethylammonio]-l-propanesulfonate(CHAPS), and the nonionic detergents Triton X-100, Triton X-114, Tween 20, and Triton X-305 similarly enhanced transferase activity and protected against thermal inactivation. Maximal enzyme activation by CHAPS was less, however, than that produced by NaCI, histones, or lysophosphatidylcholine (Moss et al., 1984a). ADP-ribosyltransferaseactivity was enhanced or inhibited by nucleoside triphosphates depending on the protein substrate (Watkins and Moss, 1982). With lysozyme or soluble proteins from thymus as substrate, the rate of ADP-ribosylation was increased 100% by 10 mM nucleoside triphosphates; ATP > ITP = GTP > CTP = UTP. ADP-ribosylation of histone fla, however, was unaffected by ATP but inhibited by GTP. Turkey erythrocyte transferase B was purified 270,000-fold by sequential chromatography on phenyl-Sepharose, concanavalin A Sepharose, carboxymethyl-cellulose, Procion red-agarose, and Ultrogel AcA 54 (Yost and Moss, 1983). The 32,000-M, enzyme had apparent Km values for NAD and arginine methyl ester of 36 p M and 3.0 mM, respectively. Transferase B differed from transferase A in that it was inhibited -40% by chaotropic salts and was not activated by histones. In addition, transferase B did not self-associate in the absence of NaCl or histone. Transferases C and A' were partially purified from the particulate fraction of turkey erythrocytes (West and Moss, 1986).Transferase C, localized to the plasma membrane, had a M, of 26,000. The Km values for NAD and agmatine was 15p M and 2 mM respectively, similar to those determined for transferases A and B. Unlike transferases A and B, however, transferase C activity was unaffected by histones or salt (West and Moss, 1986). Transferase A', a 25,000 M, protein from the erythrocyte nuclear fraction, differed from transferase A in its chromatographic behavior and subcellular localization. Like transferase A, transferase A' was stimulated by NaCl and histones. The insensitivityof transferase A' to DNA and the use of simple guanidino compounds as ADP-ribose acceptors distinguished transferase A' from poly(ADP-ribose) polymerase, a nuclear protein (West and Moss, 1986). 2. In Vitro Substrates of the Turkey Erythrocyte Transferase A

Regulation of glutamine synthetase by ADP-ribosylation was initially proposed when depletion of cellular NAD levels in Chinese hamster ovary

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cells resulted in enhanced enzyme activity of the synthetase (M. R. Purnell, and W. R. Kidwell, unpublished results). It was subsequently demonstrated that ADP-ribosylation of a critical arginine in ovine brain glutamine synthetase by turkey transferase A in vitro decreased synthetase activity (Moss et al., 1984b). Likewise, arginine 172 of glutamine synthetase of E. coli was ADP-ribosylated by turkey transferase A resulting in the parallel loss of glutamine biosynthetic activity and y-glutamyltransferase activity (Moss et al., 1990). Activity of the modified glutamine synthetase was restored by treatment with the turkey erythrocyte ADP-ribosylargininehydrolase (Moss et al., 1990). The Ha-ras protooncogene product, p21 (Tsai et al., 1985), and transducin (Watkins et al., 1987) also functioned as substrates for transferase A. Incubating Ha-rus with enzyme and NAD in the presence of lysophosphatidylcholine resulted in incorporation of -3 mol of ADP-ribose per mol of p21 and decreased by -50% GTP-binding and GTPase activity of H a w s (Tsai et al., 1985). Similarly, ADP-ribosylation of transducin by transferase A inhibited GTP-binding -60%, and GTPase activity -90%. Although the a and p subunits of transducin were modified by transferase, inhibition of GTPase activity was predominantly due to ADP-ribosylation of the asubunit. Tubulin was initially demonstrated to be a substrate for ADP-ribosylation by incubating cytosolic proteins from rat glioma cells with [32P]NAD and cholera toxin (Hawkins and Browning, 1982). Transferase A ADPribosylated the a and fi polypeptide chains of chicken red blood cell tubulin with stoichiometries of 0.8 to 1.2 mol ADP-ribose per mol tubulin dimer (Raffaelli et al., 1992). In bovine brain extracts, 2.4 rnol of ADP-ribose was incorporated per mol of tubulin and 30 mol of ADP-ribose per rnol of high molecular weight microtubule-associated proteins (Scaifeet al., 1992). ADPribosylation of tubulin inhibited microtubule assembly, and modification of assembled microtubules from bovine brain resulted in subsequent microtubule depolymerization. Another in vitro substrate of transferase A, skeletal muscle a-actin, was ADP-ribosylated on arginine-95 and arginine-372 (Just et al., 1995a). In contrast, a-actin was modified by Clostridium perfringens iota toxin, the clostridial ADP-ribosylatingtoxin, on arginine 177 (Vandekerckhove et al., 1987; Aktories, 1994). Both monomeric G-actin and polymerized F-actin were ADP-ribosylated by transferase A. Modification of G-actin by the avian enzyme retarded monomer polymerization but did not alter the final extent of F-actin formation. Actin-catalyzed ATPase activity was likewise unaffected by transferase A-catalyzed ADP-ribosylation. Iota toxin, on the other hand, preferentially ADP-ribosylated G-actin resulting in the formation of an F-actin capping protein (Wegner and Aktories, 1988) which inhibited polymer formation (Aktories et al., 1986). ADP-ribosylation of arginine177 also inhibited actin ATPase activity.

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3. Chicken Heterophil ADP-Ribosyltransferases

An ADP-ribosyltransferase from chicken heterophil granules was purified 2 19-fold by sequential chromatography on carboxymethyl-cellulose, Sephadex G-75, phenyl 5-PV, and Mono S columns, and had a specific activity of 0.4 mmolmg-'*h-' (Mishima et al., 1991). It was identical to the transferase previously isolated from hen liver (Tanigawa et al., 1984; Mishima et al., 1991). Although the molecular mass of the purified transferase was estimated at 27.5 kDa (Mishima et al., 1991), a second 28.0-kDa isoform was recently separated on SDS-PAGE (Yamada et al., 1994). The 27.5-kDa transferase utilized histones, casein, protamine, and simple guanidino compounds such as arginine methyl ester and agmatine as ADPribose acceptors (Tanigawa et al., 1984). The Km values for NAD with arginine methyl ester, histone H1, and histone H2a as acceptors were 0.07, 0.56 and 0.29 mM, respectively; values for arginine methyl ester and agmatine were 24 and 1.9 mM, respectively; all of which were higher than those for the turkey erythrocyte transferase. The chicken transferase exists as an active monomer; it was inhibited by NaCl and lysophosphatidylcholine, both of which activated turkey transferase A. In addition, unlike the turkey enzyme, CHAPS and Triton X-100 had no effect on the heterophil transferase. Enzyme activity was stimulated by sulfhydryl reagents such as P-mercaptoethanol and polyanions such as double-stranded DNA, RNA, or poly(L-glutamate)(Mishima, et al., 1989). Similar to the turkey transferase, the heterophil enzyme also had NAD glycohydrolase activity. NAD hydrolysis exceeded ADP-ribosylationwith all ADP-ribose acceptors tested; the ratio of NADase to ADP-ribosyltransferase activity varied with the ADP-ribose acceptor (Tanigawa et al., 1984). Unlike the turkey enzyme, however, the heterophil transferase was auto-ADPribosylated when incubated with [32P]NADin a zymographic analysis (Yamada et al., 1994). Degenerate oligonucleotide primers based on amino acid sequences of proteolytic fragments of the purified heterophil transferase were utilized to generate a SOO-bp nucleotide fragment by polymerase chain reaction (PCR) from bone marrow cells. A chicken bone marrow cDNA library probed with the 500-bp PCR product contained two similar but distinct transferases, AT1 and AT2 (Tsuchiya et al., 1994). The AT1 cDNA hybridized with a 1.5-kb band on Northern analysis of total RNA from chicken bone marrow but not peripheral heterophils, spleen, liver, brain, lung, heart, or skeletal muscle. Oligonucleotide probes specific for AT1 and AT2 hybridized on Northern blot to mRNAs of similar size. In the coding region, there was 89.4% nucleic acid sequence identity between AT1 and AT2 whereas identity in the 5'- and 3'-untranslated regions was 100 and 98.4'3'0, respectively. AT1 and AT2 had open reading frames of 312 amino acids with 78.3% identity. The hydrophobicity plot of the deduced amino acid sequence of

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AT1 along with direct N-terminal sequencing of purified transferase was consistent with the presence of a hydrophobic amino terminal signal peptide. Further, amino acid sequencing at the C-terminus of AT1 ended at glutamine 266 which is consistent with processing of the amino and carboxy termini to yield the mature enzyme. COS 7 cells transformed with the coding region cDNAs exhibited transferase activity; when AT1 was used, activity was detected in the culture medium, and with AT2, activity was found in both medium and cell lysate. AT1 activity required P-mercaptoethanol, and was optimal with 5 mM, whereas 200 mM NaCl inhibited activity -70%. In contrast, AT2 activity was independent of reducing agent, although enzyme activity was enhanced 100% by 5 mM P-mercaptoethanol. NaCl stimulated AT2 activity 80% over that with P-mercaptoethanol alone. Whether AT2 corresponds to the 28-kDa isoform purified from the heterophil granules has not yet been resolved (Tsuchiya et al., 1994; Yamada et al., 1994). Endogenous GTP-dependent ADP-ribosylation of Gsa was demonstrated in chicken spleen membranes (Obara et al., 1991), with a concomitant increase in adenylyl cyclase activity similar to that which occurred in the presence of cholera toxin. It was not determined whether transferase activity in chicken spleen membranes was identical to that from heterophils. 4. In Vitro Substrates of the Heterophil Transferase

Nonmuscle Ply-actin, skeletal muscle a-actin, and smooth muscle yactin were ADP-ribosylated in vitro by the heterophil transferase (Terashima et al., 1992). Polymerization of the actin isoforms was completely inhibited by ADP-ribosylation, an effect seen with Clostridium perfringens iota toxin (Scheringet al., 1988). Unlike the iota toxin, however, the heterophil enzyme also ADP-ribosylated F-actin. Incubation of permeabilized heterophils with [32P]NADresulted in labeling of Ply-actin (Terashima et al., 1992). Endogenous ADP-ribosylation of heterophil nonmuscle actin was proposed as a mechanism for modulating heterophil phagocytosis, secretion, and chemotaxis-cellular functions dependent on actin polymerization and depolymerization (Terashima et al., 1992). A 33,000-M, protein, p 33, another major in vitro ADP-ribose acceptor for the heterophil transferase, was purified from heterophil granules, and separated from the transferase by chromatography on phenyl 5-PW and Radial PAK CI8reverse phase HPLC (Mishima et al., 1991). ADP-ribosylation of p33 by the heterophil transferase incorporated 4 mol of ADP-ribose per mol of p33. In permeabilized heterophils incubated with [32P]NAD, p33 was the predominant ADP-ribosylated protein (Mishima et al., 1991). Amino acid sequences of purified peptide fragments of p33 exhibited similarities to that of mim-1, the myb-induced myeloid protein-1 (Yamada et al., 1992), which appears to be developmentally regulated and expressed in heterophil precursors (Ness et al., 1989).

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The in vitro ADP-ribosylation of several proteins by the purified heterophi1 transferase inhibited subsequent phosphorylation of the modified substrate. ADP-ribosylation of arginine 34 of histone H1 by the transferase suppressed phosphorylation of serine 38 by CAMP-dependent protein kinase (Tanigawa et d., 1983a,b; Ushiroyama et d., 1985). Likewise, ADPribosylation of arginine residues in the (Y and p subunits of phosphorylase kinase blocked CAMP-dependentphosphorylation of the enzyme thus inhibiting phosphorylase kinase activation (Tsuchiya et al., 1985). Inactivation of L-type pyruvate kinase by CAMP-dependent phosphorylation was blocked by transferase-catalyzed ADP-ribosylation (Matsuura et al., 1988). Histones ADP-ribosylated by the heterophil transferase served as an initiator for poly(ADP-ribose) synthesis catalyzed in vitro by purified poly(ADP-ribose) polymerase (Tanigawa et al., 1984). Whether ADPribosylation of histone proteins by a heterophil granule transferase occurs in vivo is unknown.

B. Mammalian ADP-Ribosyltransferases 1. Skeletal and Cardiac Muscle ADP-Ribosybansferases

A rabbit ADP-ribosyltransferase,partially purified from skeletal muscle (Peterson et al., 1990), utilized guanidino compounds as ADP-ribose acceptors. To determine its structure, the transferase was purified further, a total of 215,000-fold, by column chromatography on DE52, DEAE-cellulose, concanavalin A-agarose, and DEAE MemSep followed by gel filtration HPLC (Zolkiewska et al., 1992). The specific activity of the purified 36-kDa protein at optimal NAD concentration (2 mM), and with agmatine as ADP-ribose acceptor was 68 pg.min-'-mg-', similar to those of the avian transferases (Moss et al., 1980). Oligonucleotide primers, based on amino acid sequences of tryptic peptides, were utilized to clone the rabbit transferase from a skeletal muscle cDNA library (Zolkiewska et al., 1992). A rabbit muscle transferase-specific oligonucleotide probe hybridized on a Northern blot of total RNA from rabbit tissues, with a 3-kb mRNA from rabbit skeletal and cardiac muscle, but not with RNA from smooth muscle, brain, lung, kidney, spleen, or liver (Zolkiewska et al., 1992), demonstrating apparent tissue specificity of the muscle transferase. A truncated cDNA lacking 5'- and 3'-ends encoding apparent signal sequences and expressed in E. coli exhibited transferase activity. The human skeletal muscle ADP-ribosyltransferasewas cloned by PCR procedures from human skeletal muscle poly(A)+ RNA (Okazaki et al., 1994). On Northern blot of total RNA from skeletal and cardiac muscle, a PCR-generated human muscle transferase cDNA probe hybridized with a 1.2-kb band from mouse and rat, a major 3.0-kb and minor 4.0-kb band from rabbit, a major 3.8-kb band and minor 5.7-kb band from monkey,

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and a 5.7-kb band from human skeletal muscle, consistent with the conclusion that the muscle transferase structure is conserved among species. Crossspecies conservation was also demonstrated on immunoblot where polyclonal antirabbit skeletal muscle transferase antibodies reacted with a M, 36,000 protein from bovine, dog, and rabbit cardiac muscle and a M, 40,000 protein from human skeletal muscle (Okazaki et al., 1994). The deduced amino acid sequence of the rabbit skeletal muscle transferase revealed hydrophobic amino and carboxy termini consistent with the presence of a glycosylphosphatidylinositolanchor (Zolkiewska et al., 1992). The sequences of the signal peptides were similar to those used for secretion into the endoplasmic reticulum and attachment of a GPI-anchor (Gerber et al., 1992). Indeed, in rat mammary adenocarcinoma (NMU) cells transformed with the rabbit muscle transferase cDNA, the majority of transferase activity was localized to the membrane fraction but could be solubilized by incubating intact cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Okazaki et al., 1994). In addition, a 35-kDa protein released by PI-PLC from transformed, but not control, NMU cells reacted with antitransferase antibodies as well as with anticross-reacting determinant (CRD) antibodies which recognize the oligosaccharide inositol-1, 2-cyclic phosphate moiety exposed on GPI-anchored proteins after incubation with PI-PLC. Transferase activity in NMU cells transformed with a truncated cDNA lacking the carboxyterminal signal peptide was secreted into the medium and not retained on the plasma membrane, presumably due to the lack of the GPI-anchoring sequence. Partially purified transferases from rabbit and human skeletal muscle reacted with anti-CRD antibodies only after incubation with PI-PLC supporting the hypothesis that the ADPribosyltransferase was GPI-anchored in native tissues (McMahon et al., 1993; Okazaki et al., 1994). Although a skeletal muscle transferase exists as an exoenzyme (Zolkiewska and Moss, 1993), transferase activity was also localized to the sarcoplasmic reticulum (Soman et al., 1984) and cytoplasmic face of the sarcolemma and transverse tubule membranes by fractionation on sucrose gradients (Klebl et al., 1994). ADP-ribosyltransferase activity was detected in the C2C12 and G8 mouse skeletal muscle cell lines (Zolkiewska and Moss, 1993). The enzyme activity increased on differentiation of myoblasts to myotubes and was released from intact cells by treatment with PI-PLC. Incubating radiolabeled NAD with intact cells resulted in ADP-ribosylation of a 97-kDa membrane protein. If the cells were disrupted, different proteins were ADP-ribosylated (Piron and McMahon, 1990), suggesting that an intact cell is necessary to preserve substrate specificity. In intact cells, protein labeling was inhibited by treatment of cells with PI-PLC prior to, but not after, incubation with [32P]NAD.The radiolabel associated with the 97-kDa protein was sensitive to NaOH and NH,OH, consistent with an ADP-ribose-arginine linkage. Based on mobility on SDS-PAGEunder reducing and nonreducing conditions

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(97-kDa and 140-kDa, respectively), which was similar to that reported for integrin a7, the labeled protein was purified 150-fold on a laminin affinity column. Amino acid sequences of the N-terminal and internal peptide fragments were identical to that of an isoform of rat integrin a7 (Zolkiewska and Moss, 1993). By limited trypsin digestion of labeled integrin a7, the site of ADPribosylation was narrowed to the 39-kDa segment located between ligandbinding and transmembrane domains that includes amino acids 575 to 886 (Zolkiewska and Moss, 1993). At higher NAD concentrations (75 p M ) , integrin a7 was also modified in the 63-kDa N-terminal segment without adversely affecting a 7 P l heterodimer formation or its association with the cytoskeleton or laminin (Zolkiewska and Moss, 1995). ADP-ribosylated integrin a7 was rapidly processed by extracellular phosphodiesterases yielding phosphoribosyl-integrinand 5 'AMP.Phosphoribosyl-or ribosyl-integrin was resistant to further ADP-ribosylation for at least 1 hr. The presence of residual ribose attached to integrin a7 was verified by incubation of intact cells with nicotinamide proximal ribose-labeled NAD. Although the AMP was released from the ADP-ribosylated protein, ribose incorporation remained constant. The studies suggest that ADP-ribosylarginine hydrolase may not be involved in processing the ADP-ribosylated integrin (Zolkiewska and Moss, 1995). The expression of the ADP-ribosyltransferasein parallel with that of integrin a 7 during myogenesis (Song et al., 1992), and the demonstration of ADP-ribosylation of integrin a7 in intact cells suggested a regulatory role for such a modification in muscle cell development. Further, inhibition of proliferation and differentiation of embryonic chick myoblasts by meta-iodobenzylguanidine in vitro, an inhibitor of arginine-specific mono-ADP-ribosyltransferases (Khardia et al., 1992), was consistent with the hypothesis that ADP-ribosylation may, to some extent, modulate myogenesis. 2. Rat Brain ADP-ribosyltransferases

Four distinct ADP-ribosyltransferases were purified -3000-fold by chromatography on CM-Sepharose, Butyl-Toyopearl, and Concanavalin A-Ultrogel (Matsuyama and Tsuyama, 1991). The estimated M, of the four transferases was 66,000 by gel filtration HPLC. Using Ply-actin as ADPribose acceptor, the Km values for NAD were 17.2 p M , 12.1 pM, 24.6 p M , and 30.6 pM for transferases I, 11, 111, and IV, respectively. Phospholipids had variable effects on the activity of the brain transferases (Matsuyama and Tsuyama, 1991). Transferase I was activated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, transferase I1 by lysophosphatidylcholine,and transferase I11 by lysophosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; transferase IV was inhibited by all phospholipids tested. Activity of the brain transferases was also affected by soluble ADP-

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ribosylation factors (sARF) I and 11, 20-kDa guaninine nucleotide-binding proteins from bovine brain, which enhance in a GTP-dependent manner, cholera-toxin induced ADP-ribosylation and are involved in regulation of vesicular transport (Moss and Vaughan, 1993; Welsh et al., 1994). sARF I activated transferase 11, whereas sARF I1 enhanced the activity of transferases I and IV. Transferase I11 activity was suppressed, however, by both sARFs (Matsuyama and Tsuyama, 1991). ADP-ribose and ADP, but not nicotinamide, inhibited the brain transferases. All four transferases ADP-ribosylated nonmuscle Ply-actin and smooth muscle y-actin, whereas skeletal muscle a-actin was a substrate for transferases I and IV. Modification of nonmuscle G-actin by transferase resulted in the failure of actin to polymerize in vitro. The four transferases ADPribosylated microtubule-associated protein (MAP-2) are also critical for microtubule assembly and disassembly (Takenaka et al., 1994). Go,the other heterotrimeric GTP-binding protein that is ADP-ribosylated by pertussis toxin (Sternweis and Robishaw, 1984), and G, were also ADP-ribosylated in vitro by the brain transferases (Matsuyama and Tsuyama, 1991). Transferases I, 11, and IV modified G,, and transferases I, 111, and IV modified Go. None of the transferases, however, ADP-ribosylated histones (Matsuyama and Tsuyama, 1991). After incubation of rat brain homogenates with [3zP]NAD and 5’guanylyl-imidodiphosphate [Gpp(NH)P],ADP-ribosylation of several proteins was demonstrated on SDS-PAGE with major labeling of 20-, 42-, 4 5 , and 50-kDa proteins (Duman et al., 1991). Cholera toxin ADPribosylated the 20-, 42-, and 45-kDa proteins under similar conditions, although toxin-catalyzed labeling was considerably greater than that occurring endogenously. Endogenous and toxin-mediated ADP-ribosylation was enhanced in the presence of isoniazid and 3-acetylpyridine adenine dinucleotide, inhibitors of NAD-glycohydrolaseactivity. Triton X-1 00 also increased endogenous modification of the 20-, 42-, and 45-kDa proteins, Moreover, in brain homogenates from rats that had been treated with corticosterone for 7 days, there was increased ADP-ribosylation of the 20-, 42-, 4 5 , and SO-kDa proteins. There were significant differences in the regional distribution of endogenous ADP-ribosylation in rat brain. The highest levels of ADP-ribosylation of the 42- and 45-kDa proteins were found in the hippocampus, hypothalamus, and cerebral cortex, intermediate levels in the midbrain, thalamus, and neostriatum; the lowest levels were observed in the cerebellum. Modification of the SO-kDa protein was similar throughout the brain except for significantly higher levels of ADP-ribosylation in the neostriatum (Duman et al., 1991). One dimensional peptide maps of the labeled 42- and 45-kDa proteins were identical to those of the two major forms of G,a. The 20-kDa protein was thought to be ARF based on its modification by cholera toxin and

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increased endogenous ADP-ribosylation after chronic administration of glucocorticoids (Duman et al., 1991). Glucocorticoids have been shown to regulate the expression of G,a (Saito et al., 1989) and ARF (Duman et al., 1990) in the cerebral cortex. The SO-kDa protein has not been identified. Brain ADP-ribosyltransferases that modify actin and GTP-binding proteins suggest a critical role for ADP-ribosylation in neuronal function. 3. Other Mammalian Mono-ADP-ribosyltransferases

ADP-ribosyltransferase activity has been reported in lymphoid cells including T cell lymphomas, T cell hybridomas, and a thymoma cell line (Soman et al., 1991). The sequence of the lymphocyte transferase appears to be quite similar to its muscle counterparts (Okazaki et al., 1996).Indirect evidence of the effects of ADP-ribosylation on the function of cytotoxic T cells (Wang et al., 1994) has been described. Incubation of murine cytotoxic T lymphocytes (CTL) with 10 p M NAD inhibited CTL proliferation in a mixed lymphocyte reaction, whereas 100 p M NAD suppressed cytolytic activity. NAD had no effect on stimulator or target cells. Inhibition of CTL proliferation and cytotoxicity by NAD corresponded to inhibition of CTLtarget cell binding without affecting T cell receptor-mediated signaling. Incubation of CTL with [32P]NADresulted in the labeling of numerous membrane proteins which was not inhibited by 100-fold molar excess of the NAD metabolites, nicotinamide, ADP-ribose or cyclic-ADP-ribose, S’AMP, adenosine, or nicotinamide mononucleotide. These metabolites likewise had no effect on CTL proliferation or cytotoxicity. Label was released from the proteins by NaOH or NH,OH, but not by HgC12, consistent with an ADPribose-arginine linkage. Incubation of CTL with PI-PLC solubilized a -35-kDa protein with guanidine-specific ADP-ribosyltransferase activity. In addition, PI-PLC-treatment resulted in the partial loss of the inhibitory effect of NAD on CTL proliferation and totally eliminated the suppressive effect of NAD on cytotoxicity (Wang et al., 1994). The ADP-ribosylated membrane proteins and the physiologic mechanism of NAD-induced suppression of CTL function remain to be defined. Endogenous ADP-ribosylation of several proteins including G,a was demonstrated in permeabilized NG108-15 cells, a neuroblastoma-glioma hybrid, incubated with [32P]NAD(Donnelly et al., 1992). Incubating cells with 50 mM nicotinamide, 25 mM benzamide, or 10 mM 5-bromo-2’deoxyuridine, inhibitors of ADP-ribosyltransferases, reduced ADPribosylation of all proteins. Treatment of cells with nicotinamide for 18 h increased membrane-associated G,a and resulted in a significant increase in adenylyl cyclase activity. Endogenous ADP-ribosylation of G,a and subsequent stimulation of adenylate cyclase was similarly demonstrated in cardiac muscle membranes (Feldman et al., 1987; Quist et al., 1994) and platelets (Molina y Vedia et al., 1989).

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ADP-ribosylation of 38- and SO-kDa cytosolic proteins from rat basophilic leukemia (RBL) and Fischer rat thyroid line 5 (FRTL5) cells was observed in the presence of [32P]NADand cellular extracts (De Matteis et al., 1994). The fungal toxin brefeldin A (BFA), which inhibits constitutive protein secretion and disrupts the structure and function of organelles involved in protein trafficking (Pelham, 1991; Klausner et al., 1992), stimulated ADP-ribosylation of these proteins. The concentrations of BFA that stimulated ADP-ribosylation were similar to those that inhibited ADPribosylation factor binding to Golgi membranes (Tsai et al., 1993; De Matteis et al., 1994). The ADP-ribose-protein linkage was sensitive to treatment with HCI and NaOH but stable to NHZOH and HgC12 which was unlike that formed by the known arginine-, cysteine-, and asparaginespecific transferases (De Matteis et al., 1994), and different from the linkage resulting from nonenzymatic ADP-ribosylation of cysteine (McDonald and Moss, 1993b). The identity of the 38-kDa protein was determined to be glyceraldehyde-3-phosphatedehydrogenase by its electrophoretic behavior on 2-dimensional gels, whereas the identity of the SO-kDa protein has not yet been determined. C. Inhibitors of Mono-ADP-ribosyltransferase

In general, three enzymes appear to be affected by a common pool of inhibitors: poly(ADP-ribose)polymerase, mono-ADP-ribosyltransferase, and NAD glycohydrolase (Banasik and Ueda, 1994). Some compounds appear to be more selective in their preferential inhibition of one or more of these enzymes. Inhibitors of mono-ADP-ribosyltransferase or poly(ADPribose) polymerase activity are shown in Table I. The concentration of some of the inhibitors which is effective in blocking poly(ADP-ribose)polymerase activity is two or more orders of magnitude lower than that required to block mono-ADP-ribosyltransferase activity. For example, the concentration of benzamide and its derivatives that inhibited polymerase activity by 50% (ICsO)was 3 to 6 p M , whereas that for turkey transferase A was 2,700 to 4,100 pM (Rankin et al., 1989). 5’-Bromodeoxyuridine, the most effective inhibitor of transferase A, had ICsOvalues of 590 and 15 p M for transferase A and the polymerase, respectively. These differences may be useful in differentiating pathways involving poly(ADP-ribosy1)ationfrom those dependent on mono(ADP-ribosy1)ation. Five natural compounds, vitamins K1 (phylloquinone) and K2,20)(menaquinone), and saturated long-chain fatty acids arachidic, stearic, and palmitic acids were specific inhibitors of mono-ADP-ribosyltransferase activity with ICso values between 1.9 and 16 p M (Banasik et al., 1992). Unsaturated long-chain fatty acids, on the other hand, inhibited both the heterophil transferase and the poly(ADP-ribose)polymerase.Novobiocin was another specific inhibitor of the transferase (Table I). It is important to note that

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TABLE I 50% Inhibitory Concentration (IC,) of Compounds that Inhibit MonoADP-ribosyltransferases and Poly(ADP-ribose) Polymerase Compound

IC50 Transferase(pM)

IC,, Polymerase (pM)

5’ Bromodeoxyuridine Thymidine 3-Methoxybenzamide Theophylline 3-Aminobenzamide Nicotinamide Benzamide Arachidic acid (C20 :0) Stearic acid (C18 :0) Palmitic acid (C16: 0) Arachidonic acid (C20 :4) Linoleic acid (C18 :2) Linolenic acid (C18 :3) Palmitoleic acid (C16 : 1) Vitamin K, Vitamin Kzczo, Novobiocin

590 ? 94 1900 2 300 2700 ? 250 2800 ? 460 3000 t 970 3400 2 410 4100 2 220 4.4 6.1 16 66 90 110 200 1.9 13 280

15 2 1.3 43 2 5.2 3.4 2 0.31 46 ? 15 5.4 ? 0.40 43 2 5.2 3.3 2 0.28

a

>loo0 >loo0

>loo0 44 48 110 95 520 ND’ 2200

Nd, not determined. (From Rankin et al., 1989; Banasik et al., 1992).

these studies were conducted with a limited number of ADP-ribosylating enzymes; conceivably, effects of inhibitors on other members of these families would differ (e.g., ICso, specificity).

111. Conserved Regions among ADP-ribosyltransferases

-

Despite an overall lack of amino acid sequence identity, several bacterial toxin ADP-ribosyltransferases appear to have three noncontiguous regions of similarity (Rappuoli and Pizza, 1991; Domenighini et al., 1994). Region I contains a nucleophilic arginine or histidine possibly involved in hydrogen bonding. Region 11, located -50 to 75 amino acids downstream of region I, comprises closely spaced aromatic amino acids which form a pocket for binding of the nicotinamide moiety and adenine ring of NAD. In region 111, a glutamic acid, -100 to 150 amino acids following region 11, serves as the catalytic-site residue for NAD hydrolysis and AD€’-ribosetransfer. The crystal structures of diphtheria toxin (Choe et al., 1992), heat-labile enterotoxin of E. coli (Sixma et al., 1991), Pseudomonas aeruginosa exotoxin A (Allured et al., 1986), and pertussis toxin (Stein et al., 1994) revealed catalytic clefts formed, in part, by the three conserved regions. The localization of nicotinamide to this region was confirmed by crosslinking the group to glutamate.

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The cloned vertebrate ADP-ribosyltransferases have significant amino acid sequence identity to each other and to the RT6 family of rat T cell alloantigens, RT6.1 and RT6.2, which have NAD glycohydrolase activity (Takada et al., 1994; Tsuchiya et al., 1994; Okazaki et al., 1996). The variably glycosylated RT6.1 and nonglycosylated RT6.2 are GPI-anchored proteins with a molecular mass of 25 to 30-kDa (Thiele et al., 1986; Koch et al., 1988, 1990). The absence of RT6+ T cells has been associated with the occurrence of an autoimmune-mediated diabetes in BB/Wor rats (Greiner etal., 1986; Burstein etal., 1989; Doukas and Mordes, 1993).The vertebrate transferases and the RT6.2 enzymes also appear to share regions of amino acid sequence similarity to the bacterial toxin transferases, which is consistent with the hypothesis that many of the mono-ADP-ribosyltransferases have similar NAD-binding and catalytic sites.

A. Region I His-21 of diphtheria toxin (DT), His440 of P. aeruginosa exotoxin A (ETA), Arg7 of cholera toxin (CT) and heat-labile enterotoxin of E. coli (LT), and Arg9 of pertussis toxin (PT) are nucleophilic amino acid residues responsible for NAD binding (Fig. 2A). Incubation of DT with diethylpyrocarbonate (DEPC) converted His21 to N-carbethoxyhistidine and inhibited NAD binding and ADP-ribosyltransferase and NAD glycohydrolase activities (Papini et al., 1989). Replacement by site-directed mutagenesis of His21 with other amino acids, with the exception of the asparagine substitution, drastically reduced ADP-ribosyltransferase activity ( Johnson and Nicholls, 1994; Blanke et al., 1994a). That mutation caused only modest reductions in Km value for NAD, catalytic rate (kat), and catalytic efficiency (kcar/Km), and a Kd value for NAD ten times that of the wild-type toxin (measured by quenching of intrinsic protein fluorescence) compared to a >30-fold increase for the other mutants. Replacement of His21 in DT with the sterically similar asparagine without abolishing NAD-binding or enzyme activity is consistent with the suggestion that the n-nitrogen of the imidazole group of histidine is important in orienting NAD in the catalytic site by hydrogen bonding to the carboxamide group of nicotinamide (Johnson and Nicholls, 1994; Blanke et al., 1994a). Based on X-ray crystallography, the position of His440 of ETA corresponds to that of His21 of DT (Carroll and Collier, 1988). Replacement of His426, positioned on the external surface of the catalytic cleft (Allured et al., 1986), with tyrosine abolished transferase activity of ETA (Wozniak et al., 1988). There was, however, no difference between H426Y and wildtype ETA in fluorescence quenching, with respect to NAD binding (Wozniak et al., 1988). His440 of ETA is probably functionally analogous to His21 of DT, whereas His426 affected ETA ADP-ribosyltransferase activity without altering NAD binding.

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*

a

RMT RT6. 2 AT 1 AT 2 DT ETA LT

11 0 87 97 97 17 436

FRDEHGVALL * N - F - T -- V LIR-gai * * L R P - g a i -* SSY TKPG -VGY--TF.E 3 D-LYRaDBRP

-

-

--

-

b RMT RT6.2 AT 1 AT2 DT ETA LT C

RMT RT6.2 AT1 AT2 DT ETA LT c3 T2 T4 T6

225 LGVPIQGYSFFPGEEEVLIPP 194 *.-Y*KEf--R-Dq-..*.. g 209Y....KQf....e-d 2OgY....KQf..y.e.d...... 136 -el-FAEG-SSV----YiNNW 539 -DA-*T.PEEEg*RL.TilgW 101 .--Y----.PH.Y.g--SALQ 158 K.GY.dpI-y-..qL...l.R 581 -SLGIAT-A-.il.R 568 -SLAPSN-W**i1.R 581 -SLGIAT.A. ei1.R

......

FIGURE 2 Regions of sequence similarity among vertebrate and bacterial toxin ADPribosyltransferases and NAD glycohydrolases are aligned with presumably analogous regions in the rabbit muscle transferase. (A)RegionIcontaininga nucleophilichistidine orarginine (asterisk) residue. (B) Region II containing closely spaced aromatic amino acids (underlined). (C)Region 111 active-site glutamic acid (asterisk) among transferases is aligned. Sequences are in the single letter code with the position of the first amino acid following the name of the protein. . indicates amino acid identical to that in rabbit muscle transferase. Lower case letter indicates conservative differences from the rabbit transferase. indicates a gap to optimize alignment. RMT, rabbit muscle transferase; RT6.2, rat T cell alloantigen RT6.2; ATl, chicken ADP-ribosyltransferase 1; AT2, chicken ADP-ribosyltransferase 2; DT, diphtheria toxin; ETA, Pseudomonus uemginosu exotoxin A; LT, heat-labile enterotoxin of Escherichiu coli; C3, Clostridium botulinum C 3 exotoxin; T2, ADP-ribosyltransferase (gene product Alt) of bacteriophage T2; T4, ADP-ribosyltransferase of bacteriophage T4; T6, ADP-ribosyltransferase of bacteriophage T6.

Based on crystal structure and site-directed mutagenesis, Arg7 of LT and CT, which are structurally and immunologically related, was identified as essential for toxin activity. Substituting lysine for Arg7 in LT (Lobet et

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al., 1991) and CT (Burnette et al., 1991) resulted in loss of enzymatic activity, and in LT, increased sensitivity to trypsin digestion. Although the conservative lysine replacement had the same charge and size as Arg7, it was unable as determined by computer modeling to participate in a hydrogen bond network with Val53 and Arg54, which maintains the required conformation at the enzymatic site (Pizza et al., 1994). Likewise an Arg9 to lysine mutation in PT (Burnette et al., 1988; Kaslow et al., 1989) resulted in complete loss of activity. In the crystal structure of PT, the side chain of Arg9 projected into the active site where it formed a hydrogen bond with Ser52 holding adjacent strands of the catalytic cleft together (Stein et al., 1994). A similar histidine in RT6.2 (HisSl), and in the rabbit skeletal muscle ADP-ribosyltransferase (His114), can be aligned with the region I nucleophilic amino acid of the bacterial toxins (Fig. 2A). Like the H21N mutant of DT, the analogous H114N mutant of the muscle transferase retained activity and immunoreactivity (Takada et al., 1995). Residual activity of the H114N mutant is consistent with hypothesis that His114 is involved in hydrogen bonding at the active site (Takada et al., 1995). GlnlOl of the chicken heterophile transferases AT1 and AT2 was aligned with His114 of the muscle enzyme (Tsuchiya et al., 1994). Although the activity of a H21Q mutant of DT was markedly reduced (Blanke et al., 1994a; Johnson and Nicholls, 1994), GlnlOl of the chicken transferase may be functionally similar to His114 in the muscle species. Arg99 of AT1 and Arg98 of AT2, on the other hand, may be the critical region I residue. 6. Region II

Alignment of active site cleft amino acids of DT and ETA revealed conservation of aromatic amino acids hypothesized to be involved in hydrophobic interactions with the aromatic rings of NAD; TrpSO, Phe53, Tyr54, and Tyr65 of DT correspond to Trp466, Phe469, Tyr470, and Tyr481 of ETA (Carroll and Collier, 1988). Photolabeling of DT with 8-azidoadenine and 8-azidoadenosine suggested a stacking of the nicotinamide moiety of NAD on the phenolic ring of Tyr65 and the adenine ring on Trp5O (Papini et al., 1991). Adenylyl-3', 5'-uridine monophosphate (ApUp),a dinucleotide structurally similar to NAD, bound to the active site of DT. Crystallographic data positioned the nicotinamide ring adjacent to the imidazole ring of His21, the phenolic ring of Tyr65, and the side chain of the active site Glu148 (Choe et a/., 1992).Conservative replacement of Trp5O with phenylalanine in DT resulted in minimal reduction of NAD-binding and ADPribosyltransferase activity (Wilson et al., 1994). The replacement of T50 with alanine (TSOA),however, eliminated NAD-glycohydrolaseactivity and markedly reduced (5000-fold) transferase activity (kcat)which was 6 x 104-foldless efficient (kJKm) than wild-type. Whereas the & value for NAD

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binding by the T5OA mutant was too great to be measured by fluorescence quenching, the k, for NAD in the transferase reaction with elongation factor 2 (EF-2) as ADP-ribose acceptor was only -10-times that for wild-type DT suggesting that EF-2 stabilized NAD in the active site pocket during ADPribosylation, an effect lacking during NAD hydrolysis (Wilson et al., 1994). Replacement of T65 of DT with phenylalanine (T65F) reduced ADPribosyltransferase activity -4-fold without altering NAD binding (Blanke et al., 1994b). Substituting alanine for Tyr65, however, inhibited NAD binding and dramatically reduced transferase activity 350-fold. Whereas T65F bound adenosine with affinity equal to that of wild-type DT, the T65A mutant had a slightly higher affinity for adenosine suggesting that nicotinamide interacted with Tyr65 and not with adenine or the adenineribose moieties of NAD (Blanke et al., 1994b). Although Trpl53 is also located in the active site of DT, an alanine substitution for Trpl53 had only moderate effects on NAD glycohydrolase and ADP-ribosyltransferase activities (Wilson et al., 1994). The proposed model for NAD binding to DT positions the adenine ring of NAD adjacent to the indole ring of Trp5O and stacks the nicotinamide ring against the phenolic ring of Tyr65. These hydrophobic interactions, along with the hydrogen bonding of nicotinamide with the imidazole ring of His21, limit the rotational and translational freedom of the N-glycosidic bond providing a favorable orientation for nucleophilic attack in ADP-ribose transfer or NAD glycohydrolase reactions (Domenighini et al., 1991). The aromatic amino acid-rich segment in PT comprises amino acids 82 to 98 based on sequence and structural alignment of toxins (Rappuoli and Pizza, 1991). The active site Tyr65 of DT is aligned with Phe97 of PT and Phe95 of CT and LT (Blanke et al., 1994b). Similar regions of hydrophobic amino acids are present in the rabbit muscle ADP-ribosyltransferase and the RT6.2 NAD glycohydrolase (amino acids 145 to 165 and 120 to 136, respectively) which may serve a similar function in orienting NAD within the catalytic site (Fig. 2B). Likewise, in the chicken transferase, closely spaced aromatic and hydrophobic amino acids, located -30 to 50 residues from the region I glutamine, were aligned with those of the rabbit transferase and RT6.2 (Tsuchiya et al., 1994). C. Region 111 A strictly conserved active-site glutamic acid (Fig. 2C) in the bacterial toxins has been demonstrated on three-dimensional studies and by photoaffinity labeling and site-directed mutagenesis (Domenighini et al., 1994). Photoaffinity labeling of DT resulted in the lysis of the nicotinamide-ADPribose bond of NAD, decarboxylation of Glu148, and formation of a new bond between the y-methylene carbon of Glu148 and carbon-6 of the nicotinamide ring (Carroll et al., 1985). Replacement of Glu148 in DT with

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aspartate inhibited ADP-ribosylation of EF-2 without affecting NAD binding (Tweten et al., 1985). Glu553 of ETA was found analogous to Glu148 of DT by photoaffinity labeling (Carroll and Collier, 1987) and site-directed mutagenesis (Douglas and Collier, 1987); similar properties were demonstrated for Glu129 of PT (Barbieriet al., 1989; Pizza et al., 1988). Mutation of Glu129 to aspartate also inhibited the NAD glycohydrolase activity of PT with minimal effect on K, or kd values for NAD (Antoine et al., 1993). His35 of PT is also located in the catalytic site within hydrogen bonding distance of Glu129 (Stein et al., 1994). Substitution of asparagine for His35 drastically reduced ADP-ribosyltransferase activity (Kaslow et al,, 1989). Replacement of His35 with glutamine reduced but did not eliminate both transferase and NAD glycohydrolase activities by decreasing catalytic rates of each reaction without affecting NAD binding measured by the efficiency of photolabeling wild-type PT and the mutants with NAD (Xu et al., 1994). Proline substitution for His35, on the other hand, did not affect NAD binding, but enzyme activity was totally abolished. Since glutamine, but not proline or asparagine, can mimic the hydrogen bonding capacity of the E-N of the imidazole group, His35 appeared to enhance ADP-ribosylation by hydrogen bond formation between the acceptor protein (cysteine), or water molecule in the hydrolytic reaction. The E-N and the activated cysteine or water molecule would then function as a nucleophile to attack the Nglycosidic linkage of NAD (Xu et al., 1994; Antoine and Locht, 1994). The carboxylate group in Glu129 of PT is postulated to stabilize a transition state intermediate between the anomeric carbon of the N-glycosidic bond of NAD and the incoming substrate prior to ADP-ribose transfer (Antoine and Locht, 1994; Domenighini et al., 1994). GlullO and Glu112 of LT and CT were similar to Glu553 of ETA in crystallographic structures (Sixma et al., 1991) and resembled the glutamic acid residues in the active sites of DT and PT (Pizza et al., 1994).Mutagenesis of GlullO or Glull2 severely reduced ADP-ribosyltransferaseactivity (Tsuji et al., 1990; Lobet et al., 1991; Pizza et al., 1994). Despite loss of enzymatic and biologic activity, a mutant LT containing a lysine substitution for Glu112 demonstrated immunologic identity to wild-type LT (Tsuji et al., 1990) and remained capable of interacting with its allosteric activator, ARF (Moss et al., 1993). Other bacterial toxin ADP-ribosyltransferases also have been dernonstrated to have active site glutamates. The glutamic acid of Clostridium limosum C3-like toxin, an ADP-ribosyltransferase that modifies asparagine 41 of the Rho family of GTP-binding proteins (Sekine et al., 1989; Braun et al., 1989; Narumiya et al., 1988) that was photolabeled with NAD corresponds to Glu173 of the related C. botulinum exoenzyme C3 (Nemoto etal., 1991; Jungetal., 1993).An active site glutamate was also photolabeled in Bacillus cereus exoenzyme, a structurally distinct Rho-ADP-ribosylating toxin (Just et al., 1995b).

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Of the three glutamic acids at positions 238 to 240 of the skeletal muscle ADP-ribosyltransferase (Fig. 2C) which follow the region I1 hydrophobic/ aromatic amino acids, Glu240 and the surrounding amino acids were aligned with the region containing the catalytic glutamate of several bacterial toxins (Takada et al., 1995). Substituting aspartate or alanine for Glu240 resulted in the loss of transferase activity. Glu239 of the muscle transferase is not conserved among the bacterial toxins and a mutant with Glu239 replaced by aspartate retained activity. Mutation of Glu238, on the other hand, by replacement with aspartate or glutamine yielded inactive proteins. Glu238 and Glu240 of the muscle transferase were suggested to be functionally homologous to GlullO and Glu112 of LT and CT (Takada et al., 1995). A region containing a similar cluster of acidic amino acids in the NAD glycohydrolase RT6.2, Gln207, Glu208, and Glu209 was aligned with the region containing glutamates 238 to 240 of the transferase. Moreover, Glu222, Asp223, Glu224, and the adjacent amino acids of the chicken heterophil transferases, AT1 and AT2, have significant similarity to the active site region of the muscle transferase (Tsuchiyaet al., 1994). Of interest, the amino acid that is amino terminal to the critical glutamate was not critical for activity although in some species it too is an acidic residue. In the ADP-ribosyltransferasesof bacteriophages T2, T4, and T6 (geneproduct Alt) which ADP-ribosylate and inhibit the host E. coli RNA polymerase asubunit (Williamson and Moss, 1990; Koch and Ruger, 1994), a similar region is found although a methionine or alanine is present between two glutamates (Fig. 2C). Based on the mutagenesis studies with the rabbit transferase, the presence of a nonacidic amino acid at this position should not affect activity (Takada et al., 1995). Another potential region I11 glutamate of the muscle transferase was located in the Glu278-Tyr-Ile sequence similar to that found at the active site of DT (Tweten et al., 1985) as well as poly(ADP-ribose) synthetase (Marsischky et al., 1992). Aspartate or alanine replacement of Glu278, however, did not affect activity. Likewise, Glu282 of the muscle enzyme was aligned with Asp993 of poly(ADP-ribose) synthetase, another critical residue in its catalytic site (Marsischky et al., 1992). Unlike Asp993, replacement of which with alanine (but not glutamate) caused inactivation (Simonin et al., 1993), replacement of Glu282 of the muscle transferase with alanine or aspartate did not affect enzyme activity (Takada et al., 1995). Based on sequence similarity and site-specific mutagenesis, Glu240 in the muscle transferase appears to correspond to the critical glutamate of the bacterial toxins. IV. AD P-ribosylarginine Hydrolases Along with mono-ADP-ribosyltransferases,ADP-ribosylargininehydrolases comprise an ADP-ribosylation cycle. Hydrolases have been identified

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in mammalian, avian, and bacterial systems. They appear to be relatively widespread in eukaryotic tissues.

A. Turkey ADP-ribosylarginine Hydrolase The extensively purified turkey erythrocyte hydrolase is a soluble, M, 39,000 monomeric protein (Moss et al., 1988).The hydrolase was maximally activated by 5 to 10 mM Mg2+and 5 to 10 mM dithiothreitol, and inhibited >80% by 5 mM NaF or 200 mM NaC1; histones had no effect on activity (Moss et al., 1986). The ADP-ribose moiety, but not the arginine group, was critical for substrate recognition and degradation by the hydrolase. The hydrolase cleaved ADP-ribosylarginine, (2’phospho-ADP-ribosyl)arginine, and ADPribosylguanidine (Moss et al., 1986). The Km for ADP-ribosylarginine was 65 pM, whereas the values for (2’phospho-ADP-ribosy1)arginine and ADPribosylguanidine were 47 p M and 27 pM, respectively. Ribosylarginine and (phosphoribosyl)arginine,products of sequential degradation of ADPribosylarginine by phosphodiesterase and phosphatase, were neither substrates nor inhibitors of the hydrolase. The hydrolase was competitively inhibited by ADP-ribose > ADP > AMP; arginine, agmatine, and guanidine had no effect. Incubation of arginine produced by hydrolase-catalyzed degradation of ADP-ribosylarginine with NAD and the purified turkey ADPribosyltransferase resulted in the regeneration of ADP-ribosylarginine(Moss et al., 1985), demonstrating preservation of the guanidino moiety during hydrolysis. Moreover, the stereospecificity of the hydrolase matched that of the turkey ADP-ribosyltransferase which in the presence of P-NAD and arginine catalyzed the synthesis of the a-anomer of ADP-ribosylarginine; the hydrolase utilized a-ADP-ribosylarginine, but not the p-anomer, as substrate (Moss et al., 1986). This finding is compatible with the hypothesis that the ADP-ribosyltransferasesand ADP-ribosylarginine hydrolases act as opposing arms of an ADP-ribosylation cycle. Purification of the turkey hydrolase generated thiol-resistant and thiolsensitive species (Moss et al., 1988).Hydrolase activity in the soluble fraction of the erythrocyte crude homogenate was thiol independent. Chromatography on DE-52, phenyl-Sepharose, and hydroxylapatite generated the thiolresistant hydrolase. Following chromatography on Affi-gel 501 (organomercurial agarose) and removal of thiol on DE-52, the enzyme was thiol sensitive. Similarly, incubating the thiol-resistant hydrolase with HgC12 produced the thiol-sensitive species. The dithiothreitol concentration required for activation of the thiol-resistant hydrolase was -30-times that for the thiol-sensitive form. Both species were inactivated by N-ethylmaleimide but only after incubation with dithiothreitol; it required higher temperatures to reduce the thiol-resistant form to one readily inactivated by N-ethylmaleimide. Activation of the hydrolase by thiol appeared to be a reversible event

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(Moss et al., 1988) and may reflect conformational changes of the enzyme induced by dithiothreitol. The hydrolase was ADP-ribosylated and inactivated by incubation with NAD and the transferase. Inactivation was prevented by Mg2+or Mg2+plus dithiothreitol. Activity of the ADP-ribosylated hydrolase was not, however, restored by addition MgZ+or Mg2+and dithiothreitol nor did these agents alter ADP-ribosyltransferase activity. The hydrolase was seemingly “stabilized” by Mg2+and dithiothreitol in a conformation resistant to ADP-ribosylation of critical arginine residues by the transferase (Moss et al., 1988). 6. Mammalian ADP-ribosylarginine Hydrolases

An ADP-ribosylargininehydrolase was purified -20,000-fold from rat brain and partially purified for functional and immunological comparison from other rat tissues and mouse, guinea pig, rabbit, sheep, pig, and calf brain (Moss et al., 1992). Rat and mouse brain hydrolase activities were higher than those from the other mammalian species tested. Among rat tissues, hydrolase activity was highest in brain, spleen, and testis. Similar to the turkey hydrolase, the rat and mouse brain enzymes were synergistically stimulated by Mg2+and dithiothreitol. In contrast, the pig and calf hydrolases were stimulated by MgZ+ but not dithiothreitol. Despite differences in enzymatic properties, rabbit polyclonal antibodies against rat brain hydrolase reacted on immunoblot with 39-kDa proteins from turkey erythrocytes and mouse, rat, and calf brains. Amino acid sequence of a tryptic peptide from the purified rat hydrolase was used to design a PCR-generated cDNA probe that was used to clone a hydrolase coding region cDNA from a X ZAP cDNA library (Moss et al., 1992). Identity of the cloned hydrolase cDNA, which contained an open reading frame of 1089 bp, was confirmed by expression in E. coli as a glutathione S-transferase-linked fusion protein. The expressed protein had Mg2+-and dithiothreitol-dependent hydrolase activity and reacted on immunoblot with antihydrolase antibodies. On Northern blot, a PCR-generated, hydrolase cDNA probe hybridized with a 1.7-kb band in poly (A)+RNA from rat and mouse brain, but not with chicken, rabbit, or bovine brain or cultured IMR-32 or HL-60 cells. A hydrolase-specific oligonucleotide probe detected a 1.7-kb mRNA in total RNA from all rat tissues although expression was highest in brain, spleen, testis, and lung; the levels of mRNA correlated with amount of enzyme activity (Moss et al., 1992). Mouse and human hydrolase genes were cloned by PCR using oligonucleotide primers generated from the rat brain hydrolase cDNA (Takada et al., 1993). Nucleic acid and deduced amino acid sequences of the rat and mouse hydrolases were 92 and 94% identical, respectively. The human hydrolase, on the other hand, was 82% identical in nucleotide and 83% identical in deduced amino acid sequences to those of the rat. On Northern

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blot, human hydrolase-specific oligonucleotide probes hybridized with a 4-kb band in poly(A)+RNA extracted from human brain, lung, and placenta, but not IMR-32 or undifferentiated HL-60 cells. The positions of five cysteines were identical in the rat and mouse hydrolases, whereas only four of the five were found in the human hydrolase (Takada et al., 1993). Cysteine 108 in the rat and mouse enzymes was replaced by a serine at position 103 in the human sequence. The hydrolase in rat tissues was activated by dithiothreitol but activity in human tissues was thiol independent. Site-directed mutagenesis of the rat and human hydrolases was utilized to determine whether the differences in thiol sensitivity was affected by the position of critical cyteines (Takada et al., 1993). Expression in E. coli of mutated human hydrolase that contained cysteine at position 103 instead of serine (S103C) produced a thiol-dependent enzyme similar to the native rat hydrolase. Wild-type human hydrolase expressed in E. coli retained its thiol independence. There was no difference in Mg2+ requirement, specific activity, or Km value for ADP-ribosylargininebetween wild-type and mutant enzymes. On the other hand, a mutant rat hydrolase in which serine was substituted for cysteine at position 108 (Cl08S) expressed in E. coli demonstrated thiol independence in contrast to the similarly expressed thioldependent, wild-type rat hydrolase. Both recombinant rat proteins had Mg2+ requirements and specific activities similar to those of the native rat hydrolase. On immunoblot, antihydrolase antibodies reacted equivalently with the recombinant wild-type and native rat hydrolases, but only weakly with the C108S mutant rat protein and not at all with the wild-type (S103) or mutant (S103C) human hydrolases. Such single amino acid differences may explain the differences observed in enzymatic properties as well as immunoreactivity among native hydrolases (Takada et al., 1993). The rat ADP-ribosylargininehydrolase expressed as a fusion protein in E. coli released the ADP-ribose moiety from G,a ADP-ribosylated by cholera toxin, and the auto-ADP-ribosylated Al subunit of cholera toxin (Maehama et al., 1994). Nonmuscle actin modified on an arginine by botulinum C2 toxin (Vandekerckhove et al., 1988) also served as a substrate of the hydrolase. G,a ADP-ribosylated by pertussis toxin, EF-2 modified by diphtheria toxin, and rho GTP-binding protein modified by C3 exoenzyme, however, were not affected by the recombinant hydrolase (Maehama et al., 1994) consistent with the specificity of the hydrolase for the ADP-ribosearginine bond.

V. Summary Mono-ADP-ribosyltransferase activity has been detected in numerous vertebrate tissues and transferase cDNAs from a few species have recently

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been cloned. In vitro ADP-ribosylation has been demonstrated with diverse substrates such as phosphorylase kinase, actin, and G,cr resulting in the alteration of substrate function. ADP-ribosylationof endogenous target proteins has been observed in chicken heterophils, rat brain, and human platelets, and integrin a7 was found to be the endogenous substrate of the GPI-anchored rabbit skeletal muscle transferase. The reversibility of ADPribosylation is made possible by ADP-ribosylargininehydrolases which have been isolated and cloned from rodent and human tissues. The transferases and hydrolases could in principle form an intracellular ADP-ribosylation regulatory cycle. In the case of the skeletal muscle transferases, however, processing of ADP-ribosylated integrin a7 is carried out by phosphodiesterases and possibly phosphatases (Fig. 1). Most bacterial toxin and eukaryotic mono-ADP-ribosyltransferases, and perhaps other NAD-utilizing enzymes such as the RT6 family of proteins, share a common catalytic-site structure despite a lack of overall sequence identity. The transferases that have been studied thus far possess a critical glutamic acid and other amino acids at the catalytic cleft which function to position NAD for nucleophilic attack at the N-glycosidic linkage for either ADP-ribose transfer or NAD hydrolysis. The amino acid differences among transferases at the active site may reflect different catalytic mechanisms of ADP-ribosylation or may be required for accommodating the different ADPribose acceptor molecules.

Acknowledgment We thank Dr. Martha Vaughan for helpful discussions and critical review of the manuscript.

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Banasik, M., Komura, H., Shimoyama, M., and Ueda, K. (1992). Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosy1)transferase.J. Biol. Chem. 267, 1569- 1575. Banasik, M., and Ueda, K. (1994). Inhibitors and activators of ADP-ribosylation reactions. Mol. Cell. Biocbem. 138,185-197. Barbieri, J. T., Mende-Mueller, L. M., Rappuoli, R., and Collier, R. J. (1989). Photolabeling of Glu-129 of the S1 subunit of pertussis toxin with NAD. Infect. Immun. 57,3549-3554. Blanke, S . R., Huang, K., Wilson, B. A.,Papini, E., Covacci, A., and Collier, R. J. (1994a).Activesite mutations of diphtheria toxin catalytic domain: Role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2. Biochemistry 33,5155-5161. Blanke, S. R., Huang, K., and Collier, R. J. (1994b).Active-site mutations of diphtheria toxin: Role of tyrosine-65 in NAD binding and ADP-ribosylation. Biochemistry 33, 1549415500. Braun, U., Habermann, B., Just, I., Aktories, K., and Vandekerckhove, J. (1989). Purification of the 22-kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product. FEBS Lett. 243, 70-76. Burnette, W. N., Cieplak, W., Mar, V. L., Kaljot, K. T., Sato, H., and Keith, J. M. (1988). Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope. Science 242, 72-74. Burnette, W. N., Mar, V. L., Platler, B. W., Schlotterbeck, J. D., McGinley, M. D., Stoney, K. S., Rohde, M. F., and Kaslow, H. R. (1991). Site-specificmutagenesis of the catalytic subunit of cholera toxin: Substitution lysine for arginine 7 causes loss of activity. Infect. Immun. 59,4266-4270. Burstein, D., Mordes, J. P., Greiner, D. L., Stein, D., Nakamura, N., Handler, E. S., and Rossini, A. A. (1989).Prevention of diabetes in BBlWor rat by single transfusion of spleen cells. Parameters that affect degree of protection. Diabetes 38, 24-30. Carroll S. F., and Collier, R. J. (1987). Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. 1. Biol. Chem. 262, 8707-8711. Carroll S. F., and Collier, R. J. (1988). Amino acid sequence homology between the enzymic domains of diphtheria toxin and pseudomonas aeruginosa exotoxin A. Mol. Microbio!. 2,293-296. Carroll, S. F., McCloskey, J. A., Crain, P. F., Oppenheimer, N. J., Marschner, T. M., and Collier, R. J. (1985). Photoaffinity labeling of diphtheria toxin fragment A with NAD: Structure of the photoproduct at position 148. Proc. Natl. Acad. Sci. U.S.A. 82, 72377241. Chang, Y.-C., Soman, G., and Graves, D. J. (1986). Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle. Biochem. Biophys. Res. Commun. 139, 932-939. Choe, S., Bennett, M. J., Fujii, G., Curmi, P. M. G., Kantardjieff, K. A., Collier, R. J., and Eisenberg, D. (1992). The crystal structure of diphtheria toxin. Nature (London) 357, 216-222. Collier, R. J. (1990). Diphtheria toxin: Structure and function of a cytocidal protein. In “ADPribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss, and M. Vaughan, Eds.), pp. 3-19. American Society for Microbiology, Washington D.C. De Maneis, M. A., Di Girolamo, M., Colanzi, A., Pallas, M., De Tullio G., McDonald, L. J., Moss, J., Santini, G., Bannykh, S., Corda, D., and Luini, A. (1994). Stimulation of endogenous ADP-ribosylation by brefeldin A. Proc. Nati. Acnd. Sci. U.S.A. 91, 11141118. Domenighini, M., Montecucco, C., Ripka, W. C., and Rappuoli, R. (1991). Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding. Mol. Microbiol. 5 , 23-31.

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Domenighini, M., Magagnoli, C., Pizza, M., and Rappuoli, R. (1994). Common features of the NAD-bindingand catalyticsite of ADP-ribosylatingtoxins. Mol. Microbiol. 14,41-50. Donnelly, L. E., Boyd, R. S., and MacDermot, J. (1992). G.a is a substrate for mono(ADPribosy1)transferaseof NG108-15 cells. ADP-ribosylationregulates G p activity and abundance. Biochem. J. 288,331-336. Douglas, C. M., and Collier, R. J. (1987). Exotoxin A of Pseudomonas aeruginosa: Substitution of glutamic acid-553 with aspartic acid drastically reduces toxicity and enzymic activity. Infect. Immun. 169, 4967-4971. Doukas, J., and Mordes, J. P. (1993). T lymphocytes capable of activating endothelial cells in vitro are present in rats with autoimmune diabetes. J. lmmunol. 150, 1036-1046. Duman, R. S., Winston, S. M., Clark, J. A., and Nestler, E. J. (1990). Corticosterone regulates the expression of ADP-ribosylation factor messenger RNA and protein in rat cerebral cortex. J. Neurochem. 55, 1813-1816. Duman, R. S., Terwilfiger, R. Z., and Nestler, E. J. (1991). Endogenous ADP-ribosylation in brain: Initial characterization of substrate proteins. J. Neurochem. 57, 2124-2132. Feldman, A. M., Levine, M. A., Baughman, K. L., and Van Dop, C. (1987). NAD+-mediated stimulation of adenylatecyclase in cardiac membranes. Biochem. Biophys. Res. Commun. 142,631-637. Gerber, L. D., Kodukula, K. and Udenfriend, S. (1992). Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-Gattachment in the COOH-terminal signal peptide./. Biol. Chem. 267,12168-12173. Godeau, F., Belin, D., and Koide, S. S. (1984).Mono(adenosinediphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographiclocalization in sodium dodecyl sulfate-polyacrylamidegels. Anal. Biochem. 137, 287-296. Greiner, D. L., Handler, E. S., Nakano, K., Mordes, J. P., and Rossini, A. A. (1986). Absence of the RT-6 T cell subset in diabetes-prone BBlW rats. J. lmmunol. 136, 148-151. Hawkins, D. J., and Browning, E. T. (1982). Tubulin adenosine diphosphate ribosylation is catalyzed by cholera toxin. Biochemistry 21,4474-4479. Jacobson, M. K., Loflin, P. T., Aboul-Ela, N., Mingmuang, M., Moss, J., and Jacobson, E. L. (1990). Modification of plasma membrane protein cysteine residues by ADP-ribose in vivo. J. Biol. Chem. 265, 10825-10828. Johnson, V. G., and Nicholls, P. (1994). Histidine-21 does not play a major role in diphtheria toxin catalysis. J. Biol. Chem. 269,4349-4354. Jung, M., Just, I., van Damme, J.. Vandekerckhove, J., and Aktories, K. (1993). NAD-binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum. J. Biol. Chem. 268,23215-23218. Just, I., Sehr, P., Jung, M., van Damme, J., Puype, M., Vandekerckhove, J., Moss, J., and Aktories, K. (1995a). ADP-ribosyltransferase type A from turkey erythrocytes modifies actin at arg-95 and arg-372. Biochemistry 34, 326-333. Just, I., Selzer, J., Jung, M., van Damme, J., Vandekerckhove, J., and Aktories, K. (1995b). Rho-ADP-ribosylatingexoenzyme from Bacillus cereus. Purification,characterization,and identification of the NAD-binding site. Biochemistry 34, 334-340. Kaslow, H. R., Schlotterbeck, J. D., Mar, V. L., and Burnette, N. W. (1989). Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferaseactivity of the S1 subunit of pertussis toxin. J. Biol. Chem. 264, 6386-6390. Kharadia, S. V., Huiatt, T. W., Huang, H.-Y., Peterson, J. E., and Graves, D. J. (1992). Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture. Exp. Cell. Res. 201, 33-42. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwa-, J. (1992). Brefeldin A: Insights into the control of membrane traffic and organelle structure.]. Cell. Biol. 116,1071-1080. Klebl, B. M., Matsushita, S., and Pette, D. (1994). Localization of an arginine-specificmonoADP-ribosyltransferasein skeletal muscle sarcolemma and transverse tubules. FEBS Lett. 342, 66-70.

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Koch, F., Kashan, A., and Thiele, H.-G. (1988). The rat T-cell differentiation marker RT6.1 is more polymorphic than its alloantigeniccounterpart RT6.2. Immunology 65,259-265. Koch, F., Haag, F., Kashan, A., and Thiele, H.-G. (1990). Primary structure of rat RT6.2, a nonglycosylated phosphatidylinositol-linkedsurface marker of postthymic T cells. Proc. Natl. Acud. Sci. U.S.A. 87, 964-967. Koch, T., and Ruger, W. (1994).The ADP-ribosyltransferases(gpAlt)of bacteriophages T2, T4, and T6: Sequencing of the genes and comparison of their products. Virology203,294-298. Liu, Y., and Kahn, M. L. (1995). ADP-ribosylationof Rhizobium meliloti glutamine synthetase III in vivo. J. Biol. Cbem. 270, 1624-1628. Lobet, Y., Cluff, C. W., and Cieplak, W. Jr. (1991).Effect of site-directed mutagenic alterations on ADP-ribosyltransferaseactivity of the A subunit of Escherichia coli heat-labile enterotoxin. Infect. Immun. 59, 2870-2879. Ludden, P. W. (1994). Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes. Mol. Cell. Biochem. 138, 123-129. Maehama, T., Nishina, H., and Katada, T. (1994).ADP-ribosylarginineglycohydrolasecatalyzing the release of ADP-ribose from the cholera toxin-modified a-subunits of GTP-binding proteins. J. Biocbem. 116, 1134-1138. Marsischky, G. T., Ikejima, M., Suzuki, H., Sugimura, T., Esumi, H., Miwa, M., and Collier, R. J. (1992). Directed mutagenesis of glutamic acid 988 of poly(ADP-ribose)polymerase. In “ADP-ribosylation Reactions” (G. G. Poirier, and P. Moreau, Eds.), pp. 47-52. Springer-Verlag, New York. Matsuura, R., Tanigawa, Y ., Tsuchiya, M., Mishima, K., Y oshimura, Y ., and Shimoyama, M. (1988). ADP-ribosylation suppresses phosphorylation of the L-type pyruvate kinase. Biocbem. Biopbys. Acta 969,57-65. Matsuyama, S., and Tsuyama, S. (1991). Mono-ADP-ribosylation in brain: Purification and characterization of ADP-ribosyltransferasesaffecting actin from rat brain. J. Neurocbem. 57, 1380-1387. McDonald, L. J., Wainschel,L. A., Oppenheimer, N. J., and Moss, J. (1992).Amino acid-specific ADP-ribosylation:Structural characterization and chemicaldifferentiationof ADP-ribosecysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. Biochemistry 31,11881-1 1887. McDonald, L. J., and Moss, J. (1993a).Nitric oxide-independent,thiol-associated ADP-ribosylation inactivates aldehyde dehydrogenase. J. Biol. Cbem. 268, 17878-17882. McDonald, L. J., and Moss, J. (1993b). Stimulation by nitric oxide of a novel linkage of NAD to glyceraldehyde3-phosphate dehydrogenase.Proc. Natl. Acad. Sci. U.S.A. 90,6238-6241. McDonald, L. J., and Moss, J. (1994). Enzymatic and nonenzymatic ADP-ribosylation of cysteine. Mol. Cell. Biocbem. 138, 221-226. McMahon, K. K., Piron, K. J., Ha, V. T., and Fullerton, A. T. (1993). Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specificmono-ADPribosyltransferase. Biocbem. J. 293, 789-793. Mishima, K., Tsuchiya, M., Tanigawa, Y.,Yoshimura, Y., and Shimoyama, M. (1989). DNAdependent mono(ADP-ribosy1)ationof p33, an acceptor protein in hen liver nuclei. Eur. J . Biocbem. 179,267-273. Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K., Shimoyama, M. (1991). Arginine-specific ADP-ribosyltransferaseand its acceptor protein p33 in chicken polymorphonuclear cells: Co-localization in the cell granules, partial characterization, and in situ mono(ADP-ribosy1)ation.J. Biocbem. 110, 388-394. Molina y Vedia, L., Nolan, R. D., and Lapetina, E. G. (1989). The effect of iloprost on the ADP-ribosylation of G,a (the a-subunit of G,). Biocbem. J. 261, 841-845. Moss, J., Stanley, S. J., and Oppenheimer, N. J. (1979). Substrate specificity and partial purification of a stereospecific NAD-and guanidine-dependent ADP-ribosyltransferase from avian erythrocytes. J. Biol. Chem. 254, 8891-8894.

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Moss, J., Stanley, S. J., and Watkins, P. A. (1980). Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes. J. Biol. Chem. 255, 5838-5840. Moss, J., Stanley, S. J., and Osborne, J. C. Jr. (1981). Effect of self-association on activity of an ADP-ribosyltransferase from turkey erythrocytes. J. Biol. Chem. 256, 11452-1 1456. Moss, J., Stanley, S. J., and Osborne, J. C. Jr. (1982). Activation of NAD:arginine ADPribosyltransferase by histone. J. Biol. Chem. 257, 1660-1663. Moss, J., Osborne, J. C. Jr., and Stanley, S. J. (1984a). Activation of an erythrocyte NAD:arginine ADP-ribosyltransferase by lysolecithin and nonionic and zwitterionic detergents. Biochemistry 23,1353-1357. Moss, J., Watkins, P. A., Stanley, S. J., Purnell, M. R., and Kidwell, W. R. (1984b). Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase. J. Biol. Chem. 259,5100-5104. Moss, J., Jacobson, M. K., and Stanley, S. J. (1985). Reversibility of arginine-specific mono (ADP4bosyl)ation: Identification in erythrocytes of an ADP-ribose-L-arginine cleavage enzyme. Proc. Natl. Acad. Sci. U.S.A. 82, 5603-5607. Moss, J., Oppenheimer, N. J., West, R. E. Jr., and Stanley, S. J. (1986). Amino acid specific ADP-ribosylation: Substrate specificity of an ADP-ribosylarginine hydrolase from turkey erythrocytes. Biochemistry 25, 5408-5414. Moss, J., Tsai, S.-C., Adamik, R., Chen H.-C., and Stanley, S. J. (1988). Purification and characterization of ADP-ribosylargininehydrolase from turkey erythrocytes. Biochemistry 27, 5819-5823. MOSS,J., Stanley, S. J.. and Levine, R. L. (1990). Inactivation of bacterial glutamine synthetase by ADP-ribosylation. J. Biol. Chem. 265, 21056-21060. Moss, J., Stanley, S. J., Nightingale, M. S., Murtagh, J. J. Jr., Monaco, L., Mishima, K., Chen, H.-C., Williamson, K. C., and Tsai, S.-C. (1992). Molecular and Immunological Characterization of ADP-ribosylarginine hydrolases. J. Biol. Chem. 267, 10481-10488. Moss, J., Stanley, S. J., Vaughan, M., and Tsuji, T. (1993). Interaction of ADP-ribosylation factor with Escherichia coli enterotoxin that contains an inactivating lysine 112 substitution. J. Biol. Chem. 268, 6383-6387. Moss, J., and Stanley, S. J. (1981a). Histone-dependent and histone-independent forms of an ADP-ribosyltransferasefrom human and turkey erythrocytes. Proc. Nutf.Acad. Sci. U.S.A. 78,4809-4812. Moss, J., and Stanley, S. J. (1981b). Amino acid-specific ADP-ribosylation. Identification of an arginine-dependent ADP-ribosyltransferasein rat liver. J. Biol. Chem. 256,7830-7833. Moss, J., and Vaughan, M. (1978). Isolation of an avian erythrocyte protein possessing ADPribosyltransferase activity and capable of activating adenylate cyclase. Proc. Natl. Acud. Sci. U.S.A. 75, 3621-3624. Moss, J., and Vaughan, M. (1988). ADP-ribosylation of guanyl nucleotide-binding proteins by bacterial toxins. Adu. Enzymol. 61, 303-379. Moss, J., and Vaughan, M. (1993). ADP-ribosylation factors, 20,000 M, guanine nucleotidebinding protein activators of cholera toxin and components of intracellular vesicular transport systems. Cell. Signal. 5, 367-379. Narumiya, S., Sekine, A., and Fujiwara, M. (1988). Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. J. Biol. Chem. 263,17255-17257. Nemoto, Y.,Namba, T., Kozaki, S., and Narumiya, S. (1991). Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning sequencing, and expression of a functional protein in Escherichia coli. J. Biol. Chem. 266, 19312-19319. Ness, S. A., Marknell, A., and Graf, T. (1989). The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell 59, 1115-1125. Obara, S., Yamada, K., Yoshimura, Y.,and Shimoyama, M. (1991). Evidence for the endogenous GTP-dependent ADP-ribosylation of the a-subunit of the stimulatory guanyl-

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nucleotide-bindingprotein concomitant with an increase in basal adenylyl cyclase activity in chicken spleen cell membrane. Eur. J. Biochem. 200, 75-80. Okazaki, I. J., Zolkiewska, A., Nightingale, M. S., and Moss, J. (1994). Immunological and structural conservation of mammalian skeletal muscle glycosylphosphatidylinositol-linked ADP-ribosyltransferases. Biochemistry 33, 12828-12836. Okazaki, I. J., Kim, H.-J., McElvaney, G., and Moss, J. (1996). Molecular characterization of a glycosylphosphatidylinositol-linkedADP-ribosyltransferasefrom lymphocytes. Submitted. Oppenheimer, N. J. (1978). Structural determination and stereospecificity of the choleragencatalyzed reaction of NAD+with guanidines. J . Biol. Chem. 253, 4907-4910. Osborne, J. C. Jr., Stanley, S. J., and Moss, J. (1985).Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases:The soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cbolerae. Biochemistry 24, 5235-5240. Papini, E., Schiavo, G., Sandona, D., Rappuoli, R., and Montecucco, C. (1989). Histidine 21 is at the NAD+ binding site of diphtheria toxin. J. Biol. Chem. 264, 12385-12388. Papini, E., Santucci, A., Schiavo, G., Domenighini,M., Neri, P., Rappuoli, R., and Montecucco, R. (1991).Tyr-65 is photolabelled by 8-azido adenine and 8-azido-adenosineat the NAD binding site of diphtheria toxin. J. Biol. Chem. 266, 2494-2498. Pelham, H. R. B. (1991).Multiple targets for brefeldin A. Cell 67, 449-451. Peterson,J. E., Larew,J. S.-A., and Graves, D. J. (1990).Purificationand partial characterization of arginine-specificADP-ribosyltransferase from skeletal muscle microsomal membranes. J. Bio. Chem. 265, 17062-17069. Piron, K. J., and McMahon, K. K. (1990). Localization and partial characterization of ADPribosylation products in hearts from adult and neonatal rats. Biochem. J. 270,591-597. Pizza, M., Bartoloni, A., Prugnola, A., Silvestri, S., and Rappuoli, R. (1988). Subunit S1 of pertussis toxin: Mapping of the regions essential for ADP-ribosyltransferaseactivity. Proc. Natl. Acud. Sci. U.S.A. 85, 7521-7525. Pizza, M., Domenighini, M., Hol, W., Giannelli, V., Fontana, M. R., Giuliani, M. M., Magagnoli, C., Peppoloni, S., Manetti, R., and Rappuoli, R. (1994). Probing the structureactivity relationship of Escherichia coli LT-A by site-directedmutagenesis. Mol. Microbiol. 14,Sl-60. Quist, E. E., Coyle, D. L., Vasan, R., Satumitra, N., Jacobson, E. L., and Jacobson, M. K. (1994). Modification of cardiac membrane adenylate cyclase activity and G , by NAD and endogenous ADP-ribosyltransferase. J. Mol. Cell. Cardiol. 26, 251. Rafaelli, N., Scaife, R. M., and Purich, D. L. (1992). ADP-ribosylation of chicken red cell tubulin and inhibition of microtubule self-assemblyin vitro by the NAD+-dependentavian ADP-ribosyltransferase. Biochem. Biopbys. Res. Commun. 184, 414-418. Rankin, P. W., Jacobson, E. L., Benjamin, R. C., Moss, J., and Jacobson, M. K. (1989). Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo. J. Biol. Chem. 264,4312-4317. Rappuoli, R., and Pizza, M. (1991). Structure and evolutionary aspects of ADP-ribosylating toxins. In “Sourcebook of Bacterial Protein Toxins” (J. E. Alouf, and J. H. Freer, Eds.), pp. 1-21. Academic Press, San Diego. Saito, N., Guitart, X., Hayward, M. D., Tallman, J. F., Duman, R. S., and Nestler, E. J. (1989). Corticosterone differentially regulates the expression of G,a and Gia messenger RNA and protein in rat cerebral cortex. Proc. Natl. Acud. Sci. U.S.A. 86, 3906-3910. Scaife, R. M., Wilson, L., and Purich, D. L. (1992).Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.Biochemistry 31,310-316. Schering, B., Barmann, M., Chhatwal, G. S., Geipel, U., and Aktories, K. (1988).ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin. Eur. J. Biochem. 171,225-229. Schuman, E. M., Meffert, M. K., Schulman, H., and Madison, D. V. (1994). An ADPribosyltransferase as a potential target for nitric oxide action in hippocampal long-term potentiation. Proc. Natl. Acud. Sci. U.S.A. 91, 11958-11962.

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Sekine, A., Fujiwara, M., and Narumiya, S. (1989).Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.J. Biol. Chem. 264, 86028605. Simonin, F., Poch, O., Delarue, M., and de Murcia, G. (1993).Identification of potential activesite residues in the human poly(ADP-ribose)polymerase. J. Biol. Chem. 268,8529-8535. Sixma, T. K., Pronk, S. E., Kalk, K. H., Wartna, E. S., van Zanten, B. A. M., Witholt, B., and Hol, W. G. J. (1991).Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature (London) 351, 371-377. Smith, K. P., Benjamin, R. C., Moss, J., and Jacobson, M. K. (1985).Identification of enzymatic activities which process protein bound mono(ADP-ribose). Biochem. Biophys. Res. Commun. 126, 136-142. Soman, G., Mickelson, J. R., Louis, C. F., and Graves, D. J. (1984). NAD:guanidino groupspecific mono-ADP-ribosyltransferaseactivity in skeletal muscle. Biochem. Biophys. Res. Commun. 120,973-980. Soman, G., Haregewoin, A., Hom, R. C., and Finberg, R. W. (1991). Guanidine group specific ADP-ribosyltransferase in murine cells. Biochem. Biophys. Res. Commun. 176,301-308. Song, W. K., Wang, W., Foster, R. F., Bielser, D. A., and Kaufamn, S. J. (1992). H-36alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis. J. Cell. Biol. 117, 643-657. Stein, P. E., Boodhoo, A., Armstrong, G. D., Cockle, S. A., Klein, M. H., and Read, R. J. (1994).The crystal structure of pertussis toxin. Structure 2, 45-57. Sternweis, P. C., and Robishaw, J. D. (1984).Isolation of two proteins with high affinity for guanine nucleotides from membrane of bovine brain. J. Biol. Chem. 259, 13806-13813. Takada, T., Iida, K., and Moss, J. (1993). Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase. J. Biol. Chem. 268, 17837-17843. Takada, T., Iida, K., and Moss, J. (1994). Expression of NAD glycohydrolase activity by rat mammary adenocarcinoma cells transformed with rat T cell alloantigen RT6.2. J. Biol. Chem. 269, 9420-9423. Takada, T., Iida, K., and Moss, J. (1995). Conservation of a common motif in enzymes catalyzing ADP-ribose transfer. J. Biol. Chem. 270, 541-544. Takenaka, S., Nakano, Y., and Tsuyama, S. (1994).Mono-ADP-ribosylation of microtubuleassociated protein 2 that inhibits polymerization of rat brain microtubules. From “The 1lrh International Symposium on ADP-ribosylation. DNA Repair, Signal Transduction”. Abstract #56. Strasbourg-Bischenberg, France. Tanigawa, Y., Tsuchiya, M., Imai, Y., and Shimoyama, M. (1983a).Mono(ADP-ribosy1)ation of hen liver nuclear proteins suppresses phosphorylation. Biochem. Biophys. Res. Commun. 113,135-141. Tanigawa, Y., Tsuchiya, M., Imai, Y., and Shimoyama, M. (1983b). ADP-ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP-dependent protein kinase. FEBS Lett. 160, 217-220. Tanigawa, Y., Tsuchiya, M., Imai, Y., and Shimoyama, M. (1984). ADP-ribosyltransferase from hen liver nuclei. J. Biol. Chem. 259, 2022-2029. Tanuma, S., Kawashima, K., and Endo, H. (1987).An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes. J. Biochem. 101, 821-824. Tanuma, S., Kawashima, K., and Endo, H. (1988).Eukaryotic mono(ADP4bosyl)transferase that ADP-ribosylates GTP-binding regulatory G, protein. J. Biol. Chem. 263,5485-5489. Tanuma S., and Endo, H. (1990).Identification in human erythrocytes of mono(ADP-ribosyl) protein hydrolase that cleaves a mono(ADP-ribosyl) G,linkage. FEBS Lett. 261,381-384. Terashima, M., Mishima, K., Yamada, K., Tsuchiya, M., Wakutani, T., and Shimoyama, M. ( 1992). ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils. Ear. J. Biochem. 204, 305-311. Thiele, H.-G., Koch, F., Hamann, A., and Amdt, R. (1986). Biochemical characterization of the T-cell alloantigen RT6.2. Immunology 59, 195-201.

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Tsai, S.-C., Adamik, R., Moss, J., Vaughan, M., Manne, V., and Kung, H.-F. (1985).Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21. Proc. Nutl. Acud. Sci. U.S.A. 82, 8310-8314. Tsai, S.-C., Adamik, R., Haun, R. S., Moss, J., and Vaughan, M. (1993). Effects of brefeldin A and accessory proteins on association of ADP-ribosylation factors 1, 3, and 5 with Golgi. J. Biol. Cbem. 268, 10820-10825. Tsuchiya, M., Tanigawa, Y., Ushiroyama, T., Matsuura, R., and Shimoyama, M. (1985).ADPribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme. Eur. J. Biochem. 147, 33-40. Tsuchiya, M., Hara, N., Yamada, K., Osago, H., Shimoyama, M. (1994).Cloning and expression of cDNA for arginine-specific ADP-ribosyltransferase from chicken bone marrow cells. J. Biol. Cbem. 269, 27451-27457. Tsuji, T., Inoue, T., Miyama, A., Okamoto, K., Honda, T., and Miwatani, T. (1990).A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. J. Biol. Cbem. 265, 22520-22525. Tweten, R. K., Barbieri, J. T., and Collier, R. J. (1985).Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. J. Biol. Cbem. 260,10392-10394. Ui, M. (1990).Pertussis toxin as a valuable probe for G-protein involvement in signal transduction. In “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss, and M. Vaughan, Eds.), pp. 45-77. American Society for Microbiology, Washington D.C. Uroshiyama, T., Tanigawa, Y., Tsuchiya, M., Matsurra, R., Ueki, M., Sugimoto, O., and Shimoyama, M. (1985).Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation. Eur. J. Biocbem. 151, 173-177. Vandekerckhove, J., Schering, B., Barmann, M., and Aktories, K. (1987). Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEES Lett. 225, 48-52. Vandekerckhove, J., Schering, B., Barmann, M., and Aktories, K. (1988).Botulinum C2 toxin ADP-ribosylates cytoplasmic &actin in arginine 177. J. Biol. Cbem. 263, 696-700. Wang, J., Nemoto, E., Kots, A. Y., Kaslow, H. R., and Dennert, G. (1994). Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchoredlarginine ADP-ribosyltransferase. J. Immunol. 153, 4048-4058. Watkins, P. A., and Moss, J. (1982). Effects of nucleotides on activity of a purified ADPribosyltransferase from turkey erythrocytes. Arch. Biocbem. Biopbys. 216, 74-80. Watkins, P. A., Kanaho, Y., and Moss, J. (1987).Inhibition of the GTP-ase activity of transducin by an NAD’:arginine ADP-ribosyltransferase from turkey erythrocytes. Biochem. J. 248,749-754. Wegner, A., and Aktories, K. (1988). ADP-ribosylated actin caps the barbed ends of actin filaments. J. Biol. Chem. 263, 13739-13742. Welsh, C. F., Moss, J., and Vaughan, M. (1994). ADP-ribosylation factors: A family of -20-kDa guanine nucleotide-binding proteins that activate cholera toxin. Mol. Cell. Biocbem. 138, 157-166. West, R. E. Jr., and Moss, J. (1986).Amino acid specific ADP-ribosylation: Specific NAD:arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes. Biochemistry 25, 8057-8062. Wick, M. J., and Iglewski, B. H. (1990). Pseudomoms aeruginosa exotoxin A. In “ADPribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss, and M. Vaughan, Eds.), pp. 31-43. American Society for Microbiology, Washington D.C. Williamson, K. C., and Moss, J. (1990). Mono-ADP-ribosyltransferases and ADPribosylarginine hydrolases: A mono-ADP-ribosylation cycle in animal cells. In “ADP-

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ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss, and M. Vaughan, Eds.), pp. 493-510. American Society for Microbiology, Washington D.C. Wilson, B. A., Blanke, S. R., Reich, K. A., and Collier, R. J. (1994). Active-site mutations of diphtheria toxin. J. Biol. Chem. 269,23296-23301. Wozniak, D. J., Hsu, L.-H., and Galloway, D. R. (1988).His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor 11. Proc. Nutl. Acad. Sci. U.S.A. 85, 8880-8884. Xu,Y., Barbancon-Finck, V., and Barbieri, J. T. (1994).Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of Transducin. J. Biol. Chem. 269,9993-9999. Yamada, K., Tsuchiya, M., Mishima, K., and Shimoyama, M. (1992). p33, an endogenous target protein for arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes, is highly homologous to mim-1 protein (myb-induced myeloid protein-1). FEBS Lett. 311,203-205. Yamada, K., Tsuchiya, M., Nishikori, Y., and Shimoyama, M. (1994). Automodification of arginine-specific ADP-ribosyltransferase purified from chicken peripheral heterophils and alteration of the transferase activity. Arch. Biochem. Biopbys. 308, 31-36. Yost, D. A., and Moss, J. (1983). Amino acid-specific ADP-ribosylation. Evidence for two distinct NAD:arginine ADP-ribosyltransferases in turkey erythrocytes. J. Biol. Cbem. 258,4926-4929. Zolkiewska, A., Nightingale, M. S., and Moss, J. (1992).Molecular characterization of NAD: arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc. Nutl. Acud. Sci. U.S.A. 89, 11352-11356. Zolkiewska, A., and Moss,J. (1993).Integrin a 7 as substrate for a glycosylphosphatidylinositolanchored ADP-ribosyltransferase on the surface of skeletal muscle cells. J. Biol. Cbem. 268,25273-25276. Zolkiewska, A., and Moss, J. (1995). Processing of ADP-ribosylated integrin a 7 in skeletal muscle myotubes. J. Biol. Chem. 270, 9227-9233.

Samuel R. Denmeade" John T. Isaacs*J The John Hopkins University School of Medicine Department of Oncology* and Urology,+ Baltimore, Maryland 2 I23 I

Activation of Programmed (Apoptotic) Cell Death for the Treatment of Prostate Cancer

1. Overview of the Problem Over the last decade, prostate cancer has become the most commonly diagnosed cancer in American men. In 1995, more cases of prostate cancer (244,000) are expected in men than lung cancer (96,000) and colorectal cancer (70,000)combined (Wingo et al., 1995).These high annual incidence rates translate into the human reality that one in every six American men will be diagnosed with prostate cancer during their lifetime (Wingo et al., 1995).Of all cancers, the incidence of prostate cancer increases most rapidly with age. The average age at diagnosis is 72 with 80% of cases being diagnosed in men over 65 (Carter and Isaacs, 1988). As Americans live longer, there continues to be a corresponding rise in the number of prostate cancers with an expected 60% increase this decade alone (Wingo et al., 1995).Due to detection at an earlier age, there has also been a rapid increase Advances in Pharmacology, Volume 35 Copyright 8 1996 by Academic Press, Inc. All rights of reprodualon in any form reserved.

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in men diagnosed under the age of 65. In this age group alone more cases of prostate cancer are expected than the combined number of leukemias, Hodgkin’s disease, melanomas, and brain tumors in men of all ages. Mortality from prostate cancer has increased at a slower rate but overall has doubled in the last 50 years (Wingo et al., 1995). Although typically diagnosed in men over the age of 65, the impact of the disease is still significant in that the average lifespan of a man who dies from prostate cancer is reduced by 9 to 10 years (Horm and Sondik, 1989). In 1995, 41,000 American men are expected to die from prostate cancer makinig it the second leading cause of cancer deaths in the United States (Wingo et al., 1995). The relative incidence of prostate cancer continues to be approximately twice as high in African-American men than in whites (Wingo et al., 1995). An increase has also been observed in white men but this is thought to reflect an increase in screening and early detection of small cancers. On the contrary, African-American men are diagnosed more often with advanced disease at an earlier age and have an overall lower survival rate for all stages of disease. The reasons for this are not entirely clear and seem to be more than secondary to socioeconomic factors. What is clear is that the mortality rate of African-American men from prostate cancer remains the highest in the world. II. Androgen Sensitivity of Prostate Cancer If discovered early when it is still localized within the gland, prostate cancer can be cured surgically via a radical prostatectomy or with localized radiation therapy in approximately 90% of cases. Improved techniques have significantly decreased the complications associated with both of these procedures. Unfortunately, once the cancer metastasizes outside of the prostate, the disease is uniformly fatal since no effective curative systemic therapy currently exists. Since the work of Charles Huggins in the 1940s, it has been known that prostate cancer often retains an androgen responsiveness for stimulation of its growth. Prostate cancer is thus often highly responsive to androgen ablation therapy. Based on this, patients with nonorgan confined disease eventually require systemic androgen ablation therapy. Nearly all men with metastatic prostate cancer treated with surgically or chemically induced castration have an initial, often dramatic, beneficial response to such androgen withdrawal therapy (The Leuprolide Study Group, 1984; Crawford et al., 1989). While this initial response is of substantial palliative value, essentially all treated patients eventually relapse to an androgen-insensitive state and succumb to the progression of their cancer unless they die of intercurrent disease first; cures, if any, are rare (Lepor et al., 1982; Crawford et al,, 1989). Because of this nearly universal relapse

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phenomenon, the annual death rate from prostate cancer has not decreased at all over the subsequent SO years since androgen withdrawal has become standard therapy (Wingo et al., 1995). Over the last 50 years, the superficially benign nature of androgen withdrawal therapy has tended to disguise the fact that metastatic prostate cancer is still a fatal disease for which no therapy is available which effectively increases survival (Lepor et al., 1982; Raghavan, 1988). Studies by a series of laboratories have demonstrated that the reason for this universal relapse of metastatic prostate cancer to androgen ablation is that prostate cancer within an individual patient is heterogeneously composed of clones of both androgen-dependent and -independent cancer cells even before hormone therapy is begun (Sinha et al., 1977; Prout et al., 1976; Isaacs and Coffey, 1981). Development of such tumor cells heterogeneity can occur by a variety of mechanisms [e.g., multifocal origin of the tumor, adaptation, or genetic instability (Isaacs, 1989)]. Regardless of the mechanism of development of such cellular heterogeneity, once androgenindependent cancer cells are present within individual prostatic cancer patients, the patient is no longer curable by androgen withdrawal therapy alone, no matter how complete, since this therapy kills only the androgendependent cells without eliminating preexisting androgen-independent prostatic cancer cells. To effect all the heterogenous prostatic cancer cell populations within an individual cancer, effective chemotherapy, specifically targeted against the androgen-independent cancer cell, must be simultaneously combined with androgen ablation to affect the androgen-dependent cells (Isaacs, 1984a, 1989). While the concept of early combinational chemohormonal therapy for prostate cancer is valid, for such an approach to be therapeutically effective in humans, a chemotherapeutic agent which can effectivelycontrol the growth of the preexisting androgen-independent prostatic cancer cells must be available. There are presently no highly effective chemotherapeutic agents which can control the growth of androgenindependent prostate cancer cells (Raghavan, 1988). The inability to control androgen-independent prostate cancer cells in human and rodent tumors by standard chemotherapeutic methods has lead to a search for new approaches. 111. Cell Kinetics during Progression of Prostate Cancer

-

Growth of a cancer is determined by the relationship between the rate of cell proliferation and the rate of cell death. Only when the rate of cell proliferation is greater than cell death does tumor growth continue. If the rate of cell proliferation is lower than the rate of cell death, then regression of the cancer occurs. Metastatic prostate cancers, like the normal prostates from which they arise, are sensitive to androgenic stimulation of their

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growth. This is due to the presence of androgen-dependent prostatic cancer cells within such metastatic patients. These cells are androgen dependent since androgen stimulates their daily rate of cell proliferation (i.e., Kp) while inhibiting their daily rate of death (i.e., Kd) (Isaacs et al., 1992). In the presence of adequate androgen, continuous net growth of these dependent cells occurs since their rate of proliferation exceeds their rate of death. In contrast, following androgen ablation, androgen-dependent prostatic cancer cells stop proliferating and activate a cellular suicide pathway, termed programmed cell death or apoptosis (Isaacs et d.,1992).This activation results in the elimination of these androgen-dependent prostatic cancer cells from the patient since under these conditions their death rate value now exceeds their rate of proliferation. Due to this elimination, 80 to 90% of all men with metastatic prostatic cancer treated with androgen ablation therapy have an initial positive response. All of these patients relapse eventually to a state unresponsive to further antiandrogen therapy, no matter how completely given (Crawford et al., 1989). This is due to the heterogeneous presence of androgen-independent prostatic cancer cells within such metastatic patients. These latter cells are androgen independent since their rate of proliferation exceeds their rate of cell death even after complete androgen blockage is performed (Isaacs, 1982). Attempts to use nonandrogen ablative chemotherapeutic agents to adjust the kinetic parameters of these androgen-independent prostatic cancer cells so that their rate of death exceeds their rate of proliferation have been remarkable in their lack of success (Raghavan, 1988). The agents tested in patients failing androgen ablation have been targeted at inducing DNA damage directly or indirectly via inhibition of DNA metabolism or repair. These agents are thus critically dependent on an adequate rate of proliferation to be cytotoxic (Shackney et al., 1978). In Vitro cell culture studies have demonstrated that when androgen-independent, metastatic, prostatic cancer cells are rapidly proliferating (i.e., high Kp value); these cells are highly sensitive to the induction of programmed cell death via exposure to the same antiproliferative chemotherapeutic agent which are of limited value when used in vivo in prostatic cancer patients (Isaacs and Lundmo, 1992). The paradox between the in vitro and in vivo responsiveness to the same chemotherapeutic agents by androgen-independent prostatic cancer cells is due to major differences in the rate of proliferation occurring in the two states. Likewise, for chemotherapeutic agents to be effective, not only must the cancer cells have a critical rate of proliferation but also a critical sensitivity to induction of cell death (Tubiana and Malaise, 1976). The sensitivity to induction of cell death is reflected in the magnitude of the rate of cell death in the untreated condition. The daily rates of cell proliferation (i.e., Kp) and cell death (i.e., Kd) were determined for normal, premalignant, and cancerous prostatic cells within the prostate as well as for prostatic cancer cells in lymph node, soft

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tissue and bone metastases from untreated and hormonally failing patients (Bergeset al., 1995).These data demonstrate that normal prostatic glandular cells have an extremely low (Le., <0.20% per day), but balanced, rate of cell proliferation and death producing a turnover time of 500 2 79 day for these cells (Table I). Initial transformation of these cells into high grade intraepithelial neoplasia (PIN), the lesion believed to be precursor for prostate cancer, results in an increased Kp value with no change in the Kd value. As these early lesions continue to grow into late stage high grade PIN, their Kd increases to a point equaling Kp. This results in cessation of net growth while inducing a 6-fold increase in the turnover time (i.e., 56 2 12 days) of these cells increasing their risk of further genetic changes. The transition of late stage high grade PIN cells into growing localized prostatic cancer cells involves no further increase in Kp but is due to a decrease in Kd resulting in a mean doubling time of 479 2 56 days. Metastatic prostatic cancer cells within lymph nodes of untreated patients have a 100% increase in their Kp and 40% decrease in their Kd values as compared to localized prostatic cancer cells producing a mean doubling time of 33 2 4 days. Metastatic prostatic cancer cells in the bony untreated patients have a 36% increase in Kp and a 50% decrease in Kd, resulting in a mean doubling time of 54 5 5 days. In hormonally failing patients, there is no further change in Kp. An increase in the Kd for androgen-independent prostatic cancer cells is observed within soft tissue or bone metastases with resulting mean doubling times of 126 2 21 and 94 -f 15 days, respectively, in these metastatic sites. These data demonstrate that the proliferation rate for androgenindependent metastatic prostatic cancer cells is very low (i.e., < 3.0%/day), explaining why antiproliferative chemotherapy has been of such limited value against metastatic prostatic cells. Based on this realization, what is needed is some type of cytotoxic therapy which induces the death of androgen-independent prostate cancer cells without requiring the cells to proliferate. IV. Proliferation-Independent Therapeutic Approaches for Androgen-Independent Proliferate Cancer Cells There are at least three cell proliferation independent methods to increasing the death rates of androgen-independent prostatic cancer cells. The first approach is to stimulate the host immune system to evokelor enhance a cytotoxic antitumor response since immune killing of target cells does not require the target cells to proliferate. The second approach is to block the host development of tumor blood supply. Both the growth and metastatic ability of cancers are critically dependent on the ability of the cancer cells to induce the development of new blood vessels (i.e., termed angiogenesis)

TABLE I Kinetic Parameters of Normal and Neoplastic Prostatic Epithelial Cells

Cell type

Percent of cells Histological grade Proliferationlday (Kp) Dyinglday (Kd) Doubling time (Days) (Gleason sum)

Normal prostatic Glandular epithelial cells ( n = 27)

-

0.19 t 0.03‘ [0.05-0.301”

0.20 t 0.03‘ [0.05-0.351

High grade prostatic intraepithelial neoplastic (PIN) cells Early (n = 10)

-

1.25 ? 0.30’ [0.25-2.601 1.80 & 0.28’ [0.20-2.501

0.80 ? 0.24b,‘ [0.20-0.801 1.80 t 0.16’~‘ 0.20-4.961

154 ? 22

5.3 2 0.3 [4-61 7.9 2 0.4 [7-101

1.54 ? 0.17’ [0.40-2.601 1.46 ? 0.20’ [0.80-2.851

1.42 2 0.20’ [0.25-5.251 1.32 2 0.18’ [0.10-3.101

577 -t 68

8.1 t 0.5 [6-101 8.2 2 0.20 [6-101

2.90 2 0.30’.‘ [0.45-8.101 2.04 2 0.29’.‘ [0.1-2.851

0.85 ? 0.21’ [0.15-1.301 0.76 t 0.16’.‘ [0.10-3.601

33 t 4

10

2.77 t 0.31’,‘ [0.65-3.901 2.42 t 0.40’8‘ [0.10-4.601

2.22 ? 0.40’2‘ [1.50-3.651 1.68 ? 0.38’ [2.10-7.751

126 t 21

Late ( n = 10) Localized prostatic cancer cells within the prostate low Gleason sum (>6) ( n = 15) high Gleason sum (>6) ( n = 12) Metastatic prostatic cancer cells from hormonally untreated patients Within lymph node ( n = 30) Within the bone ( n = 13) From Hormonally failing patients Within distinct nonbone sites ( n = 5 ) Within the bone ( n = 10)

a

Values in brackets are the range for particular cell type.

’p < 0.05 compared to normal prostatic glandular epithelial cells. p < 0.05 compared to primary prostatic cancer cells.

-

[lo1 9.2 ? 0.3 [7-101

-

-

495 ? 56

54

5

5

94 t 15

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(Folkman, 1990).If successful, such an antiangiogenic approach would limit the growth of androgen-independent prostatic cancer via hypoxia-induced tumor cell death. Indeed, linomide is an orally active agent which in preclinical animal models inhibits both the development of tumor blood vessels and thus tumor blood flow (Vukanovic et al., 1993). Due to its antiangiogenic ability, linomide treatment inhibits both the growth of primary prostate cancers and also the establishment and growth of metastatic lesions (Ichikawa et al., 1992; Vukanovic et al., 1993; Vukanovic and Isaacs, 1995). Using a series of rat prostate cancer sublines which differ widely in their rate of growth, androgen sensitivity, metastatic ability, and degree of morphological differentiation, linomide’s therapeutic effects have been demonstrated to be independent of the growth rate of these cancers (Ichikawa et al., 1992). The third approach is to activate the “programmed cell death” pathway within these cells leading to their suicide. In programmed cell death, specific intracellular signals induce the cell to undergo an active energy-dependent process which does not initially require a change in the plasma membrane permeability (Wyllie et al., 1980). Once initiated, programmed cell death leads to a cascade of biochemical and morphological events that result in the irreversible fragmentation of the genomic DNA and then the cell itself (Wyllie et al., 1980, 1984; Kerr et al., 1972; Umansky et al., 1981). Both androgen-dependent normal prostatic glandular cells and androgendependent prostatic cancer cells can be induced to undergo programmed cell death following androgen ablation, and this death process does not require the cells to be in the proliferative cell cycle (Kyprianou and Isaacs, 1988a; English et al., 1989; Kyprianou et al., 1988, 1990; Furuya et d., 1995). V. Summary of the Temporal Sequences Involved in the Programmed Death of Normal Prostatic Glandular Cells Following Androgen Ablation

The programmed death induced in the prostate by androgen ablation is cell type specific. Only the prostatic glandular epithelial cells and not the basal epithelial cells or stromal cells are androgen dependent and thus undergo programmed cell death following castration (English et al., 1989). These glandular cells constitute approximately 80% of the total cells in the ventral prostate of an intact adult rat and approximately 70% of these glandular cells die by 7 days postcastration (English etal., 1989). Using the ventral prostate of the rat as a model system, the temporal sequence of events involved in the programmed cell death pathway induced by androgen ablation has begun to be defined. In the androgen-maintained ventral pros-

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Samuel R. Denmeade and john

T. lsaacs

tate of an intact adult male rat, the rate of cell death is very low (approximately 2% per day) and this low rate is balanced by an equally low rate of cell proliferation, also 2% per day (Isaacs, 1989). If animals are castrated, the serum testosterone level drops to less than 10% of the intact control value within 2 hr (Kyprianou and Isaacs, 1988a). By 6 hours postcastration the serum testosterone level is only 1.2% of intact control (Kyprianou and Isaacs, 1988a). By 12 to 24 hr following castration, the prostatic dihydrotestosterone (DHT) levels (i.e., the active intracellular androgen in prostatic cells) are only 5% of intact control values. This lowering of prostatic DHT leads to changes in nuclear androgen receptor function (i.e., by 12 hr after castration, androgen receptors are no longer retained in biochemically isolated ventral prostatic nuclei) (Kyprianou and Isaacs, 1988a). These nuclear receptor changes result in a major epigenetic reprogramming within the nonproliferating glandular cell (i.e., cell out of cycle in Go)resulting in the activation phase, termed the D1 phase of the programmed death process (Fig. 1). During this D1-activation phase, certain genes to be described later which were actively transcribed and translated before castration are rapidly turned off while other genes which initially were not actively transcribed and translated are rapidly induced when the program for cell death is activated by castration. The result of this epigenetic reprogramming is that during the D1-activation phase of the programmed death process there is a change in the profile of proteins that are synthesized, which is coupled with an inhibition of glandular cell proliferation (Kyprianou and Isaacs, 1987), a decrease in polyamine levels (Pegg et al., 1970), and increase in intracellular free Ca2+ levels (Kyprianou et al., 1988; Martikainen and Isaacs, 1990). The increase in intracellular free CaZ+occurring following castration is derived from the Epigenetic Reprogramming Protein Chanaes C

B/A

den his tone TNuclease Chromatin Changes

c

A,

Apoptotic Cell Fragmentation Pha&cytosis Of

Apoptotic Bodies E ;5DK2

FIGURE I Revised cell cycle denoting the options of a Go prostatic glandular cell. D1 denotes the period during which new gene and protein expression required for induction the DNA fragmentationperiod (denoted F) occurs as part of the programmed cell death pathway. DZdenotes the period during which the cell itself fragments into apoptotic bodies as part of its programmed death.

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extracellular Ca2+pool (Kyprianou et al., 1988). The mechanism for this induced elevation in intracellular free Ca2+is not fully known. There are indications that enhanced expression of TGFPl mRNA and protein (Kyprianou and Isaacs, 1988b) as well as the receptor for TGFfll (Kyprianou and Isaacs, 1988b) following castration are somehow involved in the elevation in the intracellular free Ca2+(Cai)level (Martikainen and Isaacs, 1990). Once the Cai reaches a critical level, CaZ+-Mg2+-dependent endonucleases present within the nuclei of the prostatic glandular cells are enzymatically activated (Kyprianou et al., 1988). Normally, histone H1 binds to genomic DNA in the linker region between nucleosomes and this binding is involved in packing of the DNA nucleosomes into solenoid structures. Likewise, DNA binding of polyamines, particularly spermine, due to their negative charge is involved in maintaining the spacial constraint of genomic DNA in a compacted form (Snyder, 1989). When the normal content of Histone H1 and polyamines are bound to genomic DNA, the DNA is compacted and is not an efficient substrate for the activated Ca2+-MgZ+ endonucleases (Brune et al., 1991). During this D1-activation phase, there is a decrease in polyamine levels (Pegg et al., 1970) and the nuclear content of histone HI (Chung and Coffey, 1971). During this phase there is also a rise in the expression of the highly acidic [pI-3.5] a-prothymosin (to be described in next section). The combined results of these changes are that the genomic DNA conformation opens up in the linker region between nucleosomes in the glandular cells. This enhances the accessibility of the linker DNA to the activated CaZ+Mg2+-dependentendonuclease. Once this occurs, DNA fragmentation begins at sites located between nucleosomal units (i.e., F-phase of the programmed death process) and cell death is no longer reversible. Recent unpublished studies using inverted pulse-gel electrophoresis have demonstrated the initial DNA fragmentation produces =300-50 kb size DNA pieces. Once formed, these 300-50 kb size pieces are further degraded into nucleosomal size pieces [i.e., >1Kb]. During this F-phase, the nuclear morphology changes (i.e., chromatin condensation with nuclear margination) even though the plasma and lysosoma1 membranes are still intact and mitochondria are still functional (English et al., 1989). During the subsequent portion of the death process, termed the D2 phase, the Ca2+-dependenttissue transglutaminase actively crosslinks various membrane proteins (unpublished data) and cell surface blebbing, nuclear disintegration, and eventually cellular fragmentation into clusters of membrane bound apoptotic bodies occur. Once formed, these apoptotic bodies are rapidly phagocytized by macrophages and/or neighboring epithelial cells (English et al., 1989; Kerr and Searle, 1973).Thus, within 7 to 10 days postcastration 4 3 0 % of the glandular epithelial cells die and are eliminated from the rat prostate (English et al., 1989).

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VI. Prostate Gene Expression during Programmed Cell Death Pathway Induced by Androgen Ablation The total RNA content per ventral prostatic cell decreased more rapidly than the loss of prostatic glandular cells (i.e., based on prostatic DNA content) following castration (Furuya and Isaacs, 1993). Thus, if accurate quantitation of the level of mRNA expression of genes on a per cell basis is to be obtained, correction for this temporal difference in the RNNcell must be performed. Using total RNA extracts, equal amounts of total RNA were loaded per lane onto gels and Northern analysis was used to quantitate the level of expression of a series of genes in the ventral prostate following castration. These results were then corrected for the substantial decrease in the total RNA content per cell occurring during prostatic regression induced by castration to allow the results to be expressed as the level of expression per cell for each gene. For comparison, the results were normalized to the expression per cell of each gene in the ventral prostate from intact (i-e., noncastrated) male rats. The expression of a series of genes are upregulated during the period of programmed death by prostatic glandular cells induced by castration. These genes include c-myc (Quarmby et al., 1987),glutathione S-transferase subunit Ybl (Chang et al., 1987), testosterone repressed prostatic message2 (TRPM-2) (also called sulfated glycoprotein-2) (Montpetit et al., 1986) transforming growth factor-pl (Kyprianou and Isaacs, 1989a) H-ras (Furuya and Isaacs, 1993), calmodulin (Furuya and Isaacs, 1993), a-prothymosin (Furuya and Isaacs, 1993), and tissue transglutaminase (Furuya and Isaacs, 1993). TRPM-2 (Buttyan et al., 1989) calmodulin (Dowd et al., 1991), and tissue transglutaminase (Fesus et al., 1989) previously have been demonstrated to be induced in a variety of other cell types undergoing programmed cell death. Several of the genes (i.e., c-myc, H-ras) previously have been demonstrated to be involved in cell proliferation. Thus, as a comparison, the relative level of expression of these same genes was determined during the androgen-induced proliferation regrowth of the involuted prostate in animals previously castrated 1 week before beginning androgen replacement. Previous studies have demonstrated that between 2 and 3 days postandrogen replacement to 1 week castrated rats the prostatic glandular cells are maximally undergoing DNA synthesis and cell proliferation (Coffeyet al., 1968). These comparative results demonstrate that the expression of c-myc, H-ras, and tissue transglutaminase are enhanced in both prostatic cell death and proliferation (Furuya and Isaacs, 1993). In contrast, the expression of calmodulin (Furuya and Isaacs, 1993) TRPM-2 (Furuya and Isaacs, 1993), TGF& (Furuya and Isaacs, 1993) glutathione S-transferase subunit Ybl (Chang et al., 1987), and a-prothymosin (Furuya and Isaacs, 1993) are enhanced only during prostatic cell death and not prostatic cell proliferation.

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Additional analysis demonstrated that the expression of a series of genes are decreased following castration. For example, the C3 subunit of the prostatein gene (i.e., the major secretory protein of the glandular cells) ornithine decarboxylase (ODC), histone-&, pS3, glucose regulated protein 78, all decrease following castration (Furuya and Isaacs, 1993). In contrast to the decrease in the mRNA expression of these latter genes during programmed cell death in the prostate following castration, the expression of each of these genes is enhanced during the androgen-induced prostatic cell proliferation (Furuya and Isaacs, 1993).

VII. Role of Cell Proliferation in the Prostatic Death Process Induced by Castration Using the terminal ttansferase end-labeling technique of Gavrieli et al. ( 1992) to histological detect prostatic glandular cells undergoing pro-

grammed death and adjusting for the half-life of detection of these dying cells, the percent of glandular cells dying per day via programmed death in the prostate of intact and castrated rats was determined (Bergeset al., 1993). In intact (noncastrated) rats, 1.2% of the glandular cells die per day via programmed death. Within the first day following castration, this percentage increases and between Day 2 and 5 postcastration; -17 to 21% of these glandular cells die per day via programmed death. These results demonstrate that both the normal constitutive and androgen ablation induced elimination of glandular cells in the prostate is due to programmed cell death and not to cellular necrosis. Using standard in vivo 3H-thymidinepulse labeling, the percent of glandular cells entering the S-phase during the period of enhanced prostatic cell death occurring during the first week postcastration was determined. In the prostates of intact, noncastrated, adult male rats there is a low level of glandular cell proliferation. Using the percent of glandular cells in S-phase and the fact that S-phase is of 9 hr duration in these prostatic cells the daily rate of glandular cell proliferation is calculated to be -1.3% per day in intact control rats (Bergeset al., 1995).This calculated daily rate of proliferation is essentially identical to the calculated daily rate of programmed death of prostatic glandular cells which is consistent with the fact that the prostates of these adult, noncastrated, male rats are neither continuously growing nor regressing. Within 1 day following castration, there is an 80% decrease ( p < 0.05) in the percent of glandular cells entering S-phase. By 4 days following castration, there is more than a 90% reduction in this value. Comparing the data demonstrates that more than 98% of prostatic glandular cells die following castration without entering the proliferative cell cycle. These results confirm the previous studies by Stiens and Helpap (1981) and

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Evans and Chandler (1987) which likewise demonstrated a decrease in the percentage of prostatic glandular cells in S-phase following castration. Since double stranded fragmentation of genomic DNA is induced during programmed cell death, this raises the issue of whether DNA repair is activated during the process and whether such a futile process is required for Gocell killing. In order to detect DNA repair in prostatic glandular cells, a high dose/long exposure bromodeoxyuridine (BrdU)labeling method was used. Instead of pulse labeling animals with a short exposure to a small dose of 3H-thymidine,animals were given 50 mgkg of BrdU and 6 hr later prostates were harvested for immunohistological detection of BrdU labeled prostatic glandular cells. This is a total dose of -42 pmoles of BrdU per rat which is 3360 times higher than the nucleotide precursor dose used in the 3H-thymidine studies reported. When animals are injected with such a high dose of BrdU, incorporation of the nucleotide precursor is not limited to the first 30 min but continues for several hours following injection (Berges et al., 1995). The use of such a high dose of BrdU and such a long period of exposure (i.e., 6 hr) before harvesting prostatic tissue coupled with the use of the highly sensitive immunocytochemicaldetection of BrdU maximizes the possibility of detecting both scheduled S-phase DNA synthesis and unscheduled DNA repair (Berges et al., 1995). The percent of glandular cells incorporating BrdU into DNA was determined on prostatic tissue removed 6 hr after IP injection of 50 mgkg of BrdU at various times following castration. These data demonstrate that there is a 3- to 4-fold increase in BrdU labeling by Day 2 postcastration. By Day 3 postcastration, there is a >lO-fold increase which peaks at a >20fold increase in BrdU labeling on Day 4 postcastration before decreasing on Day 5 postcastration. The distinguishing feature between scheduled Sphase specific DNA synthesis and unscheduled DNA repair is that during S-phase DNA synthesis there is a net accumulation of nuclear DNA content (i.e., cells have > diploid content of DNA). In contrast, during Go DNA repair, no net accumulation occurs and the cells have a diploid content of DNA. To confirm that the majority of BrdU incorporation occurred as part of a futile Go DNA repair process and not S-phase specific DNA synthesis, flow cytometry was used to sort the prostatic cells from 3 day castrated rats which have incorporated BrdU. These BrdU positively labeled prostatic cells were then propidium iodide stained and analyzed by flow cytometry for their DNA content. These studies demonstrated that 82.5 ? 6.9% of these BrdU positive prostatic cells had a diploid (Go)compliment of DNA (Berges et al., 1995). The original single cell suspension used for these analysis included prostatic stromal cells, basal epithelial cells, and intraepithelial macrophages in addition to glandular cells. Previous studies have demonstrated that these former cell types are not androgen dependent and that they continue to enter the S-phase following castration (English et af., 1985). In addition,

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Evans and Chandler (1987)demonstrated that between 2 and 3 days postcastration, there is a major increase in the proliferation of the intraepithelial macrophages. The continuing proliferation of these androgen-independent prostatic cells following castration should be detectable in this analysis. Thus the detection of -20% of the BrdU positively labeled cells having an increased compliment of DNA characteristic of S-phase cells is not unexpected. Regardless of the nature of these S-phase cells, the fact that >80% of the BrdU positively labeled cells on Day 3 postcastration have a diploid content of DNA demonstrates that the vast majority of BrdU incorporation into prostatic glandular cells following castration is due to GoDNA repair and not to entrance into S-phase.

VIII. Androgen Ablation Induced Programmed Cell Death Does not Require Recruitment into a Perturbed Cell Cycle

The previous data demonstrate that during the programmed death of the prostatic glandular cells activated by castration DNA fragmentation occurs which induces a futile process of DNA repair while these cells are in Go. This raises the issue of whether such a fertile Go DNA repair process is associated with but not causally required for prostatic cell death. To resolve this issue, rats were injected IP with 500 mg of hydroxyurea (HU)/ kg every 8 hr for 5 days. This dose of U was chosen based on previous work demonstrating that this treatment inhibited both prostatic S-phase specific DNA synthesis and unscheduled GoDNA repair by more than 90% for 8 hr following an IP injection (Berges et al., 1995). When intact male rats were treated with this tri-daily HU regimen for 1 week, there was no indication of an increase in programmed cell death in the prostate based on the lack of an increase in morphologically detectable apoptotic bodies or terminal transferase end-labeled cells, or loss of DNA content. Based on these combined results, it is clear that tri-daily treatment with 500 mg of HU/kg inhibits by at least 90% both scheduled S-phase DNA synthesis and unscheduled Go DNA repair without itself inducing programmed cell death in the prostate. Therefore, rats were castrated and either injected IP with 500 mg of H U k g every 8 hr or injected with the saline vehicle every 8 hr as a control. After 5 days of castration, the DNA content was reduced 51 2 2% in the animals untreated with HU vs 47 2 3% in castrated rats receiving the tri-daily HU treatment (Berges et al., 1995). Histological analysis also demonstrated an identical atrophic morphology for the prostates from both groups and an identical frequency of glandular cell detected as apoptotic bodies in both groups of prostates. These data demonstrate that the programmed death of prostatic glandular cells induced

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by androgen ablation does not require either progression through S-phase or Go DNA repair. To determine whether androgen ablation induced PCD of prostatic glandular cells involves recruitment of nonproliferating cells into early portion of G1 of a perturbed proliferative cell cycle, rat ventral prostates were assessed temporally following castration for several stereotypical molecular stigmata of entry into the proliferative cell cycle (Furuya et al., 1995). Northern blot analysis was used to assess levels of transcripts from genes characteristically activated: (1)during the transition from quiescence (Go) into G1 of the proliferative cell cycle (cyclin D1, and cyclin C); (2) during the transition from G1 to S (cyclin E, cdk2, thymidine kinase, and H4 histone); and (3) during progression through S (cyclin A). While levels of each of these transcripts increased as expected in prostatic glandular cells stimulated to proliferate by administration of exogenous androgen to previously castrated rats, levels of the same transcripts decreased in prostatic glandular cells induced to undergo PCD following androgen withdrawal (Furuya et al., 1995). Likewise, androgen ablation induced PCD of prostatic glandular cells was not accompanied by retinoblastoma (Rb) protein phosphorylation characteristic of progression from Gl to S. This is consistent with a decrease in the number of cells entering S cells using 3H-thymidine radioautography. Nuclear run on assays demonstrated that there is no increase in the prostatic rate of transcription of the c-myc and c-fos genes following castration. Northern and Western blot analysis also demonstrated that there is no increase in the prostatic p53 mRNA or protein content per cell following androgen ablation. Likewise, following castration there is no enhanced prostatic expression of the WAFl/CIPl gene, a gene whose expression is known to be induced by either increased p53 protein levels or entrance into G1 (Furuya et al., 1995). These results demonstrate that prostatic glandular cells undergo PCD in Go without recruitment into GI phase of a defective cell cycle and that an increase in p53 protein or its function are not involved in this death process (Berges et al., 1995; Furuya et al., 1995).

IX. p53 Expression is not Required for Androgen Ablation-Induced Programmed Death of Go Prostatic Glandular Cells In order to investigate the possible role of the p.53 gene in the programmed cell death pathway induced by androgen ablation, the extent of programmed death of androgen-dependent cells in the prostate and seminal vesicles following castration was compared between wild type and p.53 deficient mice. The mutant mice were established using homologous recombination to produce null mutation in both of the p.53 alleles (Lowe et al.,

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1993). These homozygous null mutations prevent any production of p.53 protein in these mice (Lowe et al., 1993). Wild type (i.e., p.53 expressing) mice and p.53 deficient mice were castrated and after 10 days the animals were killed and their seminal vesicles and prostates removed, weighed, and DNA content determined. Histological sections were also prepared from each of these tissues. These analyses demonstrated that there is an identical decrease in the wet weight and DNA content in both the seminal vesicles and prostate from wild type and p53 deficient mice (Berges et al., 1995). Likewise, histological analysis, demonstrated an identical degree of cellular regression in these tissues in the two types of mice (i.e., similar percent of terminal transferase end-labeled prostatic glandular cells in the two groups of animals). These studies demonstrate that androgen ablation induced programmed death of androgen dependent cells does not require any involvement of p53 protein expression.

X. Redefining the Prostate “Cell Cycle” With the realization of the importance of programmed cell death, the older idea that prostatic cell number is determined by the proliferative cell cycle alone has been modified. Based on this modification, a redefined “cell cycle” has been proposed (Bergesand Isaacs, 1993). The overall “cell cycle” controlling cell number is thus composed of a multicompartment system in which the prostatic glandular cell has at least three possible options (Fig. 1).The cell can be: (1)metabolically active but not undergoing either proliferation or death (i.e., Go cell); ( 2 ) undergoing cell proliferation (i.e., Go + GI -+ S + G2 + mitosis); or (3) undergoing cell death by either the programmed pathway (i.e., Go+ D1+ F + Dz + apoptotic cellular fragmentation) or by nonprogrammed (i.e., necrotic) pathway.

XI. Therapeutic Implication of Programmed Cell Death for Prostatic Cancer

Using the human PC-82 prostatic xenograft system as a model, Kyprianou et al. (1990)demonstrated that androgen ablation activates the pathway of programmed cell death, not only in normal androgen-dependent prostatic cells, but also in androgen-dependent human prostatic cancer cells. Using bromodeoxyuridine incorporation into DNA to label human PC-82 prostatic cancer cells undergoing entrance into the S-phase of the proliferative cell cycle, we have recently demonstrated that within 1 day following castration the number of PC-82 prostatic cancer cells entering the S phase declined from 8 to 10% to one-third these initial values (i.e., to a value of 2 to 3%) and that after 2 days, the proliferative activity declined to below 1%

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(unpublished data). The combination of these latter two studies demonstrated that the programmed death of androgen-dependent human prostatic cancer cells induced by androgen ablation does not require these cells to go through a defective cell proliferation cycle but rather that these cells die without leaving Go. Additional studies have demonstrated that androgen ablation does not induce this programmed death process in androgen-independent prostatic cancer cells due to a defect in the initiation step (Kyprianou and Isaacs, 1989b). Even with this defect, however, androgen-independent prostatic cancer cells retain the basic cellular machinery to undergo this programmed cell death pathway. This was demonstrated by using a variety of chemotherapeutic agents which arrest proliferating androgen-independent prostatic cancer cells in various phases of the proliferative cell cycle (e.g., GI, s, or G2) and which subsequently induce their programmed (i.e., apoptotic) death (Kyprianou and Isaacs, 1989b). One explanation for the inability of androgen ablation to induce programmed death of androgen-independent prostatic cancer cells is that such ablation does not induce a sustained elevation in the intracellular free Ca2+(Cai)levels in these cells. To test this possibility, androgen-independent, highly metastatic Dunning R-3327 AT-3 rat prostatic cancer cells were chronically exposed in vitro to varying concentrations of the calcium ionophore ionomycin to sustain various levels of elevation in the their Cai (Martikainen et al., 1991). These studies demonstrated that an elevation of Cai from a starting value of 35 nM to a value as small as only 3-fold above baseline (i.e., 100 nM) while not inducing immediate toxicity (i.e., death within 5 hours) can induce the death of the cells if sustained for > 12 hr. Temporal analysis demonstrated that elevation in Cai results in these cells arresting in Go within 6 to 12 hr following ionomycin exposure. Over the next 24 hr, these cells begin to fragment their genomic DNA initially into 300-50 Kb size pieces which are further degraded into nucleosome-sized pieces and during the next 24 to 48 hr these cells undergo cellular fragmentation in apoptotic bodies (Martikainen et al., 1991). Associated with this programmed cell death is an epigenetic reprogramming of the cell in which the expression of a series of genes (to be presented later) is specifically modified. These results demonstrate that even nonproliferating androgen-independent prostatic cancer cells can be induced to undergo programmed cell death if a modest elevation in the intracellular free Ca2+is sustained for a sufficient time. The combination of these latter ionomycin data with the chemotherapy data demonstrates that programmed death of androgen-independent prostatic cancer cells can be induced in any phase of the cell cycle and does not necessarily require progression through the proliferation cell cycle (i.e., proliferation independent).

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XII. Ability of Thapsigargin (TG) to Activate Programmed Cell Death Thapsigargin (Fig. 2) is sesquiterpene y-lactone isolated from the root of the umbelliferous plant, Thapsia garganica. Resin from this plant was used starting about 300 BC as a medicine for rheumatic pains by the Greeks. The resin is a skin irritant and has been used in traditional Arabian medicine for centuries (Christensen et al., 1993a). The active principles from the plant, a hexaoxygenated 6,7-guaianolide, was isolated in pure form and termed Thapsigargin (TG) in 1978 by the group of S. Brogger Christensen (Rasmussen et al., 1978). One of the mechanisms for its skin irritant effects is via its ability to induce mast cells to release histamine. A series of studies by the group of Christensen have demonstrated that this ability is due to an extracellular Ca” dependent effect which mimics the ability of calcium ionophores to induced mast cells to release histamine (Christensen et al., 1993b). Recent studies have demonstrated that the Ca2+dependence for TG effects is due to the fact that this highly lipophilic ,agent enters cells and interacts with the Ca2+-ATpasepresent in the endoplasmic reticulum (ER) and inhibits its enzymatic activity with an ICsovalue of =30 nM (Thastrup et al., 1990). Such inhibition is not only efficient but also highly specific since neither the plasma membrane nor red blood cell Ca2+ATpases are inhibited by TG even at p M concentrations (Thastrup et al., 1990). Large pools of bound calcium are sequestered in the ER of cells even though the free intracellular Ca2+(Ca,)concentration is only 30 to 40 nM. This sequestered pool of bound CaZ+can be specific and transiently liberated to elevate the Ca, level from 30 to 40 nM to several hundred nM by a variety of intracellular signals. The best characterized of these signals is the production

0

14 O

V C H 3

.I0

FIGURE 2

0

Structure of the sesquiterpene y-lactone, Thapsigargin.

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from the inositol phospholipids of inositol 1,4,5 triphosphate ( IP3). IP3 binding to its specific receptor in the ER results in the release of the sequestered Ca2+and an elevation in the Cai. The elevation in Ca; is usually transitory, however, since the elevated Ca2+is rapidly pumped either out of cell via plasma membrane Ca2+ATpase pumps or back into the ER via its Ca2+ATpase pump. Recently, however, it has been demonstrated that the sequestered Ca2+in the ER is constantly “leaking” out into the cytoplasm of the cell and that the ER-Ca2+ATpase is constantly pumping this free Ca2+ back into the sequestered stores of the ER (Thastrup et al., 1990). Thus, when the cell permeable TG inhibits the ER-Ca2+ATpase pump, the leaking Ca2+from the ER is no longer pumped back into a sequestered form resulting in the 3- to 4-fold elevation of the Cai without any requirement for IP3 production. Such a primary elevation of Cai leads to a depletion of the ER CaZ+pool and, in many cell types, this results in a signal being generated which induces a change in the permeability of the plasma membrane to extracellular divalent cations, particularly Ca2+.For many cell types this initial intracellular discharge of the ER sequestered calcium pools leads to a Ca2+influx, in keeping with the prediction from the capacitance model of Ca2+entry (Lytton et al., 1991). Thus, once these changes in the plasma membrane occurs, a Ca2+influx into the cell occurs due to the high free Ca2+ concentration extracellularly (i.e., 1 to 3 mM) (Thastrup et al., 1990). This produces a secondary elevation in the Cai which is sustainable (i.e., min-hours) if the TG inhibition is maintained (Thastrup et al., 1990). Based on this background, the ability of TG to sustain an elevation in the Cai and to activate programmed cell death in androgen-independent prostate cancer cells was tested. Initially, in vitro testing was performed on a series of androgenindependent prostatic cancer cell lines of rat (i.e., AT-3 cells) and human (i.e., TSU-pr, DU-145, and PC-3) origin. Microsomes from each of these four distinct cancer cell lines are assayed for their CaZ+ATpase activity. These studies demonstrated that each of the lines possessed ER Ca2-ATpase activity with a specific activity ranging between 12 to 40 nmoles of ATP hydrolyzed per min per mg of ER protein. Coincubation of 500 nM TG with the microsomal preparation with the assay reagents resulted in 2 95% inhibition of the ER-Ca2+ATpase activity of each of the cell lines (Furuya et al., 1994). Based on these results, each of these four cell lines was chronically exposed to 500 nM TG. Using Fura-2 fluorescence ratio measurements, such Thapsigargin treatment resulted in a 2- to 3-fold elevation in the Cai levels from baseline values within 1 to 2 min of initial exposure. This elevation is only transient (i.e., returning to baseline by 5 min of TG exposure) if the cell culture media contained 5 mM EDTA to deplete the extracellular free Ca2+level. In contrast, if no EDTA is present so that Cailevel in the extracellular media is >1 mM, then the response to TG treatment is a sustained (i.e., >24 hr) 2- to 3-fold elevation in the Ca,. These studies demonstrated that

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for all of the 4 cell lines tested, 500 nM TG treatment resulted in a sustained (i.e., > 24 hr) 2- to 3-fold elevation in Cai and that the major source of the calcium for this effect is extracellular (Furuya et al., 1994). Using 2 parameter flow cytometric analysis based on DNA content and nucleus size, 500 nM TG treatment of each of the 4 distinct androgen independent prostatic cancer cell lines was found to arrest these cells in the GdG, phase of the cell cycle. This GdGl arrest was complete by 24 hr of continuous 500 nM TG exposure. Cells from each of the 4 prostatic cancer lines were incubated with 14C-thymidineto uniformally prelabel their DNA. Prelabeled cells were then treated with 500 nM TG and the percentage of the cells undergoing DNA fragmentation (i.e., to sizes 5 300 Kb) at various times of TG treatment was quantitated using inverted pulse gel electrophoresis. These results demonstrated that after a 24 hr lag period the cells begin to fragment their DNA and that by =96 hr of treatment 2 95% of the cells have fragmented their DNA regardless of cell line tested (Furuya et al., 1994). Quantitative analysis of the DNA demonstrated the characteristic, nucleosomal ladder pattern of fragmentation, which characterizes programmed cell death. The temporal pattern of DNA fragmentation was tightly correlated with the loss of clonogenic ability by the cells for each of the 4 cell lines (i.e., 72 hr of TG treatment required for 50% of the cells to fragment their DNA and 50% loss of their clonogenic ability) (Furuya et al., 1994). In contrast to the high temporal correlation between DNA fragmentation and loss of clonogenic ability, there was more than a 24 to 48 hr lag period between the time required for 50% of the cells to fragment their DNA and lose clonogenic ability and the time required for 50% of the cells to lose their cellular viability based on plasma membrane integrity measurement either by time-lapse by videomicroscopy or trypan blue extrusion. These time-lapse videomicroscopy studies did demonstrate, however, that morphological changes begin occurring within 3 to 6 hr of initial TG exposure. These changes initially involve round-up of cells. By 24 hr of TG treatment, cells are smaller in size and rounded in morphology. Between 72 and 120 hr TG treatment, the cells undergo a period of plasma membrane hyperactivity characterized by the production of plasma membrane blebbing (Furuya et al., 1994). These surface blebs are highly dynamic, coming and going on the surface and giving the appearance of the membrane boiling previously reported for ionomycin induced programmed cell death of AT3 prostatic cancer cells (Martikainen et al., 1991). These combined results demonstrate that the initiation of DNA fragmentation is occurring in viable nonproliferating (i.e., GdG,) cells from each of the 4 distinct androgen independent prostatic cancer cell lines tested, 24 to 48 hr before these cell lyse and that this DNA fragmentation is not the result of a loss of metabolic viability (i.e., loss of mitochondria1 or plasma membrane function). In contrast, the data are consistent with the initiation of DNA fragmentation as the irreversible commitment step in the TG induced programmed death of

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nonproliferating androgen-independent rodent or human prostatic cancer cells. Analysis of a mRNA expression of the series of genes previously demonstrated to be enhanced during the programmed cell death of normal prostatic cells induced by androgen ablation demonstrated that TG treatment of androgen-independent prostatic cancer cells likewise leads to an epigenetic reprogramming of the cells. AT-3 rat prostatic cancer cells were treated from 0 to 36 hr with either 500 nM TG, 10 p M ionomycin, or 100 p M 5fluordeoxyuridine (5-FudU). Previously, we have demonstrated that prostatic cancer cells must progress through the proliferative cell cycle in order for 5-FudU to induce their programmed cell death (Kyprianou and Isaacs, 1989b).In contrast, TG and ionomycin induce the proliferation independent programmed death of Go cells. These results demonstrate that within 1 hr of either TG or ionomycin treatment expression of several of these genes is already elevated [e.g., a-prothymosine, calmodulin, ornithine decarboxylase (ODC)] and that by 6 hr additional genes expression is enhanced [e.g., glucose-regulated protein-78 (GRP), c-myc]. Many of these enhancements are acute with expression decreasing at 24 hr of treatment. There are major differences in gene expression during the proliferation independent programmed death induced by TG or ionomycin and the proliferation dependent death induced by 5-FudU (e.g. in the latter, c-myc, calmodulin, prothymosine are not induced while H-rus and TWM-2 are induced) (Furuya and Isaacs, 1994). These results demonstrate that the programmed death induced by all of these agents involves an active epigenetic reprogramming of the cell and the pathway induced by TG is essentially identical to that induced by ionomycin, but distinct from that induced by 5-FU.

XIII. Thapsigargin as Therapy for Prostate Cancer Typically, prostate cancer cells have a very low proliferative rate (Berges et ul., 1995) and thus those cancer cells are not susceptible to killing by

standard antiproliferative chemotherapeutic agents. Since thapsigargin can induce programmed cell death in prostate cancer cells while in a nonproliferative, Gostate, this agent could represent a novel approach to the treatment of prostate cancer. However, using TG as a therapeutic agent would be dikicult for two reasons. First, TG is highly lipophilic and rapidly crosses the plasma membrane of cells and would be rapidly absorbed without reaching desirable levels in the target tissue. Secondly, an agent that is capable of killing cells quiescent in Go would be difficult to administer systemically without excessive toxicity since the majority of cells in human tissues are differentiated and nonproliferating. However, if TG could be derivatized to an inactive prodrug form and targeted specifically for activation by prostatic

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30 I

cells it could possibly be useful as a therapeutic agent while avoiding significant systemic toxicity. A unique characteristic of prostate cells, both normal and cancerous, is the secretion of a protein, termed prostate specific antigen (PSA)(Papsidero et al., 1981). PSA is a serine protease (Watt et al., 1986) that has as primary substrates the major secretory proteins of the seminal vesicles (Christensson et al., 1990). These proteins, termed semenogelin I and semenogelin 11, are involved in the formation of a gel that entraps spermatozoa at ejaculation (Lilja et al., 1989). PSA mediates the liquefaction fragmentation of this gel aiding in the activation of sperm motility. Several specific PSA cleavage sites have been described for semenogelin I and I1 (Lilja et al., 1989). By utilizing the peptides proximal to the cleavage sites in semenogelin, a peptide substrate that is highly specific for PSA can be synthesized. In the normal prostate the majority of PSA is secreted through the prostatic ducts into the seminal fluid with only a small amount (54 ng/ml) entering the circulation. In prostate cancer the normal cellular architecture is distorted and PSA levels can become elevated (often > 1000 ng/ml) with local tissue levels expected to be even higher. The PSA that reaches the blood is inactivated by the excess serum alpha 1-antichymotrypsin and alpha 2-macroglobulin (Christensson et al., 1990; Lilja et al., 1991). Therefore, only the PSA secreted locally into the extracellular fluid by prostate tissues, whether normal or cancerous, would be expected to have enzymatic activity. A chemotherapeutic agent, such as thapsigargin, could be coupled to a small peptide representing a specific proteolytic site for PSA (Fig. 3). The prodrug would be inactive in the circulation where PSA is enzymatically inactive. The active drug would only be released from the peptide carrier locally by prostate cells secreting enzymatically active PSA. In this way both primary disease and distant metastases could be specifically targeted. Because most prostate cancers consist of a heterogenous mixture of PSA secreting and nonsecreting cells, this approach has the advantage that it can induce a “bystander effect” to kill the PSA negative cells in the vicinity of the PSA secreting cells (Fig. 3). To create this prodrug, a derivative of TG must be synthesized that can be coupled via an amide bond to specific peptides which can be selectively cleaved only by PSA. Christensen et al. (1993a)have synthesized and characterized a variety of TG analogues and have assessed their inhibitory effect on the ER Ca2+ATPase in a bovine cerebellar microsomal preparation. Selectively altering the side chains of thapsigargin in a variety of positions produces compounds of varying potency (Christensen et al., 1993a). Several analogues with side chains ending in free amines have been synthesized. These analogues are being converted into prodrug forms by coupling their free amine containing side chain with the carboxy terminus of the appropriate PSA hydrolyzable peptides. Thus the delivery of a prodrug able to selectively kill both proliferating and nonproliferating prostate cells by acti-

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FIGURE 3

Overview of Thapsigargin (TG) prodrug induced programmed death of PSA 0 I1

secreting and nonsecreting prostate cancer cells. Inactive TG prodrug, TG -N -C -PSA peptide; PSA, enzymatically active prostate specific antigen; TG-NH2, active tg analog contam0 II

ing an primary amine; and peptide -PSA -C -OH,peptide liberated by PSA catalyzed hy-

drolyzes.

vation of programmed cell death pathways should be possible without inducing generalized host toxicity. Presently, this possibility is being tested in a series of preclinical in vitro and in vivo model systems. References Berges, R. S., Furuya, Y., Remington, L., English, H. F., Jacks, T., and Isaacs, J. T. (1993). Cell proliferation, DNA repair, and p53 function are not required for programmed death of prostatic glandular cells induced by androgen ablation. Proc. Natl. Acad. Sci. U.S.A. 90, 8910-8914. Berges, R., and Isaacs, J. T. (1993). Programming events in the regulation of cell proliferation and death. Clin. Cbem. 39, 2. Berges, R. S., Vukanovic, J., Epstein, J. I., et al. (1995). Implication of cell kinetic changes during the progression of human prostatic cancer. Clin. Cancer Res. 1, 473-480. Brune, B., Hartzell, P., Nicotera, P., and Orrenius, S. (1991). Spermine prevents endonuclease activation and apoptosis in thymocytes. Exp. Cell Res. 195,323. Buttyan, R., Zakeri, Z., Lockshin, R., and Wolgemuth, D. (1988). Cascade induction of c-fos, c-myc and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mol. Endocrinol. 2, 650.

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Buttyan, R., Olsson, C. A., Pintar, J., Chang, C., Bandyk, M., Ng, P-Y., and Sawczuk, I. S. (1989).Induction of the TRPM-2 gene in cells undergoing programmed death. Mol. Cell Biol. 9, 3473. Carter, H. B., and Isaacs, J. T. (1988). Experimental and theoretical basis for hormonal treatment of prostatic cancer. Swin. Urol. 4, 262. Chang, C., Saltzman, A. G., Sorensen, N. S., Hiipakka, R. A., and Liao, S. (1987).Identification of glutathione S-transferase Ybl mRNA as the androgen repressed mRNA by cDNA cloning and sequence analysis. J. Biol. C h w . 262, 11901. Christensen, S. B., Andersen, A., Paulsen, J-C. J., and Treiman, M. (1993a). Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ATPase. FEBS Lett. 335, 345. Christensen, S. B., Norup, E., and Rasmussen, U. (1993b). Chemistry and structure-activity relationship of the histamine secretagogueThapsigragin and related compounds. In “Natural Products and Drug Development” (P. Krogsgaard-Larsen, S. Brogger Christensen, and H. Kofod, Eds.), pp. 405-418. Copenhagen, Denmark: Munksgaard, 1984. Christensson, A., Laurel], C. B., and Lilja, H. (1990).Enzymatic activity of the prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur. J. Biochem. 194, 755. Chung, L. W. K., and Coffey, D. S. (1971). Biochemical characterization of prostatic nuclei I. androgen-induced changes in nuclear proteins. Biochim. Biophys. Acta 247, 570. Coffey, D. S., Shimazaki,J., and Williams-Ashman, H. G. (1968).Polymerization of deoxyribonucleotides in relation to androgen-induced prostatic growth. Arch. Biochem. Biophys. 124, 184. Crawford, E. D., et al. (1989). A control randomized trial of Leuprolide with and without flutamide in prostatic cancer. N . Engl. J. Med. 321,419. Dowd, D. R., MacDonald, P. N., Komm, B. S., Haussler, M. R., and Miesfeld, R. (1991). Evidence for early induction of calmodulin gene expression in lymphocytes undergoing glucocorticoid-mediated apoptosis. J. Biol. Chem. 266, 18423. English, H. F., Drago, J. R., and Santen, R. J. (1985).Cellular response to androgen depletion and repletion in the rat ventral prostate: Autoradiography and morphometric analysis. The Prostate 7 , 41. English, H. F., Kyprianou, N., and Isaacs, J. T. (1989).Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. Prostate 15, 233. Evans, G. S., and Chandler, J. A. (1987). Cell proliferation studies in the rat prostate: II. The effects of castration and androgen replacement upon basal and secretory cell proliferation. Prostate 11, 339. Fesus, L., Thomazy, V., and Falus, A. (1989).Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett. 224, 104. Folkman, J. (1990). What is the evidence that tumors are angiogenesis-dependent? J. Natl. Cancer Inst. 82, 4. Furuya, Y., and Isaacs, J. T. (1993). Differential gene regulation during programmed death ( Apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation.Endocrinology 133, 2660-2666. Furuya, Y., and Isaacs, J. T. (1994).Proliferation-dependent us independent programmed cell death of prostatic cancer cells involves distinct gene regulation. The Prostate 25, 301. Furuya, Y., Lundmo, P., Short, A. D., Gill, D. L., and Isaacs, J. T. (1994).The role of calcium pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res. 54, 6167. Furuya, Y., Walsh, J. C., Lin, X., Nelson, W. G., and Isaacs, J. T. (1995).Androgen ablation induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction. Endocrinology 136, 1898-1906.

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Gavrieli, Y., Sherman, Y.,and Ben-Sasson, S. A. (1992). Identification of Programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493. Helpap, B., Steins, R., and Bruhl, P. (1974).Autoradiographic in vitro investigations on prostatic tissue with C-14 and H-3 thymidine double labeling method. Beitr. Pathol. Anat. Allg. Pathol. 151, 65. Horm, J., and Sondik, E. (1989). Person-years of life lost due to cancer in the United States 1970 and 1984. Am. J. Public Health 79, 1490. Ichikawa, T., Lamb, J. C., Christensson, P. J., Hartley-Asp, B., and Isaacs, J. T. (1992). The antitumor effects of the quinoline-3-carboxamide linomide on Dunning R-3327 rat prostatic cancers. Cancer Res. 52, 3022. Isaacs, J. T. (1981). Cellular factors in the development of resistance to hormonal therapy. In “Drug and Hormone Resistance in Neoplasia I” (N. Bruchovsky, and J. Goldie, (Eds.), pp. 139-156. CRC Press, Boca Raton. Isaacs, J. T. (1982). Hormonally responsive vs unresponsive progression of prostatic cancer to antiandrogen therapy as studied with the Dunning R-3327-AT and G rat prostatic adenocarcinoma. Cancer Res. 42, 5010. Isaacs, J. T. (1984a). The timing of androgen ablation therapy and/or chemotherapy in the treatment of prostatic cancer. Prostate 5, 1. Isaacs, J. T. (1984b). Antagonistic effect of androgen on prostatic cell death. The Prostate 5,545. Isaacs, J. T. (1989). Relationship between tumor size and curability of prostate cancer by combined chemohormonal therapy. Cancer Res. 49, 6290. Isaacs, J. T., and Coffey, D. S. (1981). Adaptation vs selection as the mechanism responsible for the relapse of prostatic cancer to androgen ablation as studies in the Dunning R-3327 H adenocarcinoma. Cancer Res. 41, 5070. Isaacs, J. T., and Lundmo, P. I. (1992). Chemotherapeutic induction of programmed cell death in non proliferating prostate cancer cells. Proc. Am. Assoc. Cancer Res. 33, 588. Isaacs, J. T., Lundmo, P. I., Berges, R.,Martikainen, P., Kyprianou, N., and English, H. F. (1992). Androgen regulation of programmed death of normal and malignant prostatic cells. J. Androl. 13, 457. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R.(1972).Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. BY.J. Cancer 26, 239. Kerr, J. F. R., and Searle, J. (1973). Deletion of cells by apoptosis during castration-induced involution of the rat prostate. Virchows Arch. B Cell. Pathol. 13, 87. Kyprianov, N., and Isaacs, J. T. (1987). Biological significance of measurable androgen levels in the rat ventral prostate following castration. The Prostate 10, 313. Kyprianou, N., and Isaacs, J. T. (1988a).Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122, 552. Kyprianou, N., and Isaacs, J. T. (1988b). Identification of a cellular receptor for transforming growth factor-p in rat ventral prostate and its negative regulation by androgens. Endocrinology 123, 2124. Kyprianou, N., and Isaacs, J. T. (1989a).Thymine-lessdeath in androgen independent prostatic cancer cells. Biochem. Biophys. Res. Commun. 165, 73. Kyprianou, N., and Isaacs, J. T. (1989b). Expression of transforming growth factor-p in the rat ventral prostate during castration induced programmed cell death. Mol. Endocrinol. 3, 1515. Kyprianou, N., English, H. F., and Isaacs, J. T. (1988). Activation of a CaZ+-MgZ+-dependent endonuclease as an early event in castration-induced prostatic cell death. Prostate 13,103. Kyprianov, N., English, H. F., and Isaacs, J. T. (1990).Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation. Cancer Res. 50, 37483753. Lepor, H., Ross, A., and Walsh, P. C. (1982). The influence of hormonal therapy on survival of men with advanced prostatic cancer. J. Urol. 128, 335.

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Lilja, H., Abrahamsson, P-A., and Lundwall, A. (1989).Semenogelin, the predominant protein in human semen. J. Biol. Chem. 264, 1894. Lilja, H., Christensson, C., Dahlen, V., Mukkainen, M.-T., Nilsson, O., Petterson, K., and Luvgren, T. Prostate specific antigen in human serum occurs predominately in complex with a,-antichymotrypsin. Clin. Chem. 37, 618. Lowe, S., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. (1993).p53 is required for radiation induced apoptosis in mouse thymocytes. Nature 362, 847. Lytton, J., Westlin, M., and Haley, M. R. (1991).Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 266, 17067. Martikainen, P., and Isaacs, J. (1990).Role of calcium in the programmed death of rat prostatic glandular cells. The Prostate 17, 175. Martikainen, P., Kyprianou, N., and Isaacs, J. T. (1990). Effects of transforming growth factor-0, on proliferation and death of rat prostatic cells. Endocrinology 127, 2963. Martikainen, P., Kyprianou, N., Tucker, R. W., and Isaacs, J. T. (1991). Programmed death of non-proliferating androgen independent prostatic cancer cells. Cancer Res. 51,4693. Meyers, J. S., Sufrin, G., and Maring, S. A. (1982). Proliferation activity of benign human prostate, prostatic adenocarcinoma and seminal vesicle evaluated by thymidine labeling. J . Urol. 128, 1353. Montpetit, M. L., Lawless, K. R., and Tenniswood, M. (1986).Androgen repressed messages in the rat ventral prostate. Prostate 8, 25. Nemoto, R., Hattori, K., Uchida, K., et al. (1990).S-phase fraction of human prostate adenocarcinoma studies with in vivo bromodeoxyuridine labeling. Cancer 66, 509. Papsidero, L. D., Kuriyama, M., Wang, M. C., et al. (1981). Prostate antigen: A marker for human prostate epithelial cells. J. Natl. Cancer Inst. 66, 37. Pegg, A. E., Lockwood, D. H., and Williams-Ashman, H. G. (1970).Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth. Biochem. J. 117, 17. Prout, G. R., Leiman, B., Daly, J. J., MacLoughlin, R. A., et al. (1976). Endocrine changes after diethylstilbestrol therapy. Urology 7, 148. Quarmby, V. E., Beckman, W. C., Jr., Wilson, E. M., and French, F. S. (1987). Androgen regulation of c-myc messenger ribonucleic acid levels in rat ventral prostate. Mol. Endocrinol. 1, 865. Raghavan, D. (1988).Non-hormone chemotherapy for prostate cancer: principles of treatment and application to the testing of new drugs. Semin. Oncol. 15, 371. Rasmussen, U., Christensen, S. B., and Sandberg, F. (1978).Thapsigargin and thapsigargicin, two new histamine liberators from Thapsia garganica. L. Acta. Pharm. Suec. 15, 133. Sadi, M. V., and Barrack, E. R. (1991).Determination of growth fraction in advanced prostate cancer by Ki-67 immunostaining and its relationship to the time to tumor progression after hormonal therapy. Cancer 67, 3065. Shackney, S. E., McCormack, G. W., and Cuchural, G. J. (1978). Growth rate patterns of solid tumors and their relationship to responsiveness to therapy. Ann. Intern. Med. 89,107. Sinha, A. A., Blackhard, C. E., and U. S. Seal. (1977).A critical analysis of tumor morphology and hormone treatment in the untreated and estrogen treated responsive and refractory human prostatic carcinoma. Cancer 40, 2836. Stiens, R., and Helpap (1981). Regressive changes in the prostate after castration. A study using histology, morphometrics and autoradiography with special reference to apoptosis. Pathol. Res. Pract. 172, 73. Snyder, R. D. (1989). Polyamine depletion is associated with altered chromatin structure in HeLa cells. Biochem. 1. 260, 697. Thastrup, O., Cullen, P. J., Drebak, B. K., Hanley, M. R., and Dawson, A. P. (1990).Thapsigargin, a tumor promoter, discharges intracellular CaZ' stores by specific inhibition of the endoplasmic reticulum CaZ'-ATPase. Proc. Natl. Acad. Sci. U.S.A. 87, 2466.

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The Leuprolide Study Group. (1984). Leuprolide versus diethylstilbestrol for metastatic prostatic cancer. N. Engl. J. Med. 311, 1281-1286. Tubiana, M., and Malaise, E. P. (1976). Growth rate and cell kinetics in human tumors: Some prognostic and therapeutic implications. In “Scientific Foundations of Oncology” (T. Symingtonand R. L. Carter, Eds.), pp. 126-138. Year Book Medical Publishers, Chicago. Umansky, S. R., Korol, B. A., and Nelipovich, P. A. (1981). In vivo DNA degradation in thymocytes of y-irradiated or hydrocortisone-treatedrats. Biocbim. Biopbys. Actu 655,9. Vukanovic, J., Passaniti, A., Hirata, T., et al. (1993). Antiangiogenic effects of the quinoline3-carboxamide linomide. Cancer Res. 53, 1833. Vukanovic, J., and Isaacs, J. T. (1995). Linomide inhibits ongiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers. Cancer Res. 5 5 , 1499. Watt, K. W. K.,Lee, P-J., Timkulu, T.M., Chan, W-P., and Loor, R. (1986). Human prostatespecific antigen: structural and functional similarity with serine proteases. PYOC.Nutl. Acud. Sci. U.S.A. 83, 3166. Wingo, P. A., Tong, T.,and Bolden, S. (1995). Cancer statistics, 1995. CA Cancer J. Clin. 45, 8. Wyllie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D. (1984). Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Putbol. 142, 66. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251. Zimmerman, M., Ashe, B., Yurewicz, E. C., and Patel, G. (1977). Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. A d . Biocbem. 78,47.

Howard N. Hodis University of Southern California School of Medicine Division of Cardiology Los Angeles, California 90033

Reversal of Atherosclerosis with Therapy: Update of Coronary Angiograp hic Trials

1. Overview Evidence that progression of atherosclerosis can be retarded and that atherosclerotic lesions can regress derives from several sources. These sources include studies of arterial lesions obtained at autopsy from starved human populations, animal experimentation, and clinical trials using serial angiography. There is now serial angiographic evidence for atherosclerotic lesion improvement induced by diverse modes of intervention (Blankenhorn and Hodis, 1994). The weight of evidence indicates that atherosclerotic lesions as well as clinical coronary events are reduced by intervention which supports the current position that the LDL-C level should be aggressively reduced in patients with symptomatic atherosclerotic vascular disease.

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II. Coronary Angiographic Trials Utilizing Pharmacological Intervention A. The NHLBl Type II Coronary Intervention Study

This study was a pioneering trial which included men and women with LDL-C levels exceeding the age-corrected 95th percentile (Brensike et al., 1984). Baseline and 5-year angiograms were obtained on 57 placebo and 59 cholestyramine-treated subjects. Film pairs were evaluated by 3 separate panels of 3 expert angiographers each with film order and treatment assignment masked. Although overall results did not demonstrate a significant therapy effect, subgroup analysis indicated that lesions 250% diameter stenosis ( YoS)at baseline progressed less in the cholestyramine-treated group (12% of subjects) than in the placebo-treated group (33% of subjects) ( p < 0.05). Increasing TC/HDL-C and LDL-C/HDL-C ratios were the best predictors of progression.

B. The Cholesterol-LoweringAtherosclerosis Study (CLAS) This study tested combined colestipol-niacin therapy in nonsmoking men with coronary artery bypass grafts (CABG).Angiograms were obtained in 162 subjects (80 drug group, 82 placebo group) at 2 years (Blankenhorn et al., 1987) and 103 subjects (56drug group, 47 placebo group) at 4 years (Cashin-Hemphill et al., 1990). Films were evaluated by panels of expert angiographers and by computerized image processing (Blankenhorn et al., 1992). Global Change Score (GCS), an overall assessment of angiographic change, showed treatment benefits at 2 years ( p < 0.001) and 4 years ( p < 0.0001). Regression occurred in 16.2% of subjects at 2 years and in 18.0% at 4 years in the drug group compared to 3.6% of subjects at 2 years and 6.4% at 4 years in the placebo group. In drug-treated subjects, average number of progressing lesions in native arteries was less at 2 years ( p < 0.03) and 4 years ( p = 0.0002) as was the number of subjects at 2 years ( p < 0.03) and 4 years ( p = 0.001) with new lesions. Treatment reduced new lesion formation in bypass grafts at 2 years ( p < 0.04) and 4 years ( p = 0.006). With LDL-C reduced below 100 mg/dl, apolipoprotein C-I11 in HDL (an indicator of triglyceride-rich lipoprotein metabolism) became the most significant (inversely related) risk factor for lesion progression in the drug group (Blankenhorn et al., 1990a). This was the first clear indication of the importance of triglyceride-rich lipoproteins in lesion progression-an effect manifested after LDL-C was removed as a risk factor. Although clinical events were not significantly different between drug and placebo groups during the 2 years of intervention, 10 year follow-up indicates a significantly lower clinical coronary event rate in the drug group. Both GCS and QCA

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assessment of progression at 2 years were predictive of these subsequent cardiac events (Azen et al., 1996). As reviewed below, dietary modification alone or in conjunction with other lifestyle changes results in significant improvement in coronary artery disease (CAD). CLAS results are in agreement with these findings. In the CLAS placebo group, 64 subjects who had reduced dietary fat intake also had reduced new lesion formation (Blankenhorn et al., 1990b). Eighteen placebo subjects who made no significant dietary changes except to increase polyunsaturated fat developed new lesions. On trial, lipid levels were not statistically different between the 2 groups indicating that reduction of fat intake may have direct arterial wall benefit. C. The Familial Atherosclerosis Treatment Study (FATS)

This study randomized 146 men less than 62 years of age with elevated apolipoprotein B levels (>125 mg/dl) and a family history of CAD to lovastatin-colestipol, niacin-colestipol, or conventional care (Brown et al., 1990). Angiograms were separated in time by an average 2.5 years and analyzed by QCA. In the control group, 46% of subjects had lesion progression in at least 1 of 9 proximal coronary segments. Progression was less frequent in lovastatin-coIestipo1(21%) and niacin-colestipol(25%) subjects. Regression was more frequent in lovastatin-colestipol (32%) and niacin-colestipol (39%) subjects than in controls (11Y0). Lesions S O % S at baseline showed a preferential response to therapy both in the proximal and all lesion analyses. In the lovastatin-colestipol and niacin-colestipol groups, proximal lesions 2 5 O % S at baseline regressed an average of -3.9%S and -6.5%S, respectively; lesions <5O%S progressed +0.2%S in both groups. Results were similar in the all lesion analysis. Clinical events (death, myocardial infarction, or revascularization) occurred in 10 of 52 control subjects compared to 3 of 46 lovastatin-colestipol and 2 of 48 niacincolestipol subjects. Relative risk, 0.27 (95% confidence interval, 0.10 to 0.77), of a clinical coronary event during lipid lowering therapy was significantly reduced as compared to conventional care. D. The University of California, San Francisco, Specialized Center of Research Intervention Trial (UCSF SCOR)

This study was conducted in 72 subjects (41 women), 19 to 72 years of age, with heterozygous familial hypercholesterolemia in which only 3 subjects had objective evidence of CAD prior to baseline angiogram (Kane et al., 1990). Treatment group received combinations of colestipol, niacin, and lovastatin. After 2 years of treatment, QCA demonstrated that mean

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change in percent area stenosis among controls was +0.80 (progression) while mean change for the treatment group was -1.53 (regression) ( p = 0.039). Progression occurred in 20% of drug-treated subjects and in 41% of control subjects; regression occurred in 33% and 13%, respectively. When analyzed separately, lesion change among women was significant ( p = 0.05), whereas for men it was not ( p = 0.42). Treated women had a -2.06 regression in percent area stenosis whereas men had a -0.88 change. This is the first study to demonstrate significant coronary artery lesion regression in women. E. The Monitored Atherosclerosis Regression Study (MARS) This study is the first angiographic trial to test the effects of single-drug therapy with an HMG-CoA reductase inhibitor (Blankenhorn et al., 1993). This 2-year serial coronary angiographic trial randomized 270 smoking and nonsmoking men and women (9”/0)37 to 67 years of age to diet and either lovastatin 40 mg twice daily or placebo. This is the only randomized trial to prospectively evaluate coronary angiographic films by QCA with automated edge detection algorithms (primary end-point methodology) and by panel based readings with GCS (secondary end-point methodology). Because of the minimal side effects of lovastatin, this was the first truly doubie-blind clinical imaging trial. There were 247 subjects (123 lovastatin, 124 placebo) with baseline and 2 year coronary angiograms available for interpretation. Although mean change in %S by QCA over all lesions was not significant (+l.6YOSversus +2.2%S in the lovastatin and placebo groups respectively), lesions r 5 0 % S at baseline showed a mean decrease of -4.1%S with lovastatin therapy compared to +0.9%s with placebo ( p < 0.01). There was no statistical difference between treatment groups for change in lesions <50%S at baseline, +2.6%S in the lovastatin group and +3.0%S in the placebo group. Mean change in minimum lumen diameter (MLD) for all lesions was not significant (-0.03 mm versus -0.06 mm in the lovastatin and placebo groups, respectively). For lesions C50%S at baseline, there was no statistical difference between treatment groups in change in MLD (-0.05 mm for lovastatin versus -0.07 mm for placebo). However, for lesions 2 5 0 % S at baseline, lovastatin increased mean MLD +0.13 mm compared to -0.04 mm with placebo ( p < 0.01). Assessed by QCA on a per-subject basis, one-third less lovastatin subjects progressed (29%) than placebo subjects (41%), p = 0.07. Regression was twice as frequent in lovastatin subjects (23%) than placebo subjects (12Y0), p = 0.04. By panel evaluation, mean GCS in lovastatin subjects was less (i.e., less progression) than in placebo subjects (+0.41 versus +0.88; p < 0.01); 65 lovastatin versus 43 placebo subjects regressed or stabilized (53% versus 35%; p
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tion, progression occurred in one-third less lovastatin subjects (47%) than placebo subjects (65%), p < 0.01. Regression was also considered by the panel to be twice as frequent in lovastatin subjects (23%) than in placebo subjects ( l l % ) , p < 0.02. Multivariate analysis indicates absolute change in TC/HDL-C as the best predictor of lesion change in the placebo group for lesions <5o%s and 2 5 O % S . With LDL-C reduced below 85 mg/dl in the lovastatin group, ontrial LDL-C/HDL-C was the best predictor of lesion change for lesions 2 S O % S . For lesions <SO%s, on-trial apolipoprotein C-I11 levels in LDLVLDL, a marker of triglyceride-rich lipoprotein metabolism, was the best predictor of lesion change (Hodis et al., 1994).These analyses confirm CLAS findings indicating that triglyceride-rich lipoproteins play an important role in coronary artery lesion progression; their importance unmasked once LDLC is lowered. These results are the first to indicate that lipoproteins have an important differential effect on lesion progression according to lesion size. Subgroup analyses of females in MARS indicate two trends: (1)Women may have a better lesion response to lipid-lowering than men and (2)estrogen replacement may enhance lesion response to aggressive lipid-lowering. The %S change after 2 years was +2.0%S for men on lovastatin, +0.8Y0S for women on lovastatin, and -2.1%S for women on lovastatin and estrogen. The observation in MARS that women may have a better lesion response to therapy than men has also been reported in the UCSF SCOR and Lifestyle Heart Trial. It is also consistent with evidence indicating that lesions in women appear pathologically younger than those in men (Mautner et al., 1993).Compared to men, coronary artery lesions in women consist of more cellular fibrous tissue (found at earlier stages of plaque development), and less dense fibrous tissue (found at later stages of plaque development) (Dollar et al., 1991). This difference in plaque composition suggests a pathological basis for expecting a greater potential for response to therapy in women.

F. The Canadian Coronary Atherosclerosis Intervention Trial (CCAIT) This trial obtained coronary angiograms at baseline and at 2 years in 299 subjects <70 years old randomized to dietary therapy and either placebo ( n = 153 subjects) or lovastatin ( n = 146 subjects) (Waters et al., 1994). Lovastatin was titrated to attain an LDL-C <131 mg/dl. Mean reduction in MLD, indicating progression, was -0.05 mm for lovastatin and -0.09 mm for placebo subjects ( p < 0.02). Mean change in percent diameter stenosis for all subjects was 1.66%S in the lovastatin group and 2.89%S in the placebo group. Greater treatment effects were apparent for lesions 250%S at baseline (Table I). On a per-subject basis, there was no significant difference in number of lovastatin and placebo subjects with regression.

TABLE I Comparison of Lipid Lowering Therapy on Coronary Artery Lesion Progression in Three Independent Trials Using HMG-CoA Reductase Inhibitor Monotherapy (mean 2 SD) MAAS MARS Treatment group (n)b Placebo group (n) On-Trial LDL-C Level (mgldl): Treatment group Placebo group

CCAlT

2 years"

4 years

123 124

146 153

143' 129'

93 (-38%)' 1.53

122 (-29%)' 168

117 (-31%)' 174

All Lesions Mean %S change: Treatment group Placebo group Mean MLD change (mm): Treatment group Placebo group Mean %S change: Treatment group Placebo group Mean MLD change (mm): Treatment group Placebo group

1.6 t 6.7 2.2 t 6.8

1.7 t 4.5 2.9 t 5.6

-0.03 t 0.21 -0.05 t 0.13 -0.06 2 0.21 -0.09 t 0.16 Lesions ?SO%S at baseline

0 t NA' 2.6 ? NA'

1.0 t 7.9 3.6 t 9.0

-0.02 ? NA' -0.05 t NA'

-0.04 t 0.25 -0.13 t 0.27

-4.1 t 11.0 0.9 t 11.0

-1.0 2 11.1 -0.4 t 7.9

NA' NA'

NA' NA'

+0.13 t 0.35 -0.04 t 0.36

+0.02 t 0.28 +0.01 t 0.20

NA' NA'

+0.19 t 0.44 -0.01 2 0.47

Lesions <50%S at baseline Mean %S change: Treatment group Placebo group Mean MLD change (mm): Treatment group Placebo group Percentage of subjects with new lesions: Treatment group Placebo group Percentage of subjects classified as progressors: Treatment group Placebo group Percentage of subjects classified as regressors: Treatment group Placebo group

2.6 C 7.7 3.0 t 7.7

2.2 2 4.9 3.5 +- 6.0

NA' NA'

NA' NA'

NA' NA"

-0.01 C 0.34 -0.06 ? 0.36

-0.05 t 0.21 -0.07 ? 0.21

-0.06 C 0.14 -0.11 2 0.18

19 24

16 32

28 48

29 41

33 50

43 52

23 12

10 7

25 23

Estimated 2-year values from manuscript. Treatment group, lovastatin in MARS and CCAIT and simvastatin in MAAS. Number of subjects who completed baseline and 4-year coronary angiograms. Percentage change from baseline LDL-C level. NA, data not available.

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Progression and new lesion formation occurred in less lovastatin subjects ( p < 0.05).

G. Multicentre Anti-Atheroma Study (MAAS) This study obtained coronary angiograms 2 and 4 years after intervention in 178 subjects randomized to simvastatin 20 mg daily and 167 subjects randomized to placebo; 143 simvastatin subjects and 129 placebo subjects had repeat angiograms at both 2 and 4 years (Oliver et al., 1994). Both treatment groups received dietary advice. After 2 years of therapy, there was no significant effect on the progression of coronary artery atherosclerosis. After 4 years of therapy, there was a mean reduction in MLD of -0.04 mm in the simvastatin group and a -0.13 mm mean reduction in MLD in the placebo group ( p = 0.03) as well as a mean change of +l.O%S in the simvastatin group versus a mean change of +3.6%S in the placebo group ( p = 0.006). After 2 years of intervention, there were approximate mean changes of -0.02 mm and O%S versus -0.05 mm and 2.6Y0S in the simvastatin and placebo groups, respectively. Greater treatment effects were observed for lesions 2 5 O % S at baseline (Table I ) . On a per-subject basis, progression was significantly less and regression significantly greater in subjects treated with simvastatin versus placebo ( p = 0.02). A comparison of MARS, CCAIT, and MAAS provides a unique opportunity to compare 3 similarly designed trials utilizing similar interventions, namely HMG-CoA reductase inhibitor monotherapy plus dietary intervention, to stabilize coronary artery lesion progression (Table I). The treatment effect of lipid-lowering therapy on coronary artery lesion progression is remarkably similar in all 3 independent trials. H. The Stanford Coronary Risk Intervention Project (SCRIP)

This study randomized 155 men and women (13%) less than 7 5 years of age to usual care and 145 subjects to multifactorial risk reduction with drug treatment (principally colestipol-niacin), diet, smoking cessation, weight reduction, and exercise (Haskell et al., 1994). QCA arterial diameter measurements were made in nonbypassed coronary segments at baseline and 4 years later. Annual rate of change in minimum coronary artery diameter was -0.046 mm in the control group and -0.022 mm in the risk reduction group ( p < 0.01). Regression occurred in 21 % of risk reduction subjects and 10% of controls ( p = 0.025). New lesion formation was reduced 36% in the risk reduction group versus 20% in the control group ( p < 0.01). In the first study year, there were 7 deaths or nonfatal myocardial infarctions in the risk reduction group and 14 in the control group ( p =

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0.18). Over the next 3 years of the study, there were 2 clinical cardiac events in the risk reduction group and 13 in the control group ( p < 0.006). 1. The St. Thomas' Atherosclerosis Regression Study (STARS) The study included 74 men with coronary heart disease who completed the study with paired angiograms, 24 in a usual care group, 26 in a dietary intervention group, and 24 in a diet-cholestyramine group (Watts et al., 1992). Angiograms were performed at baseline and after 39 months with QCA determination of mean absolute arterial width as the primary end point. Proportion of subjects with coronary narrowing, 15% of subjects in the diet group and 12% in the diet-cholestyramine group compared to 46% in the control group, was reduced by both interventions, ( p < 0.02 for trend). Regression occurred in 38%, 33%, and 4% of subjects, respectively ( p < 0.02 for trend). Per patient average width of coronary segments decreased 0.201 mm in controls and increased 0.003 mm with diet and 0.103 mm with diet-cholestyramine ( p = 0.012 for trend). With segments rather than subjects as the experimental unit, treatment effects were found for mean arterial width, minimum width, %S, and vessel edge irregularity ( p < 0.002 for trend in all measures). Improvement in mean absolute arterial width correlated with on-trial LDL-C levels and LDL-C/HDL-C ratios. Significant therapeutic benefit in %S was found for lesions >5o%s and <15%s at baseline but not for lesions 15-50%s.Lesions >SO%s at baseline preferentially responded to treatment relative to lesions <15%s and 15-50%s. Lesions >50%S at baseline in the diet and diet-cholestyramine groups regressed -23.3%s and - 18.4%S, respectively, compared to progression of +7.4%s in the control group. Lesions <15%s progressed +4.4%S and +2.5%s, respectively, compared to +8.8%S in the control group. Lesions 15-50%sregressed -1.2%S in both treatment groups compared to +2.3%s in the control group. Both diet and diet-cholestyramine interventions reduced frequency of total cardiovascular events (death, myocardial infarction, revascularization, or stroke). Fourteen subjects had clinical events, 10 (36%) control subjects, 3 (11'70)diet subjects ( p < 0.05), and 1 (4%) diet-cholestyramine subject ( p < 0.01).

111. Coronary Angiographic Trials Utilizing Nonpharmacological Intervention A. The Leiden Intervention Trial Although not a randomized study, this trial provided first evidence that dietary modification could beneficially effect CAD without major weight

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loss (Amtzenius et al., 1985). Vegetarian diet with a polyunsaturated saturated fat ratio >2.0 and cholesterol intake 6.9 whereas subjects with TCHDL-C ratios <6.9 at baseline or with ratios greater than 6.9 and then reduced below 6.9 had no lesion growth. Mean percent increase in vessel diameter (regression) of + 3.47% occurred in lesions 2 5 O % S at baseline, whereas mean percent decrease in vessel diameter (progression) of -4.29% occurred in lesions <50%0s at baseline. 6. The Lifestyle Heart Trial

This trial reported 22 risk reduction and 19 control men and women between 35 and 75 years of age with analyzable film pairs after the 1 year interventional period (Ornish et al., 1990). Risk reduction included low fat vegetarian diet, smoking cessation, stress management, and aerobic exercise. Dietary fat and cholesterol which averaged 31% of calories and 213 mg/ day on entry were reduced to 7% of calories and 13 mglday in the risk reduction group. Average %S measured by QCA was reduced 40.0%0Sto 37.8%S in the risk reduction group and increased 42.7%S to 46.1%S in the control group ( p = 0.001). Degree of adherence to lifestyle changes was directly correlated with extent of change in %S. Progression was reported in 18% of the risk reduction 'subjects and 52% of the control subjects; regression occurred in 82% and 42%, respectively. Five women (postmenopausal and not taking hormonal replacement therapy), 1 in the intervention group and 4 in the control group, had regression with only moderate lifestyle changes. Lesions >50%S demonstrated the greatest overall improvement. C. Heidelberg Exercise-Diet Study

This study included coronary angiographic films pairs in 40 men in an exercise-diet intervention group and 52 men in a control group (Schuler et al., 1992). Intervention included daily exercise and a low fat-low cholesterol diet. Physical work capacity improved 23% ( p < 0.001) and average total fat consumption was 45 g/day and cholesterol intake 135 mg/day in the intervention group. After 1 year of intervention, average %S measured by QCA was essentially unchanged 65%S to 64% (stabilization) in the intervention group and increased 63%S to 66% (progression) in the control group. Additionally, MLD was unchanged in the intervention group 0.92 to 0.91 and decreased 1.00 to 0.87 (progression) in the control group. The angiographic changes in the control group were significantly different from those in the intervention group ( p < 0.05). Progression, stabilization, regres-

Reversal of Atherosclerosis with Therapy: Update of Coronary Angiogtaphic Trials

3 17

sion occurred in 23%, 45%, 32% of subjects in the intervention group versus 48%, 35%, 17% in the control group ( p < 0.05). In final analysis, CAD progressed at a significantly slower rate in the exercise/diet group compared with the control group.

D. The Program on the Surgical Control of the Hyperlipidemias (POSCH) This study randomized 421 men and women with previous myocardial infarction to ileal bypass surgery for reduction of blood cholesterol and 417 to a control group (Buchwald et al., 1990). Although the primary end point, overall mortality, was not significantly reduced, overall mortality in surgery subjects with an ejection fraction 250% was 36% lower (39 control subjects versus 24 surgery subjects; p = 0.021). Death due to coronary heart disease and nonfatal myocardial infarction were 35% lower in the surgery group (125 vs 82 events in control vs surgery subjects, respectively; p < 0.001). Comparison of baseline angiograms with those obtained at 3, 5, 7, and 10 years consistently showed less CAD progression in the surgery group ( p < 0.001). GCS, as in CLAS, was predictive of clinical coronary events (Buchwald et al., 1992).

IV. Summary of the Coronary Angiographic Trials Overall, regression and stabilization are 2 and 1.4 times more common in treated than placebo subjects and progression is reduced by half in treated subjects. The common denominator of all coronary angiographic trials is reduction in LDL-C through drugs, diet, diedexercise, lifestyle changes, or surgery. Odds ratios for coronary vascular deaths, coronary vascular events, and coronary vascular events combined with revascularization are all significantly reduced 30 to 60%. The odds ratio of death from any cause is also reduced approximately 25% with therapy, but falls short of the 95% confidence level (Blankenhorn and Hodis, 1994). This demonstrates a very important point, however. Overall, mortality is not increased with aggressive LDL-C lowering in secondary prevention. Based on the evidence from coronary angiographic trials, it is now recommended that LDL-C levels be reduced below 100 mg/dl in patients with CAD (National Cholesterol Education Program Expert Panel, 1993).

V. Conclusions from Coronary Angiographic Trials Many interesting and important findings have resulted from coronary angiographic trials which need further attention if we are to increase our

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success with secondary preventive measures. The majority of the coronary angiographic trials, NHLBI Type 11, CLAS, FATS, STARS, MARS, CCAIT, MAAS, Leiden Intervention Trial, and Lifestyle Heart Trial indicate that LDL-C reduction preferentially regresses lesions 250%S and typically retards coronary artery lesion progression in only 50 to 80% of subjects. Improved therapeutic regimens to alter progression of coronary atherosclerosis may require adjunctive therapy in concert with LDL-C reduction to prevent new lesion formation or to induce early lesion regression, such as for lesions <50%S. This adjunctive therapy could be envisioned as being nonlipid altering drugs such as calcium channel blockers in which 2 trials have indicated that these agents reduce new and early lesion formation (Loaldi et af., 1989; Lichtlen et af., 1990), antioxidants such as vitamin E which has been shown to induce the regression of coronary atherosclerotic lesions <50%S in conjunction with lipid-lowering therapy (Hodis et af., 1995a), or hormonal agents such as estrogen replacement therapy in postmenopausal women as a small subgroup in MARS has suggested. Likewise, adjunctive therapy may include agents which concomitantly affect other lipoprotein particles, such as Lp(a) or triglyceride-rich lipoproteins. Both CLAS and MARS strongly indicate that triglyceride-rich lipoproteins are prominent risk factors for progression of coronary artery lesions (Hodis and Mack, 1995). In conclusion, a series of coronary angiographic trials have demonstrated that reduced mortality and morbidity from LDL-C reduction are attributable, at least in part, to stabilization and regression of coronary atherosclerosis in both native vascular beds and venous bypass grafts (Blankenhorn and Hodis, 1994). These studies provide the rationale for treatment of hyperlipoproteinemia with the goal of preventing or ameliorating coronary heart disease. The data strongly support reduction of LDL-C as a secondary preventive measure and indicate little if any cause for concern over possible increase in all cause mortality under conditions of secondary prevention.

References Arntzenius, A. C., Kromhout, D., Barth, J. D., Reiber, J. H., Bruschke, A. V., Buis, B., van Gent, C. M., Kempen-Voogd, N., Strikwerda, S., and van der Velde, E. A. (1985). Diet, lipoproteins, and the progression of coronary atherosclerosis: The Leiden Intervention Trial. N.Engl. /. Med. 312, 805-811. Azen, S. P., Mack, W., LaBree, L., Cashin-Hemphill, L., Shircore, A., Selzer, R. H., Blankenhorn, D. H., and Hodis, H. N. (1996). Progression of coronary artery disease predicts clinical coronary events: Ten year follow-up from the Cholesterol Lowering Atherosclerosis Study (CLAS). Circulation, in press. Blankenhorn, D. H., Nessim, S. A., Johnson, R. L., Sanmarco, M. E., Azen, S. P., and CashinHemphill, L. (1987). Beneficial effects of combined colestipol-niacin therapy on coronary atherosclerosis and coronary venous bypass grafts. ] A M A 257, 3233-3240.

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Blankenhorn, D. H., Alaupovic, P., Wickham, E., Chin, H. P., and Azen, S. P. (1990a). Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts: Lipid and non-lipid factors. Circulation 81, 470-476. Blankenhorn, D. H., Johnson, R. L., Mack, W. J., EIZein, H. A., and Vailas, L. I. (1990b). The influence of diet on the appearance of new lesions in human coronary arteries. JAMA 263, 1646-1652. Blankenhorn, D. H., Selzer, R. H., Mack, W. J., Crawford, D. W., Pogoda, J., Lee, P. L., Shircore, A. M., and Azen, S. P. (1992). Evaluation of colestipollniacin therapy with computer-derived coronary end point measures: A comparison of different measures of treatment effect. Circulation 86, 1701-1709. Blankenhorn, D. H., Azen, S. P., Kramsch, D. M., Mack, W. J., Cashin-Hemphill, L., Hodis, H. N., DeBoer, L. W. V., Mahrer, P. R., Masteller, M. J., Vailas, L. I., Alaupovic, P., Hirsch, L. J., and the MARS Research Group. (1993). Coronary angiographic changes with lovastatin therapy: The Monitored Atherosclerosis Regression Study (MARS). Ann. Intern. Med. 119, 969-976. Blankenhorn, D. H., and Hodis, H. N. (1994). Arterial imaging and atherosclerosis reversal, Arteriosclerosis 14, 177-192. Brensike, J. F., Levy, R. I., Kelsey, S. F., Passamani, E. R., Richardson, J. M., Loh, I. K., Stone, N. J., Aldrich, R. F., Battaglini, J. W., Moriarty, D. J., Fisher, M. R., Friedman, L., Friedewald, W., Detre, K. M., and Epstein, S. E. (1984). Effects of therapy with cholestyramine on progression of coronary arteriosclerosis: Results of the NHLBI Type 11Coronary Intervention Study. Circulation 69, 313-324. Brown, G., Albers, J. J., Fisher, L. D., Schaefer, S. M., Lin, J. T., Kaplan, C., Zhao, X. Q., Bisson, B. D., Fitzpatrick, V. F., and Dodge, H. T. (1990). Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. N. Engl. J. Med. 323, 1289-1298. Buchwald, H., Varco, R. L., Matts, J. P., Long, J. M., Fitch, L. L., Campbell, G. S., Pearce, M. B., Yellin, A. E., Edmiston, W. A., Smink, R. D., Jr., Sawin, H. S., Jr., Campos, C. T., Hansen, J. B., Tuna, N., Karnegis, J. N., Sanmarco, M. E., Amplatz, K., CastanedaZuniga, W. R., Hunter, D. W., Bissett, J. K., Weber, F. J., Stevenson, J. W., Leon, A. S., Chalmers, T. C. and the POSCH Group. (1990). Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia: Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N. Engl. J. Med. 323, 946-955. Buchwald, H., Matts, J. P., Fitch, L. L., Campos, C. T., Sanmarco, M. E., Amplatz, K., Castaneda-Zuniga, W. R., Hunter. D. W., Pearce, M. B., Bissett, J. K., Edmiston, W. A., Sawin, H. S., Jr., Weber, F. J., Varco, R. L., Campbell, G. S., Yellin, A. E., Smink, R. D., Jr., Long, J. M., Hansen, B. J., Chalmers, T. C., Meier, P., and Stamler, J., for the Program on the Surgical Control of the Hyperlipidemias (POSCH)Group. (1992).Changes in sequential coronary arteriograms and subsequent coronary events: Program on the Surgical Control of the Hyperlipidemias (POSCH) Group. JAMA 268, 1429-143.3. Cashin-Hemphill, L., Mack, W. J., Pogoda, J., Sanmarco, M. E., Azen, S. P., Blankenhorn, D. H., and the CLAS Study Group. (1990). Beneficial effects of colestipol-niacin on coronary atherosclerosis: A 4-year follow-up. JAMA 264, 3013-3017. Dollar, A. L., Kragel, A. H., Fernicola, D. J., Waclawiw, M. A., and Roberts, W. C. (1991). Composition of atherosclerotic plaques in coronary arteries in women less than 40 years of age with fatal coronary artery disease and implications for plaque reversibility. Am. J. Cardiol. 67, 1223-1227. Haskell, W. L., Alderman, E. L., Fair, J. M., Maron, D. J., Mackey, S. F., Superko, R., Williams, P. T., Johnstone, I. M., Champagne, M. A., Krauss, R. M., and Farquhar, J. W. (1994). Effects of intensive multiple risk factor reduction on coronary atherosclerosis and clinical cardiac events in men and women with coronary artery disease. The Stanford Coronary Risk Intervention Project (SCRIP). Circulation 89, 975-990.

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Hodis, H. N., Mack, W. J., Azen, S. P., Alaupovic, P., Pogoda, J. M., LaBree, L., Hemphill, L. C., Kramsch, D. M., and Blankenhorn, D. H. (1994). Triglyceride- and cholesterolrich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin. Circulation 90, 42-49. Hodis, H. N., Mack, W. J., LaBree, L., Cashin-Hemphill, L., Sevanian, A., Johnson, R., and Azen, S. P. (199Sa).Serial coronary angiographic evidence that antioxidant vitamin intake reduces progression of coronary artery atherosclerosis. JAMA 273, 1849-1854. Hodis, H. N., and Mack, W. J. (1995). Triglyceride-rich lipoproteins and progression of atherosclerosis. Curr. Opin. Lipidol. 6, 209-214. Kane, J. P., Malloy, M. J., Ports, T. A., Phillips, N. R., Diehl, J. C., and Havel, R. J. (1990). Regression of coronary atherosclerosis during treatment of familial hypercholesterolemia with combined drug regimens. JAMA 264,3007-3012. Lichtlen, P. R., Hugenhola, P. G., Rafflenbeul, W., Hecker, H., Jost, S., and Deckers, J. W. (1990).Retardation of angiographic progression of coronary artery disease by nifedipine: Results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT). INTACT Group Investigators. Lancet 335, 1109-11 13. Loaldi, A., Polese, A., Montorsi, P., De Cesare, N., Fabbiocchi, F., Ravagnani, P., and Guazzi, M. D. (1989).Comparison of nifedipine, propranolol and isosorbide dinitrate on angiographic progression and regression of coronary arterial narrowings in angina pectoris. Am. j . Cardiol. 64,433-439. Mautner, S. L., Lin, F., Mautner, G. C., and Roberts, W. C. (1993). Comparison in women versus men of composition of atherosclerotic plaques in native coronary arteries and in saphenous veins used as aortocoronary conduits. j . Am. Coll. Cardiol. 21, 1312-1318. National Cholesterol Education Program (NCEP)Expert Panel. (1993).Summaryof the second report of the National Cholesterol Education Program (NCEP)expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel 11). JAMA 269,3015-3023. Oliver, M. F., de Feyter, P. J., Lubsen, J., Pocock, S., and Simoons, M. (1994). Effect of simvastatin on coronary atheroma: The Multicentre Anti-Atheroma Study (MAAS). Lancet 344, 633-638. Omish, D., Brown, S. E., Scherwitz, L. W., Billings, J. H., Armstrong, W. T, Ports, T. A., McLanahan, S. M., Kirkeeide, R. L., Brand, R. J., and Could, K. L. (1990).Can lifestyle changes reverse coronary heart disease? The Lifestyle Heart Trial. Lancet 336,129-133. Schuler, G., Hambrecht, R., Schlierf, G., Niebauer, J., Hauer, K., Neumann, J., Hoberg, E., Drinkman, A., Bacher, F., Crunze, M., and Kubler, W. (1992).Regular physical exercise and low-fat diet: Effects on progression of coronary artery disease. Circulation 86, 1-11. Waters, D., Higginson, L., Gladstone, P., Kimball, B., Le May, M., Boccuzzi, S. J., Lespkrance, J., and the CCAIT Study Group. (1994). Effects of monotherapy with an HMG-CoA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantitative arteriography. The Canadian Coronary Atherosclerosis Intervention Trial. Circulation 89, 959-968. Watts, G. F., Lewis, B., Brunt, J. N. H., Lewis, E. S., Coltart, D. J., Smith, L. D. R., Mann, J. I., and Swan, A. V. (1992). Effects on coronary artery disease of lipid-lowering diet, or diet plus cholestyramine, in the St. Thomas’ AtherosclerosisRegression Study (STARS). Lancet 339,563-569.

Lawrence A. Loeb Joseph Gottstein Memorial Laboratory Department of Pathology University of Washington School of Medicine Seattle, Washington 98 I95

Unnatural Nucleotide Sequences in Biopharmaceutics

1. Introduction The large scale screening of natural products has provided the pharmaceutical industry with an abundant source of therapeutic drugs. Potential therapeutic drugs and lead compounds have been identified in bacterial fermentation broths, plants, marine organisms, and cultured mammalian cells. However, these sources are limited to those compounds that have survived evolutionary selective pressures; the repertoire of natural products may represent only a small fraction of biologically active molecules. With the use of the three-dimensional structure of enzymes and receptors it is now possible to model the structure of therapeutic agents from first principles. By systematically altering the primary sequence of a protein one can design and evaluate potential inhibitors. However, this rational approach is limited by the difficulties in crystallizing many proteins Advunces in Pharmacology, Volume 35 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

32 I

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and by our lack of knowledge on how to predict protein structure based on primary sequence. Only in a limited number of instances has there been success in the rational design of drugs or inhibitors based on protein structure. New techniques in biochemistry and molecular biology have made it feasible to create vast libraries of biopolymers as well as small organic molecules. These libraries are much greater in diversity than those found in natural products and moreover can be evolved in vitro to meet the needs of the investigator. This review addresses the theoretical concepts that underpin the usefulness of synthesizing vast libraries by applied molecular evolution and the methods currently being used to do so. A comparison of the different methods will be presented; however, the focus will be on proteins with biological activity. The use of random sequence mutagenesis coupled with genetic selection will be considered in greater detail based on work in the author's laboratory. Finally, the potential use of new molecules for genetic engineering will be discussed.

II. Site-Specific Mutagenesis and Rational Drug Design

-

Prior to considering the combinatorial approaches to the design of new drugs and proteins, let us consider the limitations inherent in the rational design or even the sequential chemical modification of therapeutic molecules. Given a lead small molecule, multiple derivatives can usually be obtained rapidly by chemical synthesis. The difficulty is to choose which derivative to synthesize. A knowledge of the three-dimensional structure of the cellular receptor or enzyme could be instrumental. The three-dimensional structure of receptors or enzymes and alterations in their structure has frequently guided drug development. The ability to engineer precise changes in proteins by site-directed mutagenesis provides an incisive methodology to dissect the relationship between structure and function (Carroll and Richards 1987). Site-directed mutagenesis has proved to be a decisive approach to determine those amino acids that comprise the active site of a protein and to establish those that have a direct role in binding or in catalysis. However, site-directed mutagenesis is not versatile; there are two major limitations. First, it is not feasible to study a large number of different substitutions because one constructs individual molecules with designated amino acid replacements and tests them one at a time. For example, if 10 amino acids were central to the active site of an enzyme and if our analysis were restricted even to short aliphatic substitutions, then we would have to examine some 31° permutations by site-specific mutagenesis. If we analyzed all possible substitutions by 19 amino acids, there would be some 19" permutations. A second limitation and of greater concern is the absence of rules governing the interchangeabil-

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ity of amino acids. In the absence of rules there are 19 choices to replace each amino acid residue at any position in a protein. Without rules relating structure to function, we have to consider the effects of an inordinantly large number of substitutions that might determine binding or catalytic activity.

111. Molecular Evolution and I t s Consequences Random libraries overcome the limitations of site-specific mutagenesis. It is now possible to sample within limited domains all possible permutations of amino acids and to obtain unnatural molecules that are active and different from those selected by nature. The concept of applied molecular evolution is based on the likelihood that one can identify and evolve unnatural molecules from random libraries that are more useful than those that have evolved by natural selection. Evolution may not have selected the most active proteins. This assumption is based on extrapolations from Darwinian evolution in which the surviving species is the one that is most fit. It is assumed that the selection of a species is the end result of selection of cells and ultimately of individual biologically active molecules. One premise is that the best protein would be needed only in the smallest amounts. A cell containing these proteins would have a selective advantage since less energy would be required for their synthesis and they would be able to most effectively utilize environmental constituents for the synthesis of new cells. However, the most active biological proteins are unlikely to have been selected during evolution. Having a more active enzyme in a cell may not be advantageous. Enzymes streamlined for rapid catalysis may exhibit less flexibility to respond to regulatory events. Since cells harbor interdependent metabolic pathways, a mutation that increases any single step may be detrimental; it could result in the accumulation of metabolic intermediates. In the case of receptors, higher binding affinity could result in decreased dissociation and diminished reversibility. We have speculated that many active nucleotide sequences have been discarded during prebiotic evolution (Dube et al., 1991a). Current concepts of prebiotic evolution maintain that early genetic material consisted of nucleotides linked by short carbon bridges into polymers. Further evolutionary stages utilized RNA and then DNA as early self-replicating molecules. At the time of cell compartmentalization, proteins evolved as the principal catalytic molecules. At each step in this selective process, nucleotide sequences were eliminated because they were not advantageous. These sequences would no longer be available as substrates for further evolution. As a result, many sequences were eliminated from the gene pool even though they might encode more active molecules. The ability to insert random nucleotide sequences into genes has made it possible to determine if there

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are sequences that encode proteins that are better or more useful than the ones found in nature. While natural evolution has taken billions of years to assemble biologically active natural products, scientists can now construct, evolve, and screen molecular and chemical libraries within weeks.

IV. Random Molecular and Chemical Libraries Large molecular libraries have been constructed containing RNA, DNA, protein, peptides, and small organic molecules. Each of these molecules offers unique advantages for the construction of diagnostic or therapeutic products. Table I presents a summary of the diversity that has been obtained and of the potential uses of some of these libraries.

A. Random Genetic Selection for Biologically Active Proteins The earliest combinatorial libraries were analyzed by inserting random nucleotide sequences into genes and using biological selection to identify active clones (Horwitz and Loeb, 1986). This technique was first applied to promotor sequences (Horwitz and Loeb, 1986; Oliphant and Struhl, 1987) and then to proteins (Dube and Loeb, 1989; Oliphant and Struhl, 1989). Through natural selection, proteins have been chosen to perform regulatory and catalytic functions. This suggests that proteins may be best suited to carry out these functions. Of particular importance is the diversity of amino acids and side chains present in proteins. The major limitation with genetic selection for the identification of proteins of interest is the development of positive genetic selection assays sufficiently robust to isolate rare biologically active molecules from large combinatorial libraries. Since selection ultimately requires transfection into a living cell, the size of the library is restricted by the number of cells available and the efficiency of transfection. As a result, bacteria or yeast are the host of preference based on size, growth, and genetic mutants available for complementation. B. Phage Display Libraries for Binding Proteins

Phage display libraries (Scott and Smith, 1990) have been extensively utilized in studies on binding proteins, in mimicking the vast diversity of the immunological repertoire (Winter and Milstein, 1991), and in the construction of catalytic antibodies (Lerner et a]., 1991). The combinatorial regions of antibody genes have been replaced by random amino acid sequences, and the display of these libraries within the coat proteins of phage yields a diversity of lo7. Thus, phage display libraries provide a new and less costly method for the production and screening of monoclonal antibod-

TABLE I Systems Used in “Applied Molecular Evolution” System

Advantages

Disadvantages

1. Random genetic selection

Nature’s catalysts are enzymes Define function-structure relationships

Maximum diversity is 109-10” Termination codons Require highly sensitive assays

2. Phage display

Proteins are linked to nucleic acids and therefore can be amplified

Diversity limited to 107-108 Not all epitopes are expressed

3. Peptide libraries

Ease of assembly They mimic small organic molecules

Not associated with nucleic acids Few rigid structures Easily biodegradable

4. Nucleic acids (RNA and

Screening potential is 10’’ Evolution can be continuous

Limited catalytic activity Biodegradable

Drugs are small organic molecules Resistant to biodegradation

Not linked to amplifiable nucleic acid Automation or sib selection may be required for identification

DNA)

5. Small organic molecules

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ies. The underlying principle is based on the physical connection between the protein or peptide displayed on the phage coat and the nucleic acid within the phage particle that encodes it. From a large pool of phage displaying random sequences in the exposed portion of the coat protein, individual phage can be isolated based on binding to an immobilized ligand (by chromatography or by panning) and the desired sequence can be amplified simply by phage growth. Tight binders are usually obtained by repetitive rounds of binding to the ligand followed by growth of the phage. The diversity of phage displayed antibodies that can bind to small ligands has been demonstrated most strikingly with metal ligands. Using this methodology, antibodies have been isolated that selectively bind to a variety of metal ions including nickel, lead, gold, and even magnetite (Barbas et al., 1993). Even though affinity selection of phage with immobilized ligands mimic selection during the immune response (Lerner et al., 1991), other proteins with random sequence inserts might equally serve for the binding of a variety of ligands. Alternative approaches to phage display use the same principle, physically linking the randomized protein to the encoding nucleotides, including direct screening of nascent peptides of polysomes (Tuerk and Gold, 1990) as well as fusing the gene encoding the peptides to the lac repressor (Cull et al., 1992). C. Peptide Libraries for Modeling Peptide Hormones and Drugs Peptides exhibit a diversity of structures and bind to specific cellular receptors. In principle, they can substitute for, or compete with, potent peptide hormones. Alternatively, they provide space-filling structures that can be used for the design of drugs. A variety of techniques have been utilized to construct large libraries of peptides for defining the size and types of groups that bind to antibodies and cellular receptors (Cwirla et al., 1990; Devlin et al., 1990). Since the peptides are usually not physically linked to the encoding template, the size of libraries is limited by methods available for their chemical identification. As a result, peptide libraries are usually of limited size. While the diversity of functional groups is attractive for studying drug-receptor interactions, the flexibility of the peptide linkage does not easily yield a three-dimensional structure that can be used to model potentially therapeutic drugs. The use of peptides themselves as therapeutic agents is severely restricted due to rapid biodegradation. Advances in the both the construction or synthetic peptide libraries and their display as fusion proteins has recently been summarized (Scott and Craig, 1994). D. Nucleic Acid Libraries While the structure of nucleic acids may be not as suitable as proteins for binding or catalytic activity, impressive gains have been made in redesign-

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ing these molecules to perform these functions. A detailed account of these accomplishments is beyond the scope of this review. However, I will summarize the approaches used to evolve RNA and to screen for new catalytic activities, since they are likely in the future to guide the studies on the combinatorial chemistry of proteins and small molecules. The use of nucleic acids in applied molecular evolution first focused on RNA. Single stranded random RNAs were shown to contain specific highaffinity binding sites for small molecule ligands that are immobilized on a solid support (Ellington and Szostak, 1990; Sassanfar and Szostak, 1993). Starting with a pool of l O I 4 random RNA molecules, binders to different small molecules including ATP could be isolated (Sassanfar and Szostak, 1993). These studies not only demonstrated the versatility of nucleic acids for binding, but also the important concept that within a pool of 1014 molecules one encompasses the sequence-space necessary to bind virtually any small ligand. Tuerk and Gold (1990) have taken advantage of the versatility of RNA binding by using repetitive amplification within a coupled RNA and DNA amplification system (SELEX) to identify RNA molecules that specifically bind to a large variety of small ligands and protein molecules and thus could be of diagnostic potential. The isolation of single-stranded DNA from a pool of 1013 random oligodeoxynucleotides that bind with nanomolar affinity to thrombin, an important protease in blood coagulation, has provided one of the first lead compounds from combinatorial chemistry (Bock et al. 1992). The disadvantage of RNA as a therapeutic molecule is its rapid biodegradation in humans and the remote possibility that a nucleic acid could be a trigger for autoimmune diseases such as lupus erythematosis. Darwinian evolution of RNA molecules was first demonstrated in a series of elegant experiments from Spiegelman’s laboratory on the copying of Q B RNA with Q B replicase (Mills et al., 1967). The high error rate of the replicase produced mutations during each in vitro replication cycle. Current studies have started with ribozymes, a natural RNA that is catalytically active. Using random sequences, ribozymes have been isolated that can mimic ligases, kinases, and isomerases and even can catalyze carbonnitrogen bond formation (Wilson and Szostak, 1995). Joyce and associates (Joyce, 1989; 1992; Lehman and Joyce, 1993b) have followed and directed the evolution of ribozymes in the test tube. Starting with ribozymes that contain partially random sequences, they have carried out sequential cycles of amplification, mutation, and selection for increased catalytic activity. Selection in the presence of Ca2+yielded ribozymes with a Ca2+-dependent RNA-cleaving activity, an activity not present in the wild type molecule (Lehman and Joyce, 1993a). These studies demonstrate the potential of evolving macromolecules in the test tube. Since selection is carried out in the absence of a host cell, one can start with much larger numbers of random sequences than are used for proteins by either genetic selection or phage display (Table I).

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E. Combinatorial Chemical Libraries Large combinatorial libraries have been synthesized using core compounds with multiple reactive side chains (Feng et al., 1992; Morgan et al., 1993). These libraries will become a source of diverse compounds for evaluation by the pharmaceutical industry. Enormous diversity can be obtained and the resulting compounds could be used as lead compounds for the direct development of therapeutic agents. This approach is exciting; unfortunately we must await reports to assess its feasibility. At least four different approaches have been applied to isolate desired organic molecules from combinatorial libraries. Firstly, individual molecules with desired properties can be separated physically if attached to a bead. Secondly, elaborate schemes have been proposed for tagging molecules and identifying their chemical alterations (Brenner and Lerner, 1992). Third, the library can be deconvoluted by reconstructing the different synthetic steps or by sib selection (Brenner and Lerner, 1992). Methods for the synthesis of chemical libraries starting with core compounds are already in place (Morgan et al., 1993); automated methods may be required to separate the many constitutents; and most importantly, new biological assays will need to be designed to evaluate the many candidate lead compounds that will be generated by this approach.

V. Random Sequence Selection A. General Protocol Random sequence selection is based on the hypothesis that many amino acid sequences encode biological molecules that are more useful than those selected through evolution by nature. The idea is to insert random nucleotides in DNA and then use genetic complementation to obtain clones that encode unnatural biologically active molecules (Fig. 1). A random sequence of nucleotides is substituted for a defined portion of a plasmid-encoded gene that specifies a biologically active molecule. In one application of this procedure, a double-stranded oligodeoxyribonucleotide is produced by hybridizing two oligonucleotides, one or both of which contain random sequences at specified positions. The partially double-stranded oligonucleotide is filled in by DNA polymerase, cut at restriction sites and ligated into a DNA vector in place of a gene portion excised by the same restriction enzymes. After ligation, the reconstructed plasmids, constituting a library of different sequences, are transfected into a host cell (usually a bacterium) lacking that activity. Selective conditions are chosen so that only those host cells that harbor plasmids encoding active proteins are able to grow. The investigator, by the judicious choice of a host mutant, can select sequences that encode proteins to perform functions of interest. With a robust system

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iG+'

...........................

:G *;;G*: ...........................

j i

NNNNNN

...........................

cr"I *'; 0*;;a .........................

Random Sequences

Vector

..........................

.........................

FIGURE I General method for the insertion of random nucleotide sequences (N) into a DNA vector and selection of functional molecules by genetic complementation. The vector carries a marker for antibiotic resistance and the total number of transfectants can be determined by growth on the antibiotic.

for positive genetic selection, it is theoretically possible to analyze a population of as many as lo1*recombinant plasmids for a specific encoded activity. However, so far, we have not succeeded in screening populations of greater than lo8 transfectants. This approach is probably the most demanding of those we have considered (Table I), since one is selecting on the basis of function, as opposed to binding or enhanced stability. However, new molecules that are active are likely to be the most relevant for therapuetic studies. In contrast to site-specific mutagenesis, random sequence selection does not require a detailed knowedge of the three-dimensional structure of the protein of interest.

B. Choice of a Plasmid Vector and Host In choosing a vector, it is important to consider copy number, promoters, unique restriction sites, and the presence of a separate antibiotic resistance gene. In order to determine the total number of transfectants, cells are grown on an antibiotic to which hosts harboring the plasmid are resistant. Prior selection for antibiotic resistance can be used to eliminate nontransfected cells prior to selection for the gene of interest. For detailed presentation of the construction of vectors with recombinant insertion, the reader is referred to Black et al. (1993). Based on practical considerations, most studies use E. coli mutants as the host of choice. One can grow large populations of bacteria, transfection frequencies as high as 1%can be achieved by a variety of methods, and most significantly, well-characterized mutants are frequently available for genetic complementation. A small number of studies have been carried out with yeast, but the much lower transfection efficiency with this organism is a major impediment. As mutants become available it is likely that human cells in culture may also be utilized.

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C. “Dummy” or Nonfunctional Inserts Prior to inserting an oligonucleotide with random sequences (vide infra) into a double-stranded DNA vector, it is necessary to excise the corresponding sequence by cutting with restriction enzymes. To minimize contamination by the religation of singularly cut or uncut vectors, it is advantageous to first replace the wild type segment of the gene by a larger unrelated stuffer fragment or “dummy” insert (Dube et al., 1991b).A large stuffer fragment, preferably one that is out of frame and containing a stop codon, is advantageous. Such a fragment can be easily obtained from a restriction digest of lambda or an unrelated DNA vector. The presence of a small amount of the wild type plasmid can be disasterous in screening large populations of mutants for functional complementation. Large amounts of the dummy vectors can be grown without fear of contamination or religation events restoring wild type sequence. The vector with the nonfunctional insert is the starting plasmid. The insert can be excised by the same restriction enzymes and replaced by an oligonucleotide containing random sequences.

D. Oligonucleotide Inserts Containing Random Nucleotide Sequences The most direct method for the assemblage of oligonucleotide inserts with random nucleotide sequences is to hybridize two oligonucleotides containing randomized segments and then extend them with DNA polymerase. Random sequences may be located in one or both oligonucleotides, either as a continuous stretch or dispersed in key codons (Black et ul., 1993). The double-stranded extended oligonucleotide should include terminal sites for cleavage by the same restriction enzymes used to cut the vector. It is possible to assemble large oligonucleotides by hybridizing together multiple small segments with overlapping complementary regions. Gaps in the extended product containing the random regions are filled in by DNA polymerase and sealed by DNA ligase (Munir and Loeb, unpublished results). If a large amount of insert is required, the oligonucleotide can be amplified in a polymerase chain reaction. In the latter situation, the restriction sites need not be present at the ends of the oligonucleotides, but instead can be present in the PCR primers at 5’-termini that are not complementary to the oligonucleotide. An alternative approach, which still remains to be tested, is to hybridize a pool of single-stranded randomized oligonucleotides flanked by complementary sequences directly onto the gene encoded in a vector that contains uracil as well as a stop codon in the complementary segment. After extension by DNA polymerase and ligation, transfection is carried out in bacteria that contain uracil glycosylase. As a result, only the newly synthesized strand containing the random nucleotides would be copied and expressed.

Unnatural Nucleotide Sequences in Biopharmaceutics

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E. Randomization of Oligonucleotides The number of randomized nucleotides and their positions should be considered very carefully. Consider the number of permuations that would result from a limited number of substitutions. For each triplet there are 20 possible amino acid residues. At the nucleotide level, the number of possible permutations for 6 codons (18 nucleotides is 418or 6.9 X lolo;this translates into 6.4 x 10’ amino acid substitutions, corresponding to the upper limit of the number of transfectants that have so far been screened (Munir et al., 1993).As a result, substitutions at more than 6 codons should be considered in two situtations. Firstly, if one wants to identify amino acid residues that are critical for biological activity, partial randomization at a large number of positions can diagnose whether or not different amino acid residues are required for activity. This can be achieved by biasing the nucleotide pools so that the wild type nucleotide is the predominant species at every position. The frequency of encoding the complete wild type sequence can be estimated by multiplying the percent wild type at each position. For example, 30% random substitutions at each position with a stretch of 18 nucleotides would yield wild type sequence at a frequency of 0.16%; the presence of a few wild type DNA sequences can serve as an internal control to access the extent of randomization. Secondly, a large number of mutants can be analyzed in schemes that involve sequential selections for increasing activities in liquid culture; for example, resistance to increasing concentrations of an inhibitor for growth. A potential limitation in randomization is the presence of termination codons creating a background of nonfunctional proteins. Two different approaches have been proposed to eliminate nonsense codons from a pool of random nucleotides. First, the random sequences can be designed so that the first two residues of a codon contain four nucleotides in equal proportions and the third position contains only G, C, or T. Since A is eliminated, TAA and TGA stop codons would not be present. The remaining TAG stop codon can be partially eliminated by varying the preceding nucleotide and digesting with restriction enzymes that cleave at this sequence (Little, 1990). A more powerful method, only partially developed, is to assemble random nucleotide sequences by linking 20 trinucleotide codons each of which codes for a different amino acid. Linkage can be achieved chemically by the use of trinucleotide phosphoramidites (Sondek and Shortle, 1992) or enzymatically by the use of either RNA or DNA ligase (Loeb, unpublished results). One can not only eliminate nonsense codons, but also select codons that correspond to the codon usage pattern of the bacterial host and thus increase the rate of synthesis of the encoded protein. However, termination codons are usually not of sufficient frequency in any random pool to limit the size of a library; low transfection efficiency is a much greater problem.

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F. Combinatorial Consideration

In order to increase the efficiency of selecting functional proteins from random nucleic acid sequence libraries, it is desirable to limit the number of random nucleotides in a library and to make the probability of observing each protein equal. Since we lack guidelines to predict the effects of multiple substitutions on function or to predict the essentiality of each amino acid residue, we have taken an experimental aproach to the problem. We and others have first screened an area to be randomized by evaluating a small library that contains 1 to 10% random nucleotide substitutions at each position (Black and Loeb, 1993; Dube et al., 1993; Reidhaar-Olson et al., 1991). In this way we can focus on single amino acid substitutions that yield active proteins. By using a partially random library, the probability of multiple amino acid substitutions within a segment is minimized. Codons that are inviolate are eliminated from further analysis. The finding that multiple substitutions occur with amino acids having totally dissimilar side chains suggests that these residues are not critical for activity and therefore need not be included in further studies. An analysis of these substitution patterns is used to determine the importance of the mutagenized positions for the design of experiments with 100% random substitutions at a more limited number of positions. We have used this sequential two library protocol for studies of Herpes thymidine kinase (vide infra) (Munir et al., 1992), and others have used this approach for studies on the lambda repressor gene (Bowie et al., 1990; Reidhaar-Olson et al., 1991). A computation based approach would be to optimize the randomization scheme to maximize the probability of observing the entire subset of desired amino acids and express the members of this subset with equal probability. This approach has been further developed by calculating doping schemes (the percentage of each nucleotide at each position) in order to maximize for the frequency of substituting amino acids with similar structures at each position (Arkin and Youvan, 1992). However, elaborate schemes to enhance the probability of obtaining favorable sequence cassettes may not be practical, since we currently lack oligonucleotide synthesizersfor the facile changing of nucleotide mixtures at each addition step. Several procedures have been formulated to introduce mutations within a specific segment of a gene. Each procedure introduces random mutations at low frequency and does not allow one to screen large mutant libraries. Moreover, in many of these protocols it is not possible to rigorously define the position or the types of nucleotide substitutions. Methods that have been proposed and utilized include: recombination between different plasmids containing random sequences (Caren et al., 1994); chemical mutagenesis of isolated segments of genes (Sweasy and Loeb, 1993); amplification by PCR under mutagenic conditions, usually Mn2+, and biased nucleotide pools (Cadwell and Joyce,1992); as well as repetitive copying by an error prone

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polymerase such as HIV reverse transcriptase (Ji and Loeb, 1994); and by recombination during repetitive PCR reactions (“sexual PCR’) (Stemmer, 1994).Each of these techniques for introducing random mutations is feasible and effective. However, only the chemical synthesis of oligonucleotides allows one to define precisely the frequency and position of random nucleotide substitutions.

VI. Applications of Random Sequence Selection Random sequence selection so far has been utilized for delineating the consensus sequence of regulatory proteins, modifying DNA binding proteins, and identifying mutant enzymes for gene therapy. I will consider some examples of these approaches, highlighting those from our laboratory. So far, the results obtained to date utilizing applied molecular evolution are modest when weighed against the exciting prospects for the future. This technology’s major limitation is imagination in creating positive genetic assays for selection of nucleotide sequences that encode unique proteins.

VII. Regulatory D N A Sequences and Binding Proteins Genetic regulatory events are governed by both protein-DNA and protein-protein interactions. Thus, in principle, random nucleotide substitutions can be carried out in either the regulatory region of a gene or in genes that encode regulatory proteins. In our initial studies on the selection of biologically active molecules, we choose to examine the consensus sequence that governs promoter activity of the tetracycline resistance gene. By definition, a consensus sequence tolerates multiple substitutions without complete loss of activity. In our initial studies, we targeted the -35 region of the tetracycline resistance gene. Previous studies utilizing site-directedmutagenesis (substitutions of one nucleotide at a time) indicated that nearly all substitutions within the consensus sequences resulted in decreased promoter activity (McClure, 1985). We replaced a segment of 19 base pairs at the -35 promotor region of the tetracycline resistance gene in the plasmid pBR322 with various cassettes containing randomized nucleotides at different positions (Horwitz and Loeb, 1986; 1988). After transfection of E. coli, the bacteria were plated on medium containing tetracycline. From bacteria that formed colonies in tetracycline we obtained 185 new active promoters, many of which bore little resemblance to the promoter consensus sequence and some of which were more active than either the consensus sequence or the wild type tetracycline promoter (Horwitz and Loeb, 1986; 1988). Similar studies on the tetracycline promoter were subsequently performed by Oliphant and Struhl (1987).While these early studies demonstrated the large

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diversity of sequences that can carry out specific functions, they also pointed out some of the potential limitations of this approach. Most important is a powerful system for positive genetic selection; however, this approach also requires high transfection efficiency and the absence of wild type sequence contamination. Random sequence techniques have been used to define consensus sequences of a variety of regulatory proteins. The sequence binding requirements for GCN4, a transcriptional activator that binds to the promoter regions of many yeast amino acid biosynthetic genes, were determined by coupling the protein to a Sepharose column and sorting small oligonucleotides by affinity chromatography (Oliphant et al., 1989). In other studies, random substitutions were used to define the consensus sequence for binding of MyoD, the regulatory protein for muscle differentiation (Blackwell and Weintraub, 1990) and the assymmetic nature of the canonical sequence for the binding of the myc oncogene (Blackwell et al., 1993). Conversely, the transcription factors themselves have been altered. Each of the zinc fingers of the murine transcription factor Zif268 has been modified using an in vitro selection strategy based on phage technology. Even though a variety of alterations was permitted, there was no general motif that defined the binding between the zinc finger and the DNA target sequence. An interesting further use of random sequence is to determine the consensus sequence that governs the folding of proteins (Davidson and Sauer, 1994). The identification of amino acid sequences that form stable protein libraries might be an important step in designing random libraries for the isolation of new functional enzymes.

VIII. Production of Mutant Enzymes The synthesis of enzymes with defined amino acid substitutions at the active site has enriched our understanding of how protein structure governs function (Carroll and Richards, 1987; Fersht et al., 1994). The most direct approach is to select the region of a gene that encodes the active site of an enzyme and then systematically substitute nucleotides based on knowledge of three-dimensional structure, amino acid group interactions, and the mechanism of catalysis. With only a limited number of substitutions has this approach been successful (Carter et al., 1984). Multiple substitutions are difficult, and we lack rules to predict the outcome of most substitutions. We have established an alternative strategy based on the substitution of stretches of nucleotides with random sequences and the use of biological selection to identify active mutant enzymes. We have focused on amino acids within the active site, since they are most likely to affect catalysis. A. P-Lactamase P-Lactams and cephalosporins are among the most frequently prescribed class of pharmaceuticals worldwide, and the rapid evolution of P-lactamases

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in pathogenic bacteria continues to defeat the efforts of pharmacologists to create new resistant analogs. Bacterial 0-lactamases hydrolyze the lactam ring of penicillins or cephalosporins, converting them into nonreactive metabolites. We have remodeled the active site of the 0-lactamase gene encoded in the plasmid pBR322. The replacement sequence, Phe66Xaa Xaa Xaa Ser70 Xaa Xaa L Y S ~where ~ , Xaa designates 100% random amino acid substitutions, preserves the codon for the active Ser70, but also contains 15 bp of chemically synthesized random nucleotide sequences. The nucleotide sequence and three-dimensional structure of several class A 0-lactamases are known (Herzberg and Moult, 1987), and indicate a high degree of conservation around the active serine at position 70. The five 100% random substitutions in the replacement sequence have the potential to code for 3 X lo6 different amino acid permutations. From a library of 2 X los E. coli harboring plasmids with these substitutions, we identified seven mutants that render bacteria resistant to P-lactam antibiotics. The fact that we obtained only seven new mutant enzymes could signal that this segment is essential for activity. It does not indicate that each of the amino acid substitutions directly interacts with the substrate; some may be required to maintain the structural integrity of the active site. As expected from a 100% random library, we found that each of the mutants contained multiple nucleotide substitutions, and two contained substitutions at four of the five positions. Oliphant and Struhl (1989) performed similar experiments with p-lactamase; they substituted a stretch of 17 nucleotides from Arg6’ to Cys77with sequences that were 20% random at each position. They collected a large number of active mutants and sequenced 58. In accord with our studies, they observed no substitutions at either Ser’O or Pro6’. From both studies, the following conclusions can be surmised: (1) Mutant p-lactamases can be readily obtained using random substitutions, and these enzymes exhibit altered substrate specificities for the different 0-lactam antibiotics; (2)Ser70and are required for activity, since no substitution of these residues was observed; (3)Most substitutions yielded a temperature sensitive phenotype. This result suggests that maintaining stability could have been more important in the natural selection of 0-lactamases than was obtaining high catalytic activity; (4) A surprisingly large number of substitutions within the active site of an enzyme can occur without marked reduction of catalytic activity. B. Related Studies In the case of proteases, substrate specificities can be established using peptides containing random substitutions. Mathews et al. (1993), created a library of random sequences within the carboxyl-terminal domain of M13 gene 111. They attached the amino acid terminus to an affinity support and subjected the bound phage to digestion with protease. Variants that were cleaved by the protease were released from the matrix. The sequence specific-

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ity of the protease was determined from the identification of both resistant and sensitive substrate sequences.

IX. Gene Therapy for Human Cancer There are two aspects of gene therapy for cancer for which unique enzymes may be the key to success. The first approach concerns the ablation of tumor cells. Current efforts in chemotherapy depend on the selective killing of tumor cells. Unfortunately, for many cancers this approach has not been successful; there are few exploitable biochemical and cellular differences between tumor cells and normal cells. In addition, there are extensive genetic and biochemical differences among malignant cells within the same tumor; thus, it is likely that the cancer cells surviving drug treatment are drug resistant. Gene therapy offers the prospect of selectively introducing genes into cancer cells rendering them susceptibleto specific antimetabolites. With random sequence mutagenesis, one can create mutant enzymes tailored to enhance resistance to specific drugs. The second approach is to introduce genes into bone marrow stem cells, rendering them resistant to specific drugs. The major impediment to dose escalation for most chemotherapeutic agents is bone marrow toxicity. The introduction of mutant enzymes into bone marrow stem cells could protect these cells against specific chemotherapies. Moreover, the transfected cells would have a selective growth advantage and would be able to repopulate the marrow. Based on the above considerations, it is likely that the first successes in gene therapy will be protection of bone marrow against drug-induced toxicity. A. Herpes Thymidine Kinase

Herpes simplex virus type-1 (HSV) thymidine kinase (TK) is currently being used in clinical trials for gene therapy in human cancer. The choice of this enzyme for ablative gene therapy is based on its broad substrate specificity and on the successful use of nucleoside analogs (Elion, 1980) that target this enzyme in the treatment of infections with herpes virus. HSV TK, unlike the human thymidine kinase, catalyzes the phosphorylation of a wide variety of nucleoside analogs. It even phosphorylates structurally distant analogs such as acycloguanosine (acyclovir), a guanine derivative used in the treatment of herpetic infections (Elion, 1980).After phosphorylation by HSV TK, acyclovir is metabolized to the corresponding triphosphate by cellular nucleotide kinases and incorporated into both the viral and host cell DNA. Once incorporated, it terminates DNA synthesis; it lacks a 3’hydroxyl terminus required for the addition of other deoxynucleotides during synthesis of DNA by DNA polymerases. Elegant methods have been explored to introduce the HSV tk gene into cancer cells (Moolten et al.,

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1990).Methods used include introduction of fibroblasts expressing HSV tk (Culver et al., 1992; Ram et al., 1993), recombinant virus (Caruso et al., 1993) or even the direct injection of DNA (Vile and Hart 1993). In each of these protocols, the animals were subsequently treated with the chain terminators, either acyclovir or gancyclovir, and the tumors significantly regressed. The protocol for gene therapy in brain tumors is diagrammed in Fig 2. A major limitation for this ablative gene therapy is our inability to target vectors expressing the HSV tk or other genes specifically to cancer cells. Another limitation is the toxicity of gancyclovir at tumoricidal concentrations. Perhaps this difficulty can be mitigated by tailoring the enzyme for enhanced phosphorylation of gancyclovir or related nucleoside analogs. Thus, instead of utilizing the wild-type HSV tk, one could utilize a mutant that preferentially phosphorylates gancyclovir (Fig. 2). In order to create HSV TKs to specifically phosphorylate nucleoside analogs we have remodeled the active site by substituting raridom sequences. HSV tk has been cloned (McKnight, 1980; Wagner et al., 1981), and the active site can be delineated roughly based on kinetic studies, binding studies and site directed mutagenesis (Darby et al., 1986; Liu and Summers, 1988). In the absence of a three-dimensional structure of this enzyme, there is still sufficient information to designate key residues that function in catalysis and to assess their role in substrate specificity. By aligning the nucleotide sequence of 12 Herpesviridae tks, Balasubramaniam et al. (1990)identified 6 highly conserved regions and designated them as sites 1 to 6. Two of the domains, sites 3 and 4 (HSV-1 TK residues 162-164 and 171-173, respectively), were proposed as the nucleoside binding site. We have substiTumor Cells

+Gancyclovir

Termination of DNA replication

FIGURE 2 Protocol for gene therapy for gliomas with herpes thymidine kinase and gallcyclovir. Instead of the wild type gene, we propose using a mutant gene that has been selected based on increased sensitivity to gancyclovir.

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tuted random nucleotide sequences in place of the normal nucleotides in both sites 3 and 4, individually and then in combination. From libraries with random substitutions we have obtained mutant enzymes with unique properties and substrate specificities. Sequences including and surrounding site 4 (amino acid residues 165 to 177 in HSV-tk were the focus of a series of random sequence selection studies (Dube et al., 1991a; b; Munir et al., 1993; 1994), first to identify critical amino acid residues and then to identify new mutant enzymes. In order to determine the essentiality of different amino acids within an 11 codon sequence in the putative nucleoside-binding site of HSV TK (Munir et al., 1992), we used the strategy outlined in Fig. 3. The insert contained a stretch of 33 randomized nucleotides. The extent of randomization was 20%; that is, at each position 80% was the wild type nucleotide and 20% 5‘

52-mer

3

“ 7

3-

1 1

56-mer

Extension with Pol I and PCR Amplification

5-

“ 3 3’

“r 5’

3’-

Digestion with Kpnl and Sad

-’5 KDnI

Transformation Of tk- KY895

Antibiotic selection

FIGURE 3

TK-selection

Detailed method for the insertion of an oligonucleotide with 33 random residues into the herpes thymidine kinase gene.

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consisted of the other three nucleotides in equal proportion. The bias toward the wild type sequence increases the likelihood of recovering functional mutants, since there is a greater percentage of wild type sequences at each position. In such a construct, the entire wild type sequence would be present at a frequency of 0.06% and approximately 23% of the random inserts would contain a termination codon. The oligonucleotide was assembled by hybridizing a synthetic 52-mer corresponding to the wild type HSV tk sequence with a 56-mer containing random nucleotides. The hybrid region, consisting of 11complementary nucleotides, was extended with the Klenow fragment of E. coli DNA polymerase I to produce a double-stranded DNA product. This random sequence containing fragment was amplified using the polymerase chain reaction and then inserted into the putative nucleosidebinding site of the HSV tk gene in place of a nonfunctional insert. After electroporation, 53,000 E. coli transformants were obtained, of which 190 incorporated thymidine. Sequence analyses of 90 functional variants revealed a high degree of promiscuity in accommodating different types of amino acid substitutions at most positions (Fig. 4). For example, a large number of amino acids substituted for Ala16’ and Ala’@. In contrast, only

Ala Ala Cys Ala Ala Ser Ala Thr His

Val Val Tyr Val Val Val Val Met

Gly Gly Ser Ser Arg Gly Gly Thr Ser Asp Ser Arg Gly Gly Thr Gly Ser

Thr G~Y Pro Ser G~Y G~Y Ser Ser Ser Ser Thr Val Arg Tyr Asn Tyr Tyr Thr Val Val Phe Val His Gly Phe Ser Phe Glv Phe

Phe Thr Ser Val

ASP Thr Val Thr CYS Ser Ile Val Ser

Gln Met Ile Ile Val Phe Gly Phe Met Leu Phe

Pro Ile Ala Ala Leu Leu Cys Tyr Pro Ala Ala 165

166 167

168

169

170

171

172

173

174

175

Wild-type amino acid sequence FIGURE 4 A compendium of functionally active substitutions obtained by substituting random sequences into site 4 in HSV tk. From Munir et al., (1992). With permission. The wild type amino acid residues are shown on the X-axis in bold face.

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Loeb

one mutant was identified with a substitution at Ala17’ and Cys”’; only Phe was found to substitute for Tyr’” and no changes were observed at Pro’73. To further define permissible substitutions at specified positions, a library was constructed with 100% randomization using an equimolar ratio of A, T, G, and C to synthesize codons 171,172,173, and 175. A total of 600,000 variants were tested and only 5 were active. Again, was found to be conserved, and only tyrosine and phenylalanine were found at position 172. These data indicate both the stringent requirement for certain amino acids for activity at certain positions and the exceptional flexibility for substitutions at other positions. In order to analyze mutants with multiple substitutions within the active site we (Munir et al., 1993; 1994) utilized a 100% random nucleotide segment in place of the 33 nucleotides that span codons 165 to 175 of HSV1 tk gene. From a library of approximately 2 X lo6 transformants, we obtained 1540 new active TK mutants. These mutants were subjected to a secondary screening procedure using medium containing azidothymidine (AZT), a thymidine analog terminating DNA synthesis and used in the treatment of HIV infections (Larder et al., 1989). Selective conditions were chosen so that HSV TK mutants rapidly phosphorylating AZT would not form colonies. The product, AZT monophosphate, is further phosphorylated and incorporated into bacterial DNA and terminates DNA replication. Two AZT-sensitive mutants were identified; both contain a single amino acid change for Leu17’, isoleucine, or valine. HSV TK was purified from both mutants and exhibited lower Kms for AZT compared to that of the wild type enzyme. This study demonstrates the feasibility of obtaining mutants with alterations in substrate specificity. The thermostability of 50 active HSV TK mutants from the same library was analyzed by preincubating extracts at 42°C prior to assaying for TK activity (Munir et al., 1993). While most of the mutants were thermolabile, one was found to be more thermostable at 42°C than any of the other TKs tested, including the wild type. The thermostable mutant contains three -+ His; Ala167 +. Ser and Ala’74-+ Val. That amino acid substitutions: the thermostability under selective conditions was conferred by a thermosta bile thymidine kinase was verified by measurements with the purified enzyme and with an in vitro synthesized mutant HSV TK. In the latter studies, the gene encoding the triple mutant was inserted into a vector with a promoter for T3 RNA polymerase, and the RNA produced by transcription in vitro was translated using rabbit reticulocyte lysates. After preincubation at 42°C for 45 min, the translation product of the triple mutant RNA lost less than 10% of its activity while the wild type translation product lost greater than 85% of its activity. Most interestingly, single substitutions and the double substitutions that were tested at these positions do not result in enhanced thermostability . It can be argued that this mutant would never be obtained by natural selection, since none of the individual substitutions

Unnatural Nucleotide Sequences in Biopharmaceutics

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would offer a selective advantage, and simultaneous multiple substitutions would be very infrequent. It remains to be determined if this thermostability would confer enhanced enzyme longevity in cells. To further analyze substrate specificity, we substituted random se~ ~ , and flanking amino quences for a segment of site 3 (Asp162,A I - ~ 'His'") acid residues (Phe161and (Black and Loeb, 1993) for which a structural role at the nucleoside binding site has been ascribed (Balasubramaniam et al., 1990).We substituted 18 nucleotides within this region with a duplex containing 20% random sequences. Approximately 260 mutants were screened and 32% conferred TK activity. Three of the residues, Arg163,and appear to be highly conserved, especially with respect to the type of residues able to substitute. Secondary screening studies indicated that several of the mutants have a higher affinity for acyclovir and AZT than does the wild type. An analysis was carried out on eight of the mutants using the in vitro transcription vector system and the rabbit reticulocyte lysate cell-free translation system. Two of the mutants had an elevated TK activity, two were significantly thermolabile, and three exhibited enhanced efficiency in the phosphorylation of different nucleoside analogs. All three mutants that exhibited differences in the phosphorylation of nucleoside analogs, AZT, acyclovir, and gancyclovir as well as deoxycytidine, contain substitutions at Phe16', suggestingthat phenylalanine at this position directly interacts with the substrate. One of the mutants containing two substitutions was gancyclovir sensitive in culture and exhibited a five-fold increase in the rate of phosphorylation of gancyclovir. The spectrum of changes in stability and kinetics using random sequence substitutions at site 3 and site 4 is diagrammed in Fig. 5. In studies aimed at changing substrate specificity, we simultaneously substituted random nucleotides at both sites 3 and 4 (Black and Loeb, unpublished results). From a library of more than one million transformants, 426 active mutants were obtained. Several of the mutants are more active than the wild type in phosphorylating acyclovir. More importantly, with several mutants the ratio of phosphorylation of gancyclovir to thymidine was SO-fold greater than that of the wild type and thus may kill tumor cells more efficiently. It should be noted that these mutants have between three and six amino acid alterations within the nucleoside binding site; these combinations would not likely have been chosen by rational remodeling of the active site of HSV TK. 6. Protection of Bone Marrow

The tailoring of enzymes to preferentially metabolize specific drugs may provide a method to protect bone marrow precursors against the toxicity of chemotherapeutic agents. The major limitation in dose escalation for most chemotherapeutic agents is bone marrow toxicity. Overexpression of

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5

fold increase over wl

b

Km

(pM)

Vmax (pmollmin)

r-iw wl

I

0.5

1.600

7.800

dT ACV GCV AZT

I

?F

I

Mutant

..Y*

c

Heat stability 42°C

0

5

10 i7me

15

20

FIGURE 5 A summary of different mutants and their properties obtained from random substitutions within sites 3 and 4 of HSV tk. The amino acid sequence of the wild type is given large capital letters. The properties diagrammed were obtained from studies on purified HSV TKs obtained from each of the mutants. References for the original studies are given in the text. (A) Increased activity in the rate of phosphorylation of ACV (acyclovir), GCV (gancyclovir) and AZT (azidothymidine). (B) K, and V,,, calculated from double reciprocal plots. (C) Rate of phosphorylation of dT. (D) In Vitro synthesized enzymes were preincubated at 42°C for times indicated, then assayed at 3 P C .

the human DNA repair protein 06-methylguanine DNA methyltransferase in hematopoietic cells via retrovirus-mediated gene transfer has been shown to protect murine bone marrow progenitor cells against the methylating drug BCNU (Moritz et al., 1995). Moreover, protection was exhibited in vivo in mice exposed to BCNU. These studies establish the feasibility of inserted DNA repair proteins into bone marrow progenitors for protection against marrow toxicity. Might it be possible to reengineer DNA repair proteins or detoxifying enzymes to protect bone marrow cells against specific agents? A first step in this direction has been carried out by substituting random nucleotide sequences in the substrate binding site on glutathione S transferase (Gulick and Fahl, 1995).Bacteria harboring the randomized plasmids were subjected

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to eight rounds of enzyme induction and treatment with increasing concentrations of mechlorethamine, an alkylating agent used to treat Hodgkin’s disease and lymphomas. Mutant enzymes were isolated from resistant bacteria and were shown to exhibit up to a 15-foldincrease in kcat for mechlorethamine conjugation. It remains to be determined if these mutant enzymes will protect human bone marrow cells from the toxicity of mechlorethamine.

X. Status, Summary, and Future Prospects Traditionally, the goal of chemical pharmacology has been to identify a new lead compound and then carry out sequential modifications to improve its therapeutic efficacy. Similarly, the goal of enzymology has been to obtain homogeneous preparations of enzymes and to analyze their mechanism of catalysis. For both sciences the “holy grail” was to obtain a single molecular species. The multiple chemical reaction products that constituted the tar of the organic chemist was the least desired outcome of a synthetic procedure. The goal was purity, not diversity. In contrast, the success of combinatorial chemistry and molecular evolution requires the attainment of enormous diversity. With the use of new molecular and analytic techniques, we possess the tools to handle this diversity and to select and even evolve individual species to target pathological processes. Random sequence selection, the focus of this review, has made it feasible to select enzyme molecules with enhanced catalytic efficiency from a pool of random sequences in the absence of a detailed knowledge of the threedimensional structure of the enzyme. In principle, it should be possible to select individual nucleotide sequences that encode specific biologically active proteins from as many as 10l2transformed E. coli. Moreover, it soon may be feasible to evolve protein molecules to carry out specific catalytic function through repetitive cycles of mutagenesis and selection. This methodology has been most extensively applied to HSV-1 TK with the goal of creating mutant enzymes that exhibit enhanced activity with gancyclovir and other chain terminators of DNA replication. From a total of more than 3000 active HSV-1 tk mutants, we have identified mutant enzymes that have markedly different properties than that of the wild type. Some mutant enzymes preferentially phosphorylate nucleoside analogs, including AZT, acyclovir, and gancyclovir; others are more active in phosphorylating thymidine; and one maintains activity at elevated temperatures. This methodology has been applied to several enzymes and proteins and should be applicable to any enzyme for which genetic selection assays can be devised. Since random sequence selection is based on functional complementation, it should be possible to use this technique to create new enzymes that are not present in nature. In its simplest form, one could transfect bacteria with random sequences and select for the presence of activities that enable

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the bacteria to utilize specific substrates for growth. Random sequence selection is unique in that selection is based on function and not just simply changes in binding affinity. It seems a reasonable expectation that this method, combined with other methods collectively referred to as “applied molecular evolution,” will result in the production of new enzymes and other biological molecules that are entirely different from those present in nature. The widespread application of techniques of applied molecular evolution could add a new dimension to pharmacology and related sciences. Acknowledgements These studies were supported by grants from the National Institutes of Health, OIGR35-CA39903 and AG-01751. I thank the following members of this laboratory who in the past 10 years have increased our understanding of random sequence mutagenesis: M. S. Z. Horwitz, D. K. Dube, K. M. Munir, and M. E. Black, and to F. Christians, M. Suzuki, and A. Skandalis who continue. I also thank Mary Whiting for editorial assistance. This review is dedicated to the memory of Hal Weintraub, whose unique insights into biology continuously encourage our efforts.

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Honvitz, M. S., and Loeb, L. A. (1988). DNA sequences of random origin as probes of Escherichia coli promoter architecture. J. Biol. Chem. 263, 14724-14731 Ji, J., and Loeb, L. A. (1994).Fidelity of HIV-1reverse transcriptase copying a hypervariable region of the HIV-1 env gene. Virology 199, 323-330. Joyce, G. F. (1989). RNA evolution and the origins of life. Nature 338, 217-224. Joyce, G. F. (1992). Selective amplification techniques for optimization of ribozyme function. Antisense RNA and DNA, 353-372. Larder, B. A., Darby, G., and Richman, D. D. (1989).HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science, 243, 1731-1734. Lehman, N., and Joyce, G. F. (1993).Evolution in vitro of an RNA enzyme with altered metal dependence. Nature 361, 182-185. Lehman, N., and Joyce, G. F. (1993). Evolution in vitro: analysis of a lineage of ribozymes. Curr. Biol., 3, 723-734. Lerner, R. A., Benkovic, S. J., and Schultz, P. G. (1991). At the crossroads of chemistry and immunology: Catalytic antibodies. Science 252, 659-668. Little, J. W. (1990). Saturation mutagenesis of specific codons: elimination of molecules with stop codons from mixed pools of DNA. Gene 88, 113-1 15. Liu, Q., and Summers, W. C. (1988).Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. Virology 163, 638-642. Matthews, D. J., and Wells, J. A. (1993).Substrate phage: Selection of protease substrates by monovalent phage display. Science 260, 11 13-1 117. McClure, W. R. (1985). Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem 54,171-204. McKnight, S . L. (1980). The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 8 , 5949-5964. Mills, D. R., Peterson, R. L., and Spiegelman, S. (1967).An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc. Natl. Acad. Sci. U.S.A. 58, 217-224. Moolten, F. L., Wells, J. M., Heyman, R. A., and Evans, R. M. (1990).Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene. Hum. Gene Therap. 1, 125-134. Morgan, C. M., Deslongchamps, G., de Mendoza, J., and Rebek, J. (1993). High affinity complexation of adenosine derivatives within induced binding pockets. (Convergent functional groups, part 13). J. Am. Chem. SOL.115, 3548-3558. Moritz, T., Mackay, W., Glassner, B. J., Williams, D. A., and Samson, L. (1995).Retrovirusmediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitorsourea-induced toxicity in vitro and in vivo. Cancer Res. 55,2608-2614. Munir, K. M., French, D. C., Dube, D. K., and Loeb, L. A. (1992). Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected] [published erratum appears in 1. Biol. Chem. 261, 15258, 1992,l. J. Biol. Chem. 267, 6584-6589. Munir, K. M., French, D. C., Dube, D. K., and Loeb, L. A. (1994).Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection. Protein Eng. 7, 83-9. Munir, K. M., French, D. C., and Loeb, L. A. (1993).Thymidine kinase mutants obtained by random sequence selection. Proc. Natl. Acad. Sci. U.S.A. 90, 4012-4016. Oliphant, A. R., and Struhl, K. (1987). The use of random-sequence oligonucleotides for determining consensus sequences. Methods Enzymol. 155, 568-582. Oliphant, A. R., Brandl, C., and Struhl, K. (1989).Defining the sequence specificity of DNAbinding proteins by selecting binding sites from random-sequence oligonucleotides: Analysis of yeast GCN4 protein. Mol. Cell. Biol. 9, 2944-2949. Oliphant, A. R., and Struhl, R. (1989). An efficient method for generating proteins with altered enzymatic properties: application to b-lactamase. Proc. Natl. Acad. Sci. U.S.A. 86, 9094-9098.

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Ram, Z., Culver, K. W., Walbridge, S., Frank, J. A., Blaese, R. M., and Oldfield, E. H. (1993). Toxicity studies of retroviral-mediated gene transfer for the treatment of brain tumors. J . Neurosurg. 79,400-407. Reidhaar-Olson, J. F., Bowie, J. U., Breyer, R. M., Hu, J. C., Knight, K. L., Lim, W. A., Mossing, M. C., Parsell, D. A., Shoemaker, K. R., and Sauer, R. T. (1991). Random mutagenesis of protein sequences using oligonucleotide cassettes. Methods Enzymol. 208, 564-586. Sassanfar, M., and Szostak, J. W. (1993).An RNA motif that binds ATP. Nature 364,550-553. Scott, J. K., and Craig, L. (1994).Random peptide libraries. Cum. Opin. Biotechnol. 5,40-48. Scott, J. K., and Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386-390. Sondek, J., and Shortle, D. (1992). A general strategy for random insertion and substitution mutagenesis: Substoichiometric coupling of trinucleotide phosphoramidites. Proc. Natl. Acad. Sci. U.S.A. 89, 3581-3585. Stemmer, W. P. C. (1994).DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. U.S.A. 91, 10747-10751. Sweasy, J. B., and Loeb, L. A. (1993). Detection and characterization of mammalian DNA polymerase-fl mutants by functional complementation in Escherichia coli. Proc. NatL Acad. Sci. U.S.A. 90, 4626-4630. Tuerk, C., Eddy, S., Parma., Gold, L. (1990). Autogenous translational operator recognized by bacteriophage T4 DNA polymerase. J. Mol. Biol. 213, 749-61. Vile, R. G., and Hart, I. R. (1993).Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer Res. 53, 3860-3864. Wagner, M. J., Sharp, J. A., and Summers, W. C. (1981).Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc. Natl. Acud. Sci. U.S.A. 78,1441-1445. Wilson, C., and Szostak, J. W. (1995).In vitro evolution of a self-alkylating ribozyme. Nature 374, 777-782. Winter, G., and Milstein, C . (1991). Man-made antibodies. Nature 349, 293-299.

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Simon P. Aiken* Robert Zaczekt Barry S. Brown$' *Department of Pharmacology Zeneca Pharmaceuticals Wilmington, Delaware I9850 Departments of +Central Nervous System Diseases Research and 'Preclinical Pharmacology, The DuPont Merck Pharmaceutical Company Wilmington, Delaware I9880

Pharmacology of the Neurotransmitter Release Enhancer Linopirdine (DuP 996), and Insights into I t s Mechanism of Action

1. Introduction A. The Unmet Medical Need of Alzheimer's Disease The fact that the treatment of Alzheimer's disease represents one of the foremost challenges at present to the pharmaceutical industry is not in question. In economic terms, the annual cost of this disease has been estimated at $67.3 billion in the United States for 1991 (Ernst and Hay, 1994), and it can be predicted that the prevalence of the disease will increase drastically as populations in Western countries age. The nature of the disease causes it to severely impact the families of patients, as well as the patients themselves, and it is currently the fourth leading cause of death in the United States. Clinical management of Alzheimer's disease is handicapped by the To whom correspondence should be addressed. Advances tn Pharmacology, Volume 35 Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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heterogeneity of the illness, and by the difficulty of detecting it in the earlier stages. The etiology of Alzheimer’s disease remains poorly understood in spite of a number of interesting lines of study, such as the role of P-amyloid, tau protein, and genetic influences. While our understanding of the pathology of Alzheimer’s disease is steadily increasing, choices for drug treatment remain extremely limited, and only modest improvements or changes in the progression of the disease can normally be achieved.

B. The Cholinergic Hypothesis of Alzheimer’s Disease Current therapies and also previous strategies for the treatment of Alzheimer’s are based on the cholinergic hypothesis of Alzheimer’s disease. This hypothesis claims that the neurological signs of Alzheimer’s disease (memory loss, cognitive dysfunction) are due, at least in part, to impairment of cholinergic pathways in the central nervous system (CNS). Evidence for a role of the cholinergic system in Alzheimer’s disease includes the following. Firstly, brains of Alzheimer’s patients are found at postmortem to be depleted in choline acetyltransferase and acetylcholinesterase [the enzymes responsible for synthesis and degradation of acetylcholine (ACh), respectively] in the cortex, hippocampus, and amygdala (Daviesand Maloney, 1976).In experimental animals, cognitive function can be compromised by lesioning cholinergic cell bodies of the nucleus basalis magnocellularis (Hepler et al., 1985) or the cholinergic septohippocampal neurons following fimbria-fornix transection (Olton et al., 1991).Furthermore, Winkler etal. (1995)demonstrated that cognitive deficits induced by nucleus basalis magnocellularis lesions could be reversed by neocortical grafts of ACh-releasingfibroblasts. Finally, the fact that some acetylcholinesteraseinhibitors, most notably tacrine (tetrahydroaminoacridine; CognexB) have clinical efficacy, albeit limited, in Alzheimer’s disease, can be taken as evidence for the involvement of the cholinergic system in this disease. Attempts to treat Alzheimer’s disease by stimulating the cholinergic system have so far met with only modest success. The use of acetylcholine precursors (e.g., choline, a-glycerylphosphoryIcholine, lecithin) appears to have no therapeutic benefit (Vogel-Scibilia and Gershon, 1989; Davidson and Stern, 1991; Sarter, 1991). The use of acetylcholinesterase inhibitors such as physostigmine has been pursued with the intention of raising ACh levels in the synaptic cleft and thus improving the transmission by neurons in a compromised pathway. Such drugs suffer the liabilities of minimal efficacy and a low therapeutic ratio due to the possibility of neuronal overload, receptor desensitization, and hepatotoxicity. The acetylcholinesterase inhibitor tacrine has, however, been shown to have beneficial effects, at least in some patients (e.g., Summers et al., 1986), even though very careful

Pharmacology ofthe Neurotransmitter Release Enhancer Linopirdine (DuP 996)

35 I

patient selection may be required to demonstrate the therapeutic value. A third strategy for improving cholinergic function would be the administration of muscarinic agonists (e.g., arecoline, oxotremorine, bethanecol), but such agents have not yet demonstrated the required specificity of effect to be of more than marginal benefit (McKinney and Coyle, 1991). C. Rationale behind the Use of Linopirdine Drugs that enhance the release of neurotransmitters, or which otherwise affect neurotransmission via a presynaptic action, have potential use in a wide range of neurological diseases, including perhaps Alzheimer's disease, Parkinsonism, and depression. The discovery of linopirdine [DuP 996; 3,3bis(4-pyridinylmethyl)-l-phenylindolin-2-0ne; AVIVATM;Fig. 11 offered a new approach to the improvement of cholinergic function, and the drug has subsequently been used in clinical trials in Alzheimer's patients. Linopirdine was found to increase the release of ACh and other neurotransmitters in response to a depolarizing stimulus (reviewedby Zaczek and Saydoff, 1993). This mode of action, with no effect on basal release, suggests that linopirdine would not be prone to the same toxicity effects and low therapeutic ratio as acetylcholinesteraseinhibitors or muscarinic agonists. By facilitating neurotransmission only during neuronal firing, it might be expected that the functioning of impaired cholinergicpathways in the CNS could be improved. Such an approach has been reviewed by De Souza (1993). Figure 2 represents two of the traditional approaches to improving cholinergic function-cholinesterase inhibition and muscarinic stimulation-and compares them to the effects of linopirdine at the synapse. Since the postsynaptic receptors are not being continually stimulated (either by prolonged exposure to ACh in the absence of degrading enzyme or by

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FIGURE I Structure of linopirdine (DuP 996). Molecular weight 391.5 (as free base). Almost insolublein water (75 mg/liter),very sparingly soluble in hexane (148 rngfliter),sparingly soluble in 0.1 N HCl (21.8 g/liter), and soluble in alcohols.

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Normal Cholinergic Transmission

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Enhanced Stimulated Release

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exposure to a nonmetabolized agonist), linopirdine could be expected to have a different toxicity profile from cholinesterase inhibitors or muscarinic agonists. There is no reason to suppose that the cholinergic approach to the treatment of Alzheimer’s disease would reverse the gross histological manifestations of the disease: the neurofibrillary tangles and neuritic (senile) plaques. Continued studies on the etiology of Alzheimer’s are clearly of the utmost importance in determining new strategies for drug treatment, and possibly also for prevention. However, the fact that an acetylcholinesterase inhibitor such as tacrine has been demonstrated to be of some therapeutic value is sufficient indication that we should seek improved ways of enhancing cholinergic function in patients. Such treatment strategies could be best used in the early stages of the disease when a higher proportion of central cholinergic neurons remain intact. An improvement in cholinergic function would be expected ultimately to raise the quality of life for patients in the early or middle phases of the disease, rather than to reverse the disease itself. Given the extreme debilitating nature of Alzheimer’s disease and its prevalence in the population, such a goal is indeed worthy of pursuit. We will review the pharmacological actions of linopirdine with a particular emphasis on understanding the mechanism by which these effects are achieved. It is hoped that a better understanding of linopirdine’s action will lead to new improved compounds, and perhaps will suggest new target sites for the treatment of Alzheimer’s disease and other neurological disorders.

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II. Pharmacology of Linopirdine

A. Enhancement of Evoked Neurotransmitter Release by Linopirdine 1. Characteristics of ACh Release Enhancement

Aspects of linopirdine’s release-enhancing activity have been reviewed by Zaczek et al. (1994, 1995). Nickolson et al. (1990) first demonstrated that linopirdine enhanced the ACh release evoked by stimulation with a high K+ concentration in rat brain slices (hippocampus, cortex, caudate nucleus). The results of a typical experiment are shown in Fig. 3a. Brain slices suspended in a medium containing 3 mM K+ are first loaded with [3H]choline.The release of radioactivity is determined in response to raising the K+ concentration to 20 mM or 25 mM for a 4-min period. Pohorecki et al. (1988) have demonstrated that at least 90% of this radioactivity is associated with ACh. Release enhancement is quantified by applying a test compound (linopirdine) after this “Sl” K+ pulse, and then repeating the high K+ pulse (“S2”). Release can be expressed in terms of an S2/S1 ratio. As seen in Fig. 3a, linopirdine at 10 p M causes an approximately threefold increase in the S2/S1 ratio in rat cortical slices. Figure 4 shows the concentration-response relationship for enhancement of ACh release. An ECSOof approximately 5 p M for this release-enhancing effect has been calculated (Zaczek et al., 1993b). It is very important to note that linopirdine has no effect on the basal release. That is, when the drug is applied (Fig. 3a, fraction 5), release is not enhanced until the K+ concentration is raised. The use of a high K+ pulse to depolarize cells can be considered in some ways as a “model” for nerve-evoked release. Thus, linopirdine has been termed a “depolarization-activated release enhancer” (Zaczek and Saydoff, 1993). Vickroy (1993) demonstrated enhanced release of ACh from rat hippocampal synaptosomes by linopirdine in response to a raised K+ or Ca2+ concentration. This effect was found to be strongly dependent on membrane potential with linopirdine failing to significantly enhance Ca2+-stimulated release if the external K+ concentration was either too low (4.7 to 9.4 mM) or too high (30 to 40 mM). Linopirdine also enhances the release of ACh due to the Na+ channel opener veratridine (Zaczek et al., 1993b). Interestingly, however, it has no effect on electrically induced ACh release in brain slices (Smith et al., 1993a; Zaczek et al., 1993b). Although this finding might provide clues to the mechanism of action of linopirdine, there have to date been no satisfactory explanations presented as to why electrically induced release is not affected. Linopirdine also enhances the ACh release stimulated by the A-type K+ channel blocker 3,4-diaminopyridine (3,4-DAP; Zaczek et al., 1995), but not the release stimulated by the excitatory amino acid N-methyl-D-aspartate (Zaczek et al., 1993b).

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Fraction Number FIGURE 3 (a) Stimulation of [’HIACh release from rat cortical slices by raised K+, and the effect of linopirdine. Release during 4 min time bins is expressed as a percentage of the total radioactivity.K+ concentration was raised to 20 mM during fraction 4 (the “Sl” response) and again during fraction 8 (“S2”). In the control, S2 is slightly smaller than S1. When linopirdine (10 p M ) is present during fractions 5 to 8, the S2 response is significantlyenhanced. Data are means from at least 4 determinations. (b) 3,4-DAP stimulates basal release of ACh, but not K+-stimulatedrelease. Experimental details as for (a), 3,4-DAP (10p M )present during fractions 5-8. Adapted from Maciag et al. (1994).

2. K+ Channel Blockers as Release Enhancers

The activity of linopirdine in the ACh release enhancement assay shown in Fig. 3a is an interesting contrast to the activity of 3,4-DAP. The action of 3,4-DAP, shown in Fig. 3b, is to enhance basal release of ACh, with no effect on the K+-stimulatedrelease. In investigating the mechanism by which linopirdine increases neurotransmitter release, we may wish to consider the action of K+ channel blockers, since there is ample evidence that such compounds enhance release. Blockers of A-type K+ current (IA) such as 4-aminopyridine (4-AP) and a-

Pharmacology of the Neurotransmitter Release Enhancer Linopirdine (DuP 996)

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[Linopirdine] (pM) FIGURE 4 Concentration-response relationship for the enhancement of K+-evokedACh release by linopirdine in rat hippocampal slices. Release is expressed as a percentage increase relativeto the control S2h1 ratio (means ? SEM, n = 4).Data are from Nickolson et al. (1990).

dendrotoxin are known to increase the release of glutamate from guineapig cerebrocortical synaptosomes (Tibbs et al., 1989). The compound tetraethylammonium (TEA), which blocks a number of K+ channels, has been shown to enhance neurotransmitter release from synaptosomes (Bowyer and Weiner, 1990) and at the neuromuscular junction (Saint, 1989).In addition, Amoroso et al. (1990) demonstrated enhanced release of gamma-aminobutyric acid (GABA)by block of an adenosine triphosphate-sensitive K+ channel in substantia nigra. Likewise, increased transmitter release at the neuromuscular junction has been produced by block of Ca2+-gatedK+ channels (Robitaille et al., 1993). 4-AP has actually been used in a small clinical trial of Alzheimer’s patients, and deterioration appeared to be less pronounced in patients receiving the drug than in the placebo group (Wesseling et al., 1984). However, if linopirdine were to enhance release via a mechanism involving a K+ channel, the action is clearly distinct from that of the aminopyridines. As stated earlier, it is this ability of linopirdine to enhance the evoked release, without affecting basal release, which makes it potentially a far more useful therapeutic agent than drugs such as I* blockers (Wesseling et al., 1984), or indeed muscarinic agonists and acetylcholinesterase inhibitors. 3. Enhancement of Multiple Neurotransmitter Systems by Linopirdine

Although much of the study of linopirdine is concerned with its enhancement of ACh release, an interesting feature of the pharmacology of this compound is that it also enhances the release of dopamine, glutamate, aspartate, GABA, and serotonin in rat brain slices (Nickolson et al., 1990; Zaczek et al., 1993a). Sensitivity to linopirdine varies according to the

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neurotransmitter, as shown in Fig. 5, with the greatest effect being on dopamine. The effect of linopirdine is clearly not universal to all transmitter systems, however, since there was found to be no effect on release of norepinephrine from rat cortical slices (Nickolson et al., 1990; Zaczek et al., 1 9 9 3 ~ )Moreover, . the effect on norepinephrine release has been shown to be anatomically specific, with hippocampus and cortex being insensitive to linopirdine (i.e., neurons arising in the locus coeruleus) but hypothalamus being sensitive to linopirdine-induced enhancement of norepinephrine release (i.e., neurons arising in the lateral tegmentum; Zaczek et al., 1 9 9 3 ~ ) . Whereas the ability of linopirdine to interact with multiple transmitter systems might at first sight appear to be problematic for its clinical use, this property may actually offer a significant advantage over more traditional approaches to Alzheimer’s therapy. A growing body of evidence suggests the involvement of several neurotransmitter systems in the pathology of Alzheimer’s disease (e.g., Cross et al., 1981; Gottfries, 1985), and so the ability to improve functioning of both cholinergic and other pathways may be advantageous. It remains to be seen whether neurotransmitter release enhancers can be produced with varying selectivity for different transmitters, and whether this approach will be of clinical significance. 4. Linopirdine Enhances Release of Endogenous Neurotransmitters

The release enhancement experiments discussed so far have been concerned with the release of radiolabeled transmitter from tissue loaded with Dopemino

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Linopirdine enhances the K+-evoked release of a variety of neurotransmitters. Release as a percentage above control is shown for dopamine (rat caudate nucleus) and serotonin, ACh, GABA, glutamate, and norepinephrine (all rat cortex) in response to 10 pM linopirdine (means ? SEM, n = 4). Data are from Nickolson et al. (1990).

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a tritiated precursor in vitro. Since data from this type of experiment may represent release of metabolites of the transmitter, Saydoff and Zaczek (1993a) studied the release of endogenous dopamine from slices of rat striatum. It was found that linopirdine enhanced the K+-evoked release of dopamine with similar potency and efficacy to that measured using [ 3H]dopamine. Release of endogenous ACh is also enhanced by linopirdine (Saydoff and Zaczek, 1993b). 5. Linopirdine Enhances Transmitter Release in Viva

In addition to the in vitro studies described above, linopirdine-induced enhancement of neurotransmitter release has been studied in vivo. Nickolson et al. (1990) demonstrated increased cortical levels of extracellular ACh in freely moving rats treated with linopirdine (85 to 850 p g k g , s.c.), using the epidural cup technique. Marynowski et al. (1993) and Smith et al. (1993b) used in vivo microdialysis to demonstrate enhanced ACh release from the hippocampi of freely moving rats following adminstration of linopirdine (10 mg/kg, p.0.; see Fig. 6). These experiments demonstrate the neurotransmitter release-enhancing activity of linopirdine in a whole animal.

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Time (minutes) FIGURE 6 Linopirdine enhances the in vivo release of ACh from rat hippocampus. Dialysis probes were placed into the ventral hippocampi of freely moving rats, and perfused with an Either saline or artificial cerebrospinal fluid solution containing physostigmine (100 linopirdine (10 mgkg) was administered orally at time zero (arrow), and ACh was assayed in 20 min fractions for comparison to baseline measurements. Asterisks denote statistically sign& cant differences from baseline (n = 9). From Zaczek et al. (1995) and Marynowski et al. (1993). Reprinted by permission of Plenum Publishing Corporation.

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B. Behavioral Effects of Linopirdine 1. Linapirdine lmproves Performance in Animal Models of Learning and Memory

Linopirdine at low doses improves performance in a wide range of animal models of cognitive and mnemonic function (reviewed by Zaczek et af., 1995). It should be mentioned that there has been no positive demonstration that these behavioral effects result from neurotransmitter release enhancement. Whether or not the release effects relate directly to the behavioral effects, the latter suggest that linopirdine has the potential to improve cognitive function, perhaps in humans. Linopirdine improved performance of rats in an active avoidance paradigm (Cook et af., 1990). Rats were placed in a cage with a shock grid floor and a wooden pole. Shocks to the grid were preceded by illumination of the cage 10 s previously, and the acquisition of a pole-climbing response to illumination was studied. Treatment with linopirdine (250 or 850 p g k g , s.c.) prior to each block of five trials significantly improved the mean number of shock avoidances (Fig. 7a). When rats were treated with linopirdine (250 or 850 pg/kg, s.c.) after each block of five trials, performance in subsequent trials was also improved (Fig. 7b). These results suggest that linopirdine has effects on both task acquisition and on consolidation of information. The biphasic response to linopirdine seen in Fig. 7 is also seen in several other behavioral tests. While it is not known why higher doses of linopirdine are less effective, it can be speculated that some other counterproductive effect of the drug is becoming apparent at the higher doses. Linopirdine has also been shown to improve performance in both hypoxia-induced and C02-induceddeficitsin passive avoidance in rats (Cook et af., 1990).The rats’ ability to learn a simple procedure was compromised by exposure to either reduced oxygen (6.5%) before, or raised C 0 2 (80%) after the training session. The hypoxia-induced amnesia was attenuated by linopirdine or physostigmine (10,30, or 100 pgikg, S.C. of either drug), and by tacrine (3 or 5 mgkg, s.c.) In rats exposed to C 0 2 after the learning trial, linopirdine (85 or 250 pgikg, s.c.) caused a significant reversal of the C02-induced deficit (Fig. 8). Linopirdine enhanced the performance of mice in an active avoidance task (lever press to escape shock) at doses in the range 0.085 to 2.5 mg/kg, S.C. (Cook et af., 1990). Linopirdine also enhanced lever press acquisition for food in rats at 30 and 100 pgkg, P.o., whereas neither physostigmine (0.3to 100 pgkg, p.0.) nor tacrine (0.6 to 3.0 mgkg, p.0.) had any significant effect (Cook et al., 1990). This activity of linopirdine at much lower doses than physostigmine or tacrine suggests that linopirdine is potentially a far less toxic cognitive enhancer, since mortality in rats was estimated to be equivalent for linopirdine and tacrine (40 mg/kg, s.c.) and physostigmine proved lethal at only 800 p&g, S.C. (DeNoble et af., 1990; lethal dose

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Dose Linopirdine (mglkg) FIGURE 7 Linopirdine improves performance in an active avoidance test (pole jump). The mean numbers of avoidances in the last 25 of a 50-trial test were compared with results from a vehicle-treated control group. Tests were performed in blocks of 5, twice per day for 5 days (morning and afternoon). Asterisks denote statistically significant differences from control (n = 9 in each group, p < 0.05). (a) Linopirdine administered p.0. 30 min prior to each test. (b) Linopirdine administered S.C. immediately after testing. From Cook, L., Nickolson, V. J., Steinfels, G. F., Rohrbach, K. W., and DeNoble, V. J.), (1990). “Cognition enhancement by the acetylcholine releaser DuP 996” Drug Dev. Res. (01990, Wiley-Liss, Inc.) Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

defined as the minimum required to kill at least 2 of 10 rats). Thus, behavioral studies affirm the potential utility of linopirdine as a cognitive enhancer. 2. Linopirdine Improves Learning in Lesioned and Aged Rodents

Linopirdine has been shown to improve spatial learning (Morris water maze) in rats with lesions of the medial septa1 nucleus (Brioni et al., 1993). Linopirdine (10or 30 pgkg, i.p.) improved the retention of a spatial discrimination task in lesioned rats to levels equivalent to sham-operated controls (Fig. 9). This effect of linopirdine was seen when the drug was administered 15 min prior to testing for 4 days, and in the drug-treated group on a final day when no linopirdine was given. Baxter et al. (1993, 1994) used aged

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0

0 2 5 8 5 2 5 0 8 5 0 Dose Linopirdine (pglkg) FIGURE 8 Linopirdine reverses C02-induced deficit in a passive avoidance test. Rats were placed in the light compartment of a standard passive avoidance box. When the rats entered the dark compartment, they received a foot shock. They were then exposed to 80% COZ for 2 min, and were retested 24 hr later. If a rat did not enter the darkened compartment within 3 min on the second test, this was defined as “optimal retention.” Linopirdine was administered 30 min before the first test. Rats exposed to CO2 showed much reduced retention compared with “air” controls. Linopirdine improved retention in the CO2-exposed animals. Asterisks represent significant differences from the C02/saline group ( p < 0.05),with n = 8 for all groups. Adapted from Cook et al. (1990).

0 10 30 Dose Linoplrdlne (pg/kg) FIGURE 9 Linopirdine improves retention of a learned spatial discrimination task in septallesioned rats. Rats were trained in a Morris water maze to find a raised platform, and then were presented with a choice between the normal platform (in a known location) and an “unstable” one of the same appearance. The number of errors (ix., the rat touching the wrong platform) was recorded during a I-min test. Linopirdine was administered i.p. 15 min before the trial on each of 4 days, and then the “retention” trial was performed on the next day (without drug). Lesioned rats made significantly more errors in this retention test than the sham-operated rats, but linopirdine-treated lesioned animals performed comparably with the sham-operated ones. Means ? SEM; the asterisk represents statistically significant difference ( p < 0.05) from the other 3 groups. Adapted from Brioni et al. (1993). 0

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36 I

rats (24 months) in a similar test and showed that linopirdine improved spatial discrimination at 0.25, 2.5, or 8.5 mg/kg, p.0. Although there are no satisfactory animal models of Alzheimer’s disease, the activity of linopirdine in these 2 tests does auger well for the clinical efficacy of linopirdine or a similarly acting compound. 3. Conclusions on the Behavioral Activity of Linopirdine

The work cited above demonstrates that linopirdine is a very potent cognitive enhancer in a variety of animal models. It should be remembered that while the enhancement of ACh release has received the most study, enhancement of any of a range of neurotransmitters (dopamine, serotonin, GABA, glutamate, aspartate) could also underlie the behavioral effects of linopirdine. It has been noted (Zaczek and Saydoff, 1993) that the effective doses of linopirdine in most of the behavioral assays are significantly lower than the doses required to demonstrate in vivo enhancement of ACh release (5 to 10 mg/kg; Marynowski et al., 1993). While this may simply reflect the lack of sensitivity of the in vivo release assay, it may also indicate that enhancement of ACh release, or of other neurotransmitters, may not be directly related to the behavioral effects. For the clinical use of a drug of this type, it is not clear whether behavioral effects in animals or neurochernical effects (e.g., release enhancement) would be the better indicator of therapeutic efficacy. C. Other Effects of Linopirdine 1. Electroencephalographic Efects

Preclinical studies in a variety of models and assays have sought evidence for the utility of linopirdine as a cognitive enhancer in humans. A simple measurement of CNS activity can be provided by electroencephalographic (EEG) studies. Animal EEG studies have demonstrated that linopirdine increases cortical activity. In rats, Cook et al. (1990)reported increased alpha and beta activity and also small (nonsignificant)decreases in delta and theta activity in response to 0.3, 1.0, and 3.0 mgkg, S.C. doses of linopirdine. As seen in certain other paradigms (e.g., Figs. 7 and 8), this effect of linopirdine appeared to be biphasic with the maximal effect on alphdbeta activity being at 1.0 mgkg. Physostigmine had similar effects to linopirdine, but at lower doses (10 to 100 pglkg, s.c.) Similar results were obtained in the rat, rabbit, and dog by Alberici et al. (1991), although the rabbit exhibited decreased alpha activity. In clinical studies, Saletu et al. (1989) suggested that a 30 mg p.0. dose of linopirdine improved vigilance in healthy male volunteers (aged 18 to 40 years). This suggestion was based on the findings of augmented total power and increased absolute power in the alpha and alpha-adjacent beta activity. It was noted that the EEG effects of linopirdine were most pronounced in

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the temporo-occipital, temporo-frontal, parietal, and frontal regions, which are areas expected to be the most afflicted in Alzheimer’s disease. Similar results were also obtained by Saletu et al. (1991)in an EEG study of healthy elderly male subjects (aged 60 to 80 years). Dosing for 10 days with 20 mg linopirdine p.0. twice daily resulted in augmented total power, increased alpha and beta activity, and decreased delta and theta activity. These characteristics are opposite to those associated with Alzheimer’s disease, and are indicative of enhanced vigilance. Again, EEG changes were greatest in those areas that are compromised most in Alzheimer’s patients. 2. Metabolic Eficts

Dent et al. (1993a) studied the effect of linopirdine on brain metabolic function, by measuring rat cerebral glucose metabolism with the 2deoxyglucose (2-DG) technique. In normal rats, linopirdine did not affect 2-DG metabolism in doses of 0.01, 0.1, or 1.0 mgkg, S.C. However, care should be taken in interpreting this result, since the highest dose used was below threshold for demonstrating enhanced in vivo release of ACh (Marynowski et al., 1993). 2-DG metabolism was also studied in hypoxia-exposed rats, since linopirdine has been shown to reverse hypoxia-induced deficits (Cook et al., 1990). Rats made hypoxic by exposure to 6.5% oxygen for 30 min showed slight (but not statistically significant) increases in 2-DG metabolism relative to controls (Dent et al., 1993a). Previous studies have suggested that hypoxia does significantly increase brain glucose metabolism (e.g., Pulsinelli and Duffy, 1979).Linopirdine (1.0 m&g, s.c.) caused signficant decreases in glucose metabolism (to below control levels) in discrete brain regions in hypoxia-exposed animals (Dent et al., 1993a). The effects of linopirdine were seen in several brain regions thought to be associated with learning and memory leading the authors to suggest that linopirdine may enhance cognition by restoring metabolic activity toward normal levels. Induction of the immediate-early gene c-fos has been studied in animals treated with linopirdine. Dent et al. (1993b) reported no differences in c-fos induction in brains of 2 month old rats treated with linopirdine (10 mglkg, i.p.) 2 hr prior to sacrifice, compared with vehicle-injected controls. However, in 30 month old rats linopirdine caused an increase in fos staining. This effect was most noticeable in the anterior cingulate, somatosensory, piriform, visual, and auditory cortex. It could be speculated that such effects of linopirdine in aged rats could be related to the cognitive effects of the drug (e.g., Baxter et al., 1994). Cholinergic activators such as pilocarpine (Hughes and Dragunow, 1993) and oxotremorine (Bernard et al., 1993) have been shown to increase fos expression in rat brain. 3. Effect on Neuritic Branching

One further action of linopirdine that could be related to its cognitiveenhancing property is the effect of the drug on neuritic branching. In cultured

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embryonic hippocampal neurons, linopirdine (10 p M ) increased the number of neurites per cell after 3 days, and also the number of neuritic branches (M. J. Riggot and W. D. Matthew, unpublished data). This indicates that linopirdine can increase the complexity of neuronal processes; more complex processes have the ability to make an increased number of synaptic contacts. It is possible that an increase in the number of synaptic contacts may have the effect of slowing or reversing the loss of function associated with neuronal trauma of disease. 4. Anticholinesterase Activity

Linopirdine was assayed for anticholinesterase activity by Nickolson et al. (1990).Acetylcholinesterase from rat cortex was inhibited approximately 50% by 100 p M linopirdine (higher concentrations not tested). At 10 p M linopirdine there was approximately 15 % inhibition of acetylcholinesterase, whereas at 1 pM there was no inhibition. Since the effect of linopiridine on in vitro ACh release has an ECSOof approximately 5 p M (Zaczek et al., 1993b) and is near maximal at 10 p M , the slight anticholinesterase activity of this compound is not likely to contribute significantly to its other known neurochemical effects. The anticholinesterase activity should, however, be borne in mind where studies have used very high concentrations of linopirdine (e.g., Tsai et al., 1992). 5. Neuromuscular Effects

All of the effects of linopirdine considered hitherto have been confined to the central nervous system. Pharmacological studies on the peripheral effects of this drug have been performed using neuromuscular junction preparations. Tsai et al. (1992) showed that linopirdine increased twitch tension in the in vitro mouse diaphragm preparation, whether twitch was elicited by direct stimulation (of the muscle) or indirect stimulation (via the nerve). However, an appreciable effect was not seen until a concentration of 64.5 p M , which is much higher than concentrations required for most other in vitro effects. Linopirdine (64.5 to 215 p M )increased the amplitude of the endplate potential and also increased quantal content. The same authors report an increase in miniature endplate potential (MEPP) frequency in the mouse diaphragm in response to linopirdine (143.3 p M ) , and an effect on MEPP amplitude that was biphasic: increased amplitude at 64.5 pM linopirdine, but decreased amplitude at 215 pM. Due to the high concentrations used in this study, the observed effects of linopirdine may be via a different mechanism from those involved in neurotransmitter release. At the frog neuromuscular junction, Provan and Miyamoto (1994) also reported an increase in quantal content with 10 p M linopirdine. This was suggested to result from both an increase in the number of release sites for neurotransmitter and an increase in the probability of release. MEPP frequency was also increased by linopirdine (10 pM).The authors suggest

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that linopirdine may be acting via a Ca2+channel or at a site “upstream” of Ca2+influx since linopirdine in the presence of tetrodotoxin (TTX) and cobalt, or in the presence of omega-conotoxin, failed to increase MEPP frequency. However, this conclusion could be questioned due to the marked decrease in MEPP frequency when TT.X and cobalt, or omega-conotoxin, alone were added. Provan and Miyamoto (1994)demonstrated that linopirdine still increased MEPP frequency in the presence of 4-AP (100 pM). This suggests that the drug is not acting via 4-AP-sensitive K+ channels to affect MEPP frequency. At the concentration used in this study (10 pM), linopirdine did not affect MEPP amplitude, rise time, or decay characteristics, as would be expected if the drug were blocking acetylcholinesterase. The two studies summarized here demonstrate that linopirdine does have peripheral as well as central effects. Having examined the observed effects of linopirdine both in vitro and in vivo, we must now consider the mechanism by which linopirdine enhances neurotransmitter release. The initial leads in identifying this mechanism have come from neurochemical studies.

111. Mechanistic Studies on Linopirdine A. Neurochemical Studies 1. Identification of a Linopirdine Binding Site

Early studies on the mechanism of action of linopirdine centered on the discovery of a high-specificity binding site for the drug in brain (Rominger and Tam, 1991; De Souza et al., 1991; Tam et al., 1991). [3H]Linopirdine was found to bind to rat brain membranes with a Kd of 19 nM and a B,,, of 102 fmol/mg protein (Tam et al., 1991). This binding was saturable, reversible, and time-, pH-, and temperature-dependent. Trypsin treatment decreased binding suggesting that the site was a protein. The binding could also be decreased by elevating the Ca2+concentration (5 to 50 mM), while elevated Mn2+(6 to 12 mM) appeared to enhance the binding. Changes in other ionic concentrations (Na+, K+, Mg2+)did not significantly alter binding. The binding site for linopirdine does not appear to correlate with the binding of any other known pharmacological agent. A wide range of receptor-binding drugs and channel blockers were tested for their ability to displace linopirdine from its binding site (Tam et al., 1991), but none was found to have appreciable effect (see Table I). This suggests that linopirdine has a novel site of action. Tam et al. (1991) pointed out that the fact that a range of channel blockers do not displace linopirdine binding does not preclude the possibility of linopirdine binding on an ion channel. The exact nature of this binding site has not yet been identified.

Pharmacology of the Neurotransmitter Release Enhancer Linopirdine (DuP 996)

TABLE I Pharmacological Agents That Do N o t Bind to the Linopirdine Binding Site on Rat Brain Membranesab

Adrenergic Norepinephrine Isoproterenol Yohimbine Amino acid-related N-methyh-aspartate Strychnine Benzodiazepine Diazepam Cholinergic Atropine Nicotine Dopaminergic SCH 23390 Amphetamine Haloperidol GABAergic GABA Histaminergic Cimetidine Pyrilamine Opioid Naloxone ( +)-SKF-10,047 Phencyclidine Peptidergic Adrenocorticotropic hormone (1-24) Angiotensin I1 Bradykinin Cholecystokinin Cortisol-releasing factor LHRH Neurokinin A Neurokinin B Neuropeptide Y Substance P Thyrotropin-releasing hormone Purinergic Adenosine Serotoninergic Mianserin Haloperidol Ritanserin Cognition-related Caffeine Piracetam Tacrine (continues )

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TABLE I (continued) Reuptake inhibitor Amitriptyline Channel blockers Diltiazem Verapamil Phencyclidine Apamin Veratridine 3,4-DAP ~~

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The binding of [3H]linopirdineto rat brain has been shown to display a heterogenous distribution, using an autoradiographical technique (De Souza et al., 1991) or by microdissection of brain regions (Rominger and Tam, 1991; Tam et al., 1991).The highest specific binding to membrane homogenates was found in the striatum and hypothalamus, whereas cerebellum had the lowest binding. Among peripheral tissues, kidney and ileum were noted to bind linopirdine (Tam et al., 1991). Autoradiography (De Souza et al., 1991) revealed highest specific binding in the cerebral cortex, hippocampus, and amygdala-brain regions that are involved with cognitive function. In particular, the pyramidal cell layers of CA1 and CA3 showed highly localized [3H]linopirdine binding. As yet, no positive link has been established between the high-specificity linopirdine binding site and a pharmacological action of linopirdine. Therefore, it is appropriate to critically assess the relevance of this binding site to the mode of action of the compound. The discrepancy between the Kd for this binding (19 nM) and the ECSofor enhancement of ACh release (5 p M )is puzzling. However, as has been pointed out, the in vivo doses of linopirdine required to show behavioral effects are significantly lower than those that produce measurable enhancement of brain ACh release. Consequently, an effective concentration to improve cognitive performance may in fact correlate with the binding constant. Whether the release enhancement is an unrelated phenomenon, or whether an artifact of this assay necessitates the use of higher drug concentrations, are matters for speculation. One intriguing observation suggesting that binding to the linopirdine binding site is functionally related to release enhancement was provided by Tam et al. (1991). A range of 30 structural analogs of linopirdine were assayed for release-enhancing activity and for their ability to displace [3H]linopirdine from its binding site. The results show a strong positive correlation between binding and release enhancement with a correlation coefficient of 0.96. However, these results should be interpreted with cau-

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tion, owing to the expression of release enhancement as the concentration required to double release, rather than as an ECSO.It is hoped that further studies using other techniques will eventually establish the molecular nature of this binding site, and its exact role in the pharmacological actions of linopirdine. 2. Interactions between Linopirdine and other Pharmacological Agents

While a range of pharmacological agents do not bind to the linopirdine binding site, the study of ACh release in the presence of linopirdine and other drugs in combination has yielded important clues as to the mechanism of action of linopirdine. The use of various pharmacological agents and ion channel blockers has ruled out the involvement of a number of systems, at least as regards the release-enhancing activity of linopirdine. Maciag et al. (1994)investigated whether the ACh release enhancement by linopirdine was due to an action on ion channels. Linopirdine’s effect was studied in the presence of channel blockers, and a lack of inhibition of linopirdine’s release enhancement was taken to suggest that the drug was not acting via that ion channel. 4-AP did not inhibit the release enhancement due to linopirdine, the release in response to raised K+ being observed over and above the basal release enhancement caused by 4-AP. Thus, the A-type K+ current ( IA) seems an unlikely means for linopirdine to enhance release. Another K+ channel blocker, charybdotoxin, also did not affect linopirdine’s ability to enhance ACh release, arguing against the involvement of large conductance Ca2+-activedK+ channels (responsible for the C-current, L,and blocked by charybdotoxin). Likewise, TTX failed to block linopirdine’s effect, suggesting no involvement of Na+ channels in the mechanism of action. The use of chloride-deficient media or of the anion channel blocker 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid (SITS)did not affect the release enhancement due to linopirdine, suggesting that the drug does not act via these C1- channels. Maciag et al. (1994)also investigated the possible involvement of cholinergic receptors in the mechanism of action of linopirdine, since presynaptic muscarinic receptors are recognized to play a role in the modulation of neurotransmitter release. The muscarinic agonist carbachol itself decreased ACh release, but the release enhancement due to linopirdine was still seen in the presence of carbachol. Release enhancement by linopirdine in synaptosomes was not affected by the muscarinic antagonist atropine (Vickroy, 1993). A possible role of the GABA-sensitive C1- channel in the action of linopirdine was investigated by Maciag et al. (1994). Picrotoxin (0.1 to 100 p M ) , a blocker of this channel, attenuated the release enhancement due to linopirdine, and also enhanced K+-stimulated ACh release itself. While this result could suggest the involvement of a GABA-sensitive C1-

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channel in the action of linopirdine, definite conclusionscannot be drawn due to the lack of concentration-dependence of this effect of picrotoxin, plus the finding that the endogenous agonist, GABA, also enhanced release and attenuated the effects of linopirdine in this preparation (Maciag et al., 1994). The most positive indication that linopirdine acts via an ion channel comes from work with TEA, a K+ channel blocker with relatively low selectivity. TEA (30 mM) alone enhances the release of ACh in response to raised K+ (Maciag et al., 1994), and this effect is very similar to that of linopirdine, i.e., basal release is not affected (Fig. 10). When linopirdine is used in combination with varying concentrations of TEA, it is found that at the highest TEA concentrations (30 to 50 mM) the enhancement due to linopirdine is attenuated. Figure 11shows that linopirdine (10 p M ) enhances ACh release in the presence of TEA concentrations up to 10 mM, and TEA itself will enhance release at 1 mM and above. The fact that the effects of linopirdine and TEA are not additive at 30 mM TEA, and that with 50 m M TEA linopirdine causes no further enhancement, can be interpreted as evidence that TEA blocks the effect of linopirdine. This would then suggest a possible common mechanism of action, which is a particularly attractive proposition given the similar release profiles of linopirdine and TEA (Figs. 3a and 10). It cannot be ruled out, however, from the present data that there is a “ceiling” effect for this method of assaying release, and that the “attenuation” of linopirdine’s action in the presence of high TEA concentrations is simply because no further release can be produced. In spite of this proviso, TEA appears to show a clearer interaction with linopirdine than any other pharmacological agent tested hitherto. It is 8-

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(effectof TEA compared to zero TEA) and t (effectof linopirdineat a given TEA concentration). Adapted from Maciag et al. (1994).

therefore suggested (Maciag et al., 1994) that linopirdine may act to enhance release via a TEA-sensitiveK+ channel. TEA is known to block at least three K+ currents: the delayed rectifier (IK); the fast Ca2+-activatedcurrent (Ic); and the muscarine-sensitive current (IM; see Storm, 1990 for review). Ic would be expected to be blocked by relatively low TEA concentrations (Lancaster and Nicoll, 1987). Given the high TEA concentrations required to show any effect on the action of linopirdine (Fig. ll),it seems unlikely that linopirdine could be acting via Ic. Much higher concentrations are required to block IK and IM. Either or both of the K+ currents IK and IMmay therefore be of significance in the mechanism of action of linopirdine. 3. Possible Involvement of Ca" in the Release Enhancement due to Linopidine

The nonselective Ca2+channel blocker verapamil has been found to attenuate linopirdine-induced release enhancement at concentrations of 50 to 200 pM,and also to block K+-evokedrelease alone (Saydoff and Zaczek, 1993b). These results may suggest some involvement of Ca2+in linopirdine's action on release. The L-type Ca2+channel blockers nifedipine (Maciag et al., 1994) and nitrendipine (Saydoff and Zaczek, 1993b; Vickroy, 1993) did not block linopirdine's effect on release enhancement however, and

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neither did the N-type Ca2+ blocker omega-conotoxin nor the P/Q-type blocker omega-agatoxin (Saydoff and Zaczek, 1993b). It is possible that verapamil may be having effects at high concentrations (50 to 200 p M ) that are unrelated to CaZ+channel block. For instance, verapamil has been shown to inactivate (Jacobs and DeCoursey, 1990) and to block (Pappone and Ortiz-Miranda, 1993; Thomine et al., 1994)K+ channels. Fairhurst et al. (1980)have also shown that methoxyverapamil (D600)binds to muscarinic receptors and to a-adrenoceptors at these concentrations. Presumed depletion of endoplasmic reticular Ca2+stores with caffeine (20 m M ) attenuated both release and the release-enhancingeffect of linopirdine (Saydoff and Zaczek, 1993b) suggesting some sensitivity of linopirdine to caffeine. Caffeine is known to have multiple effects, however, especially at this high concentration, and so this result, while interesting, cannot be used to make a conclusion about the mechanism of action of linopirdine. to eliminate Saydoff and Zaczek (1993b) used 3-isobutyl-1-methylxanthine the possibility that the actions of caffeine to block adenosine receptors (Daly et al., 1984) or to inhibit phosphodiesterase (Choi et al., 1988) were responsible for its interaction with linopirdine. The possibility that caffeine attenuates the effect of linopirdine by blocking the M-current, I, (Pfaffinger etal., 1988; Schafer etal., 1991; Robbins etal., 1992),will be discussed later. The positive conclusions from these pharmacological studies are that the release enhancement due to linopirdine can be blocked by the following: 1. TEA: most likely via block of IK or I,. 2. Verapamil: probably via an effect unrelated to Ca2+channel block. 3. Caffeine: via an unidentified mechanism. 4. Effect of K+Concentration on Release Enhancement due t o Linopirdine

Apart from the apparent sensitivity of linopirdine’s release-enhancing effect to TEA (Fig. ll), there is other evidence that the mechanism of action of linopirdine is dependent on K+. Vickroy (1993) found release enhancement by linopirdine in rat synaptosomes to be dependent on the extracellular K+ concentration. Maciag et al. (1994) found that linopirdine shifted the K+ concentration-response curve for release to the left, perhaps suggesting an enhancement of cell excitability (Fig. 12). Taken together, these results lead to the hypothesis that linopirdine raises the level of neuronal excitability, an action that could in turn be responsible for the pronounced behavioral effects already discussed. Different K+ currents are recognized to play major roles in controlling neuronal excitability, in determining neuronal firing patterns, and in controlling the shape of the action potential (AP). Therefore, electrophysiological studies have examined the effects of linopirdine on cell excitability directly, and on specific K+ currents.

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0 ) 15

. . . . , . . . . , . . , . , . . . , , , . . . , 20

25

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FIGURE I 2 Linopirdine causes a left-ward shift of the K+ concentration-response curve for in uitro ACh release. Release of ACh from rat cerebral cortical slices preloaded with ['Hlcholine was assayed in response to varying K+ concentrations. Linopirdine (10 p M )caused an apparent increase in the sensitivity to raised K+ but did not enhance release in excess of the maximum achievable with K' alone. Adapted from Maciag et al. (1994).

B. Electrophysiological Studies 1. Linopirdine Reduces Spike Frequency Adaptation

Spike frequency adaptation (SFA; accommodation) can be defined as the ability of a neuron to suppress its firing under conditions of prolonged depolarization. SFA is an important physiological means by which neuronal firing patterns are modulated, and it plays a role in the integration of neuronal circuitry. Studying the firing characteristics of a neuron in vitro in response to a relatively long depolarizing pulse provides a convenient way to study the actions of a drug on neuronal excitability. Using hippocampal slices, SFA has been shown to be reduced by linopirdine in rat CA1 pyramidal neurons at concentrations that parallel the effective concentrations for enhancing ACh release in vitro (Lampe and Brown, 1991; Aiken etal., 1995). This reduction, in response to 10 p M linopirdine, is illustrated in Fig. 13. Whereas under control conditions the initial firing of APs is followed by a period of quiescence, in the presence of linopirdine firing is maintained throughout the duration of the depolarization. This effect is not blocked by atropine, suggesting that it is not due to an action of linopirdine at muscarinic receptors. (Nickolson et al. 1990 showed that linopirdine did have weak affinity for the muscarinic receptor. However, this concentration, 10 pM,caused only 5% displacement of [3H]3-quinuclidinyl benzilate from its binding site on rat brain membranes.)

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(a) Control

(b) Linopirdine

600 pA

(c)

FIGURE 13 Block of spike frequency adaptation by linopirdine. (a) A CAI pyramidal neuron is depolarized for 500 ms by injection of a 600 pA current pulse, causing initial firing of APs followed by a period of quiescence. (b) After exposure to linopirdine (10 pM), the depolarizing injection causes continuous firing for the duration of the pulse. (c) The current pulse used. Conventional microelectrode (4 M potassium acetate) recording from Aiken et a/. (1995).

This effect on SFA was the first in vitro electrophysiologicaldemonstration of linopirdine enhancing neuronal activity. Several K+ currents are believed to be involved in SFA, including Im, IM, Ic, and perhaps IA (see Storm, 1990 for review). To seek evidence for the involvement of one or more of these currents in the action of linopirdine, the effects on SFA were analyzed in more detail (Aiken et af., 1995). Figure 14a shows that the number of spikes during a 500 ms pulse was increased by linopirdine (10 p M ) irrespective of the amplitude of the depolarizing current injection. However, the time to first spike (Fig. 14b) was not affected by linopirdine at any depolarizing current amplitude used. This perhaps suggests that the action of linopirdine on SFA is not significant in the earliest phases of adaptation. Further evidence for this hypothesis can be gained from Fig. 14c, which shows the effect of linopirdine on inter-spike interval plotted as a function of interval number. Linopirdine reduced the mean inter-spike intervals, except for the first (i.e., time between the first and second spikes). These analyses provide circumstantial evidence for an action of linopirdine on a current involved in SFA in the mid and/or late phases. IA and Ic are thought to be involved in only the earliest phase of SFA (Storm, 1990). ,I and IMtherefore present themselves as the most likely targets for linopirdine. This deduction is in agreement with ACh release studies (Maciag

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15 CI

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

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Interval Number FIGURE 14 Analysis of the block of SFA by linopirdine (10 pM).(a) Number of spikes during a 500 ms pulse, as a function of depolarizing pulse amplitude. (b) Time to first spike as a function of depolarizing pulse amplitude. (c) Interspike interval as a function of interval number. Asterisks represent statistically significant ( p < 0.05) differences from control ( a = 9 in each group). From Aiken et al. (1995).

et al., 1994), which suggested that linopirdine does not act via Ic (since charybdotoxin does not attenuate the effect) or through IA(since 4-AP does not attenuate the effect). Murphy and Brown (1992) showed that blockade of synaptic transmission in a slice preparation with omega-conotoxin (300 nM) did not alter the block of SFA by linopirdine. This suggests that linopirdine is not exerting its effect on the impaled neuron via actions on other neurons in the slice. However, the same authors found that the use of KC1-filled electrodes, instead of potassium acetate, completely eliminated the effect of linopirdine on SFA. Muscarine also blocks SFA (presumably via block of IMand Im), but the block of SFA by muscarine was still seen using KCl electrodes. These experiments can be interpreted to imply the role of a chloride conductance in the electrophysiologicalactions of linopirdine. It is also possible to envision the altered chloride equilibrium potential having some unidentified effect that prevents the action of linopirdine. In light of these SFA studies, the muscarine-sensitive M-current, IM, seems to be the most likely target for linopirdine. This conclusion is based on the following:

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1. The ACh release enhancement by linopirdine is blocked by TEA at concentrations that are known to block IM. 2. IMis involved in SFA, which is also blocked by linopirdine.

No other K+ current is known to be both blocked by TEA and be involved with SFA. The block of IMhas accordingly been investigated directly in rat CA1 pyramidal neurons by Aiken and Brown (1994) and by Aiken et al. (1995), using the single electrode voltage-clamp technique. 2. Effect of Linopirdine on M-Current

The M-current, IM, is a slowly activating, noninactivating, voltagesensitive outward K+ conductance, first described by Brown and Adams (1980). It is blocked by muscarinic agonists such as carbachol-hence its designation “M”-via a somewhat poorly defined pathway which involves a pertussis toxin-insensitive G-protein and possibly phospholipase C and inositol triphosphate (for review, see Brown, 1988).IMis thought to contribute to the maintenance of resting membrane potential (RMP), as well as playing a role in SFA. The activation of IMon depolarization from RMP tends to return the cell toward RMP, and thus acts as a “damper” on neuronal activity. Thus, block of IM by muscarinic agonists results in increased firing (Madison and Nicoll, 1984). Aiken et al. (1995)measured M I in voltage-clamped rat CA1 hippocampal neurons. Linopirdine at 10 pM caused significant block of IM,as seen in Fig. 15. This block was reversible on washout of the drug, and was not

r L 4 ......

M

t

....

FIGURE I 5 Block of IM by linopirdine. (a) A CAI pyramidal neuron is repolarized from -30 to -50 mV for 1 sec, and the deactivation of M-current is seen as an exponential decay. (b) In the presence of linopirdine (10 p M ) ,IM is reduced by approximately 60% in this cell. Note the change in holding current at -30 mV, which may also represent block of IM. (c) Washout. (d) The voltage protocol used. From Aiken et al. (1995).

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reversed by atropine. Thus, linopirdine is not likely to be acting via a muscarinic receptor to block 1,; nor is the effect recorded in one neuron likely to be secondary to effects of the drug on other neurons~inthe slice, since synaptic transmission had been blocked with TTX.Linopirdine caused complete block of IMat 100 pM, and Fig. 16 shows the concentrationresponse relationship for IMblock. Overlaid on this figure are data for the ACh release enhancement by linopirdine (R. Zaczek, unpublished data). The two curves are strikingly similar in terms of slope, maximally effective I block concentration, and the ICsdECso values (8.5 p M and 5.4 pM, for M and release enhancement, respectively). This relationship can be taken as evidence that the ability of linopirdine to block M-current is responsible for its enhancement of ACh release in vitro. A close structural analog of linopirdine that was without effect on ACh release has been found to also have no effect on M I (S. P. Aiken, B. S. Brown, and R. Zaczek, unpublished data). It would be interesting to test compounds with similar release-enhancing properties to linopirdine (Earl et al., 1992), to further establish a relationship between IMblock and release enhancement. The fact that 20 mM caffeine blocks IMin these same neurons (S. P. Aiken and B. S. Brown, unpublished data) is also of interest, since caffeine is one of a very limited number of compounds that are known to attenuate linopirdine’s effect on ACh release. It can be speculated, therefore, that the interaction of linopirdine and caffeine is due to a common action on IM.

Release + enhancement

+

0

I

100 nM

1 PM

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[Linopirdine] FIGURE 16 Blockade of IM by linopirdine correlates closely with in vitm ACh release enhancement. Concentration-response curves are shown for IM block in hippocampal CA1 neurons (n = 3 to 9) and for release enhancement in rat hippocampal slices (n = 4), plotted as a percentage of maximal effect (means 2 SEM). Data are from Aiken et al. (1995) and R. Zaczek (unpublished).

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The apparent interaction of verapamil with linopirdine (Saydoff and Zaczek, 1993b) could also be due to an effect of the two drugs on a K+ channel, since high concentrations of verapamil are known to affect K+ channels (Jacobs and DeCoursey, 1990; Pappone and Ortiz-Miranda, 1993; Thomine et al., 1994), and since the close verapamil analog, methoxyverapamil, has affinity for the muscarinic receptor (Fairhurst et al., 1980). (This finding by the latter authors should not be taken to imply that methoxyverapamil is a muscarinic agonist, however.) Blockage of IM by linopirdine has also recently been reported in rat sympathetic ganglion cells, by Lamas and Brown (1995), with an ICSO of 2.5 to 3.4 p M . It will be interesting to see whether the drug affects M I in other cell types, and whether it shows specificity for brain regions, etc. It should be remembered that the hippocampal neurons studied by Aiken et al. (1995)were glutaminergic, whereas most of the work on neurotransmitter release has been on ACh. Also, it is not known how IM recordings such as these from the soma would differ from IM at the nerve terminals, which would presumably be more relevant to the release process. For technical reasons, IMhas not yet been demonstrated in nerve terminals. Another electrophysiological effect noted by Aiken et al. (1995)was a small depolarization in cells with relatively depolarized initial RMPs (positive of -65 mV). This phenomenon was also explained in terms of IMblock, since the slightly more depolarized cells would be expected to have more M I active at rest, and thus would depolarize in the presence of an IMblocker. The voltage-dependence of this effect argues against linopirdine affecting a non-specific K+ conductance, such as the “leak” conductance described by Benson et al. (1988). The data produced so far argue for an involvement of IMin the mechanism of action of linopirdine, at least for its release-enhancing effects. The evidence can be summarized as follows: 1. TEA and caffeine appear to attenuate the release enhancement due to linopirdine at concentrations that also block IM. 2. Concentration-response curves for release enhancement and for IM block are very similar in terms of half-maximal concentration and slope (Fig. 16). 3. There is a limited correlation between drug efficacy at release enhancement and IMblock (the inactive linopirdine analog). Some other more circumstantial evidence can also be suggested: 1. Since IM underlies SFA, block of IMexplains SFA and hence suggests a mechanism by which linopirdine increases neuronal excitability. 2. From our understanding of the physiological role of IM,we would expect the block of IMto produce increased neuronal activity in vivo. This could perhaps explain the EEG effects, and even the behavioral effects of linopirdine.

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3. The release enhancement by linopirdine requires depolarization, which would be expected to activate IM.

One anomaly in the argument that linopirdine acts via block of IMto enhance ACh release is that carbachol does not attenuate the effect of linopirdine on release (Maciag et al., 1994). This could perhaps be due to multiple actions of carbachol, leading to masking of the anticipated effect. The considerable depression of ACh release caused by carbachol alone (67% at 10 p M )also adds to the difficulty in interpreting this result. While much of the work on release enhancement with linopirdine has focused on ACh release, due to the clear involvement of central cholinergic pathways in Alzheimer’s disease, it should be borne in mind that in the electrophysiological characterization of cholinergic basal forebrain neurons (Griffith, 1988) IM-like current relaxations have not been recorded (W. H. Griffith, personal communication). The presence of IMin the nerve terminals or dendrites of these neurons remains to be determined, however. Whether or not IMblockade is eventually proved to be the mechanism of action of linopirdine, the fact that the drug blocks IMwith reasonable potency is of great significance to electrophysiologists. Although a range of compounds are known to block M I (reviewed by Brown, 1988), none are useful in selectively blocking this current. Hitherto identified blockers of M I include receptor agonists (muscarinic agonists and also serotonin, bradykinin, and luteinizing hormone-releasing hormone), nonselective K+ channel blockers (TEA, Ba2+)and compounds such as caffeine that have mixed pharmacological actions. As electrophysiological tools, these compounds are very difficult to work with, and in the case of receptor agonists it is likely that IMblock is only a secondary action. Even the muscarinic agonists, after which IMwas named, block other K+ conductances (e.g., Imp; Benardo and Prince, 1982) and activate intracellular second messengers at lower concentrations than those required for IMblock (Dutar and Nicoll, 1988). It is worth noting that tacrine also blocks IM at high concentrations in rat sympathetic ganglion cells (Marsh et al., 1990; ICso 0.5 to 1.0 mM). Using NG108-15 neuroblastoma cells, Robbins et al. (1992) calculated the ICsofor tacrine’s block of the “M-like current” to be 8 pM, thus suggesting similar potency to linopirdine in hippocampal neurons (Aiken et al., 1995). While block of IMis not thought to explain the behavioral and clinical effects of tacrine, it would certainly be interesting to know what contribution, if any, this property makes. The cognitive enhancer HP 749 (HoechstRoussel Pharmaceuticals) has also been reported to block M I (Huger et al., 1990), although again the relevance of this action to the drug’s cognitive effects is not known. Kishida et al. (1995) have reported IMblock by 3(m-henoxybenzy1idene)-quinuclidine,and suggested that amelioration of amnesia with this drug may be a result of this action on IM. Block of M-current by linopirdine at present offers the most plausible explanation for the drug’s effects, not only on release but also on cognitive

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function. Further work with linopirdine will hopefully establish a more direct link between the electrophysiological and the neurochemical effects. An important approach to this will be to identify the molecular mechanism by which linopirdine blocks IM. At the moment, several possible sites cannot be ruled out: the M-channel itself; a G-protein; or any of a number of second messenger systems. Correlation of a molecular site of action with the highspecificity linopirdine binding site (Tam et al., 1991) would be of particular importance in characterizing this drug. 3. Selectivity of the M-Current €flea: Other Currents

The demonstration of the ability of linopirdine to block one K+ conductance, IM (Aiken et a/., 1995), leads to the need to examine the drug’s selectivity for this current and to consider its action on other K+ currents. Some work on this specificity of action has been performed, and will now be discussed. However, the electrophysiological effects of linopirdine have not yet been characterized in enough detail to rule out actions at a number of other currents. Frey et al. (1991), Schnee and Brown (1995), and Lamas and Brown (1995) showed block of both IKand IAby linopirdine in cultured rat neocortical neurons, rat hippocampal CA1 neurons, and dissociated rat superior cervical sympathetic ganglion cells, respectively, but at significantly higher concentrations than those shown to block IM.The ICsovalues reported in I and A I (Frey et al., 1991); these cells were: approximately 300 pM for K 60 to 70 pM (Lamas and Brown, 1995) and >300 pM (Schnee and Brown, I (Schnee and Brown, 1995). 1995) for 1,; and approximately 100 pM for A Thus, linopirdine does affect other K+ currents, although these high ICsos make it more difficult to attribute a significant role to IK or A I blockade in explaining the effects of linopirdine on neurotransmitter release. It is worth noting that the study that obtained the lowest ICsovalues for delayed rectifier and IAby Lamas and Brown (1995) also reported a more potent block of IMin these cells (ICso= 2.5 to 3.34 p M ) than did Aiken et al. (1995) in = 8.5 p M ) . hippocampal CA1 neurons (Go IAHp,the current underlying the slow afterhyperpolarization (sAHP), plays an’important role in SFA. Aiken et al. (1995) showed by an indirect means that the sAHP is not affected by linopirdine (10 pM).The sAHP that follows a depolarizing pulse (in the absence of TTX) is dependent on the number of APs fired during the pulse. Therefore, with linopirdine, the sAHP appeared to be larger than controls. A possible block of the sAHP by linopirdine could thus be “masked” by this effect of the number of APs, and so Aiken et al. (1995) analyzed their data by comparing pretreatment and linopirdine-treated recordings in which like numbers of APs were produced (by varying stimulus intensity). When compared in this way, linopirdine was shown to have no effect on the sAHP. The mixed Na+/K+current

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termed 1, has also been shown to be unaffected by linopirdine (Aiken et

al., 1995). The characteristics of the AP itself have been examined as a means of possibly identifying electrophysiological effects of linopirdine. Aiken et al. (1995) showed that linopirdine (10 p M ) did not alter AP duration or other characteristics of the Ap. A low concentration of TEA, 1 mM, in contrast caused a pronounced prolongation of the AP. It therefore seems that linopirdine at this concentration is not affecting conductances that bring about spike repolarization. The studies of ion channels that may potentially be affected by linopirdine are incomplete. In particular, the possibility of an action at a C1channel needs to be examined because of the suggestion by Murphy and Brown (1992) that the reduction in SFA by linopirdine was abolished by altering the chloride equilibrium potential. Actions at Ca2+channels still deserve study due to the attenuation of the effect of linopirdine by high concentrations of the nonselective blocker verapamil (Saydoff and Zaczek, 1993b). Ligand-gated channels have received almost no attention, and so definitive conclusions about the mechanism of action of linopirdine cannot yet be made.

IV. Conclusions Linopirdine is a novel compound that enhances the depolarizationinduced release of ACh and other neurotransmitters in vitro and also enhances ACh release in vivo. Since basal release in vitro is unaffected, linopirdine has potential therapeutic utility in treating diseases where neurotransmission is impaired or where neuronal pathways are compromised, such as in Alzheimer’s disease. This theory of the cognitive-enhancing effect of linopirdine is borne out by behavioral studies in a wide range of animal tests. However, it must be noted that the actual clinical efficacy of linopirdine was not demonstrated in Phase I11 clinical trials (see Scrip, 1992, Vol. 1775, p. 25),nor has this approach (enhancement of transmitter release) been proven to be effective in the clinic. A limited trial conducted by van Dyck et al. ( 1995)did demonstrate significant improvement in Alzheimer’s patients receiving linopirdine as measured by one test of cognitive function, while two other tests showed nonsignificant “trends” towards an improvement. Other compounds that stimulate the cholinergic system, most notably tacrine, do have therapeutic benefit in the treatment of Alzheimer’s disease, and so an analog of linopirdine (e.g., see Earl et al., 1992) may eventually be shown to be an improvement over currently used cholinergic stimulants. Effects on several different transmitter systems (ACh, serotonin, dopamine, glutamate) may make this approach to Alzheimer’s therapy more successful.

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Linopirdine binds with high specificity to a binding site in brain, but the nature and significance of this binding site remain poorly defined. The K+ channel blocker TEA blocks the release enhancement due to linopirdine, and the suggestion that linopirdine acts by blocking a TEA-sensitivechannel has been confirmed by the finding that linopirdine blocks the M-current. Block of IMis presently the most plausible mechanism of action of linopirdine, at least on neurotransmitter release. Other ionic mechanisms (Cl-, Caz+) still need to be explored, however, and causative links between release enhancement and behavioral effects need to be established. Linopirdine is the first relatively selective blocker of the M-current to be described, and a characterization of the mechanism of M-current block may lead to a better understanding of the physiology of this current. New target sites for possible intervention may be revealed by increased knowledge of second messenger systems since the M-current is thought to be an important physiological means by which neuronal activity is regulated. Acknowledgments We wish to thank Reinhard Grzanna and Ken Rohrbach (DuPont Merck) and Marcia Riggott and William Matthew (Duke University Medical Center) for their help in preparing this article.

References Aiken, S. P., and Brown, B. S. (1994). Linopirdine (DuP 996), a neurotransmitter release enhancer, blocks M-current in rat CA1 hippocampal neurons. Biophys. J. 66, A210. Aiken, S. P., Lampe, B. J., Murphy, P. A., and Brown, B. S. (1995).Reduction of spike frequency adaptation and blockade of M-current in rat CAI pyramidal neurones by linopirdine (DuP 996), a neurotransmitter release enhancer. Br. J. Pharmacol. 115, 1163-1168. Alberici, G. P., Cook,L., Gaskill, J.-L., and Steinfels, G. F. (1991).The electroencephalographic (EEG)effects of DuP 996, a compound with cognitive enhancingproperties. SOC.Neurosci. Abstr. 17, 1588. Amoroso, S., Schmid-Antomarchi, H., Fosset, M., and Lazdunski, M. (1990).Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels. Science 247, 852-854. Baxter, M. G., Rohrbach, K. W., Tam, S. W., Zaczek, R., Frick, K. M., Golski, S., Wan, R.-Q., and Olton, D. S. (1994). Effects of linopirdine (DuP 996) and X9121 on agerelated memory impairments and on the cholinergic system. Drug Deu. Res. 31,186-196. Baxter, M. G., Rohrbach, K. W., Tam, S . W., Zaczek, R., and Olton, D. S. (1993). Effects of linopirdine and DuP 921 on age-related impairments in memory and on the cholinergic system. Soc. Neurosci. Abstr. 19, 1041. Benardo, L. S., and Prince, D. A. (1982).Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells. Brain Res. 249, 333-344. Benson, D. M., Blitzer, R. D., and Landau, E. M. (1988). An analysis of the depolarization produced in guinea-pig hippocampus by cholinergic receptor stimulation. J. Physiol. (London) 404,479-496.

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Bernard, V., Dumartin, B., Lamy, E., and Bloch, B. (1993).Fos immunoreactivity after stimulation or inhibition of muscarinic receptors indicates anatomical specificity for cholinergic control of striatal efferent neurons and cortical neurons in the rat. Eur. J. Neurosci. 5, 1218-1225. Bowyer, J. F., and Weiner, N. (1990).Alpha-2 adrenergic inhibition of Ca++-evoked[3H]norepinephrine release from synaptosomes is blocked by depolarization. J. Pharmacol. Exp. Thm. 253, 1063-1069. Brioni, J. D., Curzon, P., Buckley, M. J., Arneric, S. P., and Decker, M. W. (1993).Linopirdine (DuP996) facilitates the retention of avoidance training and improves performance of septal-lesioned rats in the water maze. Pharmacol. Biochem. Behav. 44, 37-43. Brown, D. (1988). M-currents: an update. Trends Neurosci., 11, 294-299. Brown, D. A., and Adams, P. R. (1980). Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature (London)283, 673-676. Choi, 0.H., Shamim, M. T., Padgett, W. L., and Daly, J. W. (1988).Caffeine and theophylline analogues: correlation of behavioral effects with activity as adenosine receptor antagonists and as phosphodiesterase inhibitors. Life Sci. 43, 387-398. Cook, L., Nickolson, V. J., Steinfels, G. F., Rohrbach, K. W., and DeNoble, V. J. (1990). Cognition enhancement by the acetylcholine releaser DuP 996. Drug Dev. Res. 19, 301-314. Cross, A. J., Crow, T. J., Perry, E. K., Perry, R. H., Blessed, G., and Tomlinson, B. E. (1981).Reduced dopamine-beta-hydroxyylase activity in Alzheimer’s disease. Br. Med. J. 282,93-94. Daly, J. W., Butts-Lamb, P., and Padgett, W. (1984). Subclasses of adenosine receptors in the cortical nervous system: interaction with caffeine and related methylxanthines. Cell Mol. Neurobiol. 3, 69-80. Davidson, M., and Stern, R. G. (1991).The treatment of cognitive impairment in Alzheimer’s disease: beyond the cholinergic approach. Psychiatr. Clin. North Am. 14, 461-482. Davies, P., and Maloney, A. J. F. (1976). Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet ii, 1403. DeNoble, V. J., DeNoble, K. F., Spencer, K. R., Johnson, L. C., Cook, L., Myers, M. J., and Scribner, R. M. (1990). Comparison of DuP 996, with physostigmine, THA and 3,4-DAPon hypoxia-induced amnesia in rats. Pharmacol. Biochem. Behav. 36,957-961. Dent, G. W., Rule, B. L., Tam, S. W., and De Souza, E. B. (1993a). Effects of the memory enhancer linopirdine (Dup 996) on cerebral glucose metabolism in naive and hypoxiaexposed rats. Brain Res. 620, 7-15. Dent, G., Tam, S. W., and Grzanna, R. (1993b). The memory enhancer linopirdine increases c-fos expression in cerebral cortex of aged rats. SOC.Neurosci. Abstr. 19, 1040. De Souza, E. B. (1993). Preclinical strategies for symptomatic treatment of cognitive deficits seen in Alzheimer’s disease: focus on cholinergic mechanisms. In “Alzheimer’s Disease: Advances in Clinical and Basic Research” (B. Corain, K. Iqbal, M. Nicolini, B. Winblad, H. Wisniewski, and P. Zatta, Eds.), pp. 539-548. Wiley, Chichester. De Souza, E. B., Rule, B. L., and Tam, S. W. (1991). [3H]DuP 996 labels a novel binding site in rat brain involved in the enhancement of stimulus-induced acetylcholine, dopamine and serotonin release: autoradiographic localization studies. SOC. Neurosci. Abstr. 17, 1588. Dutar, P., and Nicoll, R. A. (1988). Classification of muscarinic responses in hippocampus in terms of receptor subtypes and second-messenger systems: electrophysiological studies in vitro. J. Neurosci. 8, 4214-4224. Earl, R. A., Myers, M. J., Johnson, A. L., Scribner, R. M., Wuonola, M. A., Boswell, G. A., Wilkerson, W. W., Nickolson, V. J., Tam, S. W., Brittelli, D. R., Chorvat, R. J., Zaczek, R., Cook, L., Wang, C., Zhang, X., Lan, R.,Mi, B., and Wenting, H. (1992).Acetylcholinereleasing agents as cognition enhancers. Structure-activity relationships of pyridinyl pendant groups on selected core structures. Bioorg. Med. Chem. Lett. 2, 851-854.

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Acetylcholine, release enhancement characteristics, 353-355 a-Actin, ADP-ribosylation, 253 ADP-ribosylarginine hydrolases, 268-271 mammalian, 270-271 turkey, 269-270 ADP-ribosyltransferases avian, 250-256 chicken heterophil, 254-255 in vitro substrates, 255-256 conserved regions, 262-268 region I, 263-265 region 11,265-266 region 111, 266-268 inhibitors, 261-262 mammalian, 256-261 rat brain, 258-260 skeletal and cardiac muscle, 256-258 T cells, 260 turkey erythrocyte, 250-252 transferase A, in vitro substrates, 252-253 Alcoholism, chronic, abnormal drug metabolism, 12-13 Alloantibodies, sponge matrix allografts, 132 Allograft rejection cardiac, 129 ciclosporin therapy, 203 chronic, ciclosporin, 194-204 clinical situation, 194-195 experimental approaches, 195-199 factors involved in, 199-204

Alzheimer’s disease, see also Linopirdine cholinergic hypothesis, 350-351 medical need, 349-350 Androgen ablation induced apoptosis, role of recruitment into perturbed cell cycle, 293-294 induced apoptosis, p53 expression, 294-295 normal prostatic glandular cell apoptosis, 287-289 prostate gene expression during apoptosis, 290-291 Androgens, sensitivity of prostate cancer, 282-283 Angiogenesis, nitric oxide in, 54-55 Antacids, drug interactions, 16 Anticholinesterase activity, linopirdine, 363 Antifungal properties, ciclosporin, 190-191 Antigen-presenting cells, ciclosporin effects, 180-183 Antigens, self, discrimination from nonself antigens, 165-166 Antigen-specific functional unresponsiveness adoptive transfer of spleen cells, 147 canine renal allograft model, 148 cardiac allografts, 146-147 criteria for, 143 donor-specific, 144-146 induction and ciclosporin, 143-154 long-term functioning grafts, 152-153 lymphohematopoietic chimerism induction, 151

385

386

Index

Antigen-specific functional unresponsiveness (continued)

persistant Borna disease virus in rats, 144- 145 porcine renal allograft model, 148-150 rat heart allograft model, 144 renal allograft model of LEW, 146 skin allografts, 147-148 stepwise drug weaning, 153 Antihelmintic activity, ciclosporin, 192-1 93 Antimicrobials, nutrient-drug interactions, 10-12 Antiparasitic effects, ciclosporin, 191-193 Apoptosis, 281-302 activated Ca’+-Me endonucleases, 289 activation by thapsigargin, 297-300 androgen ablation-induced p53 expression, 294-295 prostate gene expression, 290-291 androgen-dependent prostate cancer cells, 284 androgen-independentprostate cancer cells, 285, 287 cell proliferation role, induced by castration, 291-293 ciclosporin, 170-171 D1-activation phase, 288-289 DNA repair during, 292-293 F-phase, 289 normal prostatic glandular cells, temporal sequences following androgen ablation, 287-289 prostate cancer, therapeutic implication, 295-296 S-phase, 291-292 Arachidonic acid, metabolism, 28-29 Arginine, metabolism at inflammatory sites, 48-50 Aspirin, 39 Asthma, ciclosporin therapeutic effects, 175-1 79 Atherosclerosis, see Coronary angiographic trials Autoimmunity, ciclosporin-induced, 167-168

B cells, inhibition of activation, ciclosporin, 172 Bile-acid sequestrants, 81-87 Cholesterol Lowering Atherosclerosis Study, 86-87 clinical trials, 83-87

efficacy, 82 Familial Atherosclerosis Treatment Study, 85-86 Lipid Research Clinics Coronary Primary Prevention Trial, 83-84 mechanism of action, 81-82 National Heart, Lung, and Blood Institute Type I1 Coronary Intervention Study, 84-85 side effects and drug interactions, 82-83 St. Thomas’ Atherosclerosis Regression Study, 85 Bioavailability food-induced changes, 5 metabolism and, 8 Bone marrow, protection, gene therapy in cancer, 341-343 Brain ADP-ribosyltransferases, rat, 258-260 CaZ+,in release enhancement due to linopirdine, 369-370 Calcineurin A, 126-127 Canadian Coronary Atherosclerosis Intervention Trial, 92, 311-314 Cancer, see also Prostate cancer gene therapy, 336-343 bone marrow protection, 341-343 herpes thymidine kinase, 336-342 Cancer cells, reversal of multidrug resistance, ciclosporin, 187-188 Cardiac allograft rejection, 129 ciclosporin therapy, 203 Cells, kinetics during prostate cancer progression, 283-286 Central nervous system, ciclosporin interactions, 156-158 Chemical libraries, combinatorial, unnatural nucleotide sequences, 328 Chicken heterophil ADP-ribosyltransferases, 254-255 in vitro substrates, 255-256 Cholera toxin Arg7,264-265 GlullO and Glu112, 267-268 Cholesterol agents lowering, 80-98 bile-acid sequestrants, 81-87 estrogen-replacement therapy, 96-98 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitions, 87-94 probucol, 94-96 production, inhibition of enzymes, 106

Index

Cholesterol-Lowering Atherosclerosis Study, 86-87, 308-309 Cholestyramine, 82-85 Cholinergic function, improving, 351 Cholinergic hypothesis, Alzheimer’s disease, 350-351 Ciclosporin, 115-208 adverse reaction profiles, 160, 162 immunosuppressive and nonimmunosuppressive, 164 antibiotic effects, 189-193 antifungal properties, 190-191 HIV-1 replication inhibition, 189-190 antiparasitic effects, 191-193 apoptosis, 170-171 asthma, therapeutic effects, 175-179 biological effects correlation with immunosuppressive activity, 121 separable from immunosuppressive activity, 122 cell-mediated suppressor function, 137-143 chronic allograft rejection, 194-204 clinical situation, 194-195 experimental approaches, 195-199 factors involved in, 199-204 chronic inflammation, 154 clinical chemistry profiles, dose dependence, 159-161 clinical pharmacokinetics, 204-207 combination therapy, 153-154 dose-dependence of adverse reaction profiles and exposure values, 160, 163 effects hair follicles, 179 morphine withdrawal syndrome, 157 nonlymphoid cells, 180-187 antigen-presenting cells, 180-1 83 epidermal Langerhans cells, 183 keratinocyte proliferation, 183-185 other cell types, 185-187 galenical formulation, 123, 207-208 graft arteriosclerosis, 195-196, 198-199, 204 immunosuppression activity, 121 mechanism, 117 molecular mechanism, 124, 126-127 preventing arterial and venous allograft rejection, 196

387

inhibition of B cell activation, 172 interactions with CNS, 156-158 interference in regulation of tolerance to self and nonself, 165-172 intracellular targets, 124, 126 list of compounds, 158-159 major congresses and symposia, 125-126 mechanism of action, in vivo, 119-120 mismatched renal allografts, 149-150 mode of action, 117-118 nephrotoxicity, 117 overview of disrribution and fate, 205 pharmacological properties, 119-121 prolactin antagonism, 154-156 psoriasis, therapeutic effects, 172-175 reversal of multidrug resistance in cancer cells, 187-188 selective formylpeptide receptor antagonism, 188-189 side effects, 158-165 limiting, 123 stepwise drug weaning, 153 structure, 115-116 suppressor cell development, 165 T cells functions for help, memory, and delayed-typed hypersensitivity, suppression, 133- 137 mediated cytotoxicity, 128-133 topical application effect, 130-131 variability in response, 122-123 Clofibrate, 101-103 Colestipol, 82-83, 85-87 coronary angiographic trials, 308-310, 314 Compactin, 87-88 Coronary angiographic trials, 307-318 Canadian Coronary Atherosclerosis Intervention Trial, 92, 311-314 Cholesterol-Lowering Atherosclerosis Study, 86-87, 308-309 conclusions from, 317-318 Familial Atherosclerosis Treatment Study, 85-86,309 Heidelberg Exercise-Diet Study, 316-317 Leiden Intervention Trial, 315-316 Lifestyle Heart Trial, 316 Monitored Atherosclerosis Regression Study, 91, 310-311 Multicentre Anti-Atheroma Study, 93, 314 NHLBI Type I1 Coronary Intervention Study, 84-85, 308

388

Index

Coronary angiographic trials (continued) Program on the Surgical Control of the Hyperlipidemias, 317 Stanford Coronary Risk Intervention Project, 3 14-3 15 St. Thomas’ Atherosclerosis Regression Study, 85, 315 University of California, San Francisco, Specialized Center of Research Intervention Trial, 309-310 Coronary Drug Project fibric-acid derivatives, 103 nicotinic acid, 100-101 Coronary heart disease, triglycerides and risk, 98-99 Crohn’s disease, nitric oxide in, 58 Cyclo-oxygenase, 28-30, see also Inflammation inflammation, 40-41 interaction with nitric oxide synthase pathway, 59-61 isoforms, 29-30 pharmacological inhibition, 41-44 regulation, 30-32 Cyclophilin A, 124, 126 Cyclosporin A, see Ciclosporin Cysteine, mono-ADP-ribosylation, 249-250 Cytokines, proinflammatory, 48-49 Cytolysis, ciclosporin and, 186 Cytotoxicity nitric oxide, 50-51 T cell-mediated, ciclosporin, 128-133 Cytotoxic T cells ciclosporin and, 128-133 precursor-effector, 131 veto cell-mediated suppression, 141 Diphtheria toxin Glu148,266-267 His21, 263 Tyr65,265-266 DNA regulatory sequences, binding proteins, 333-334 repair, during apoptosis, 292-293 Drug, see also Nutrient-drug interactions absorption decreased by food or nutrients, 6 delayed by food or nutrients, 4-5 enhanced by food or nutrients, 4 common therapies in elderly, risk and adverse effects, 21-22

design, rational, unnatural nucleotide sequences, 322-323 disposition, factors affecting, 7 eliminated by organic transport systems, 9 ethanol interactions, 11-14 excretion and elimination, 8-10 mechanism of action, 2 mineral interactions, 15-16, 18-19 vitamin interactions, 14-15 Duchenne muscular dystrophy, ciclosporin therapy, 186 DuP 996, see Linopirdine Electroencephalographic effects, linopirdine, 361-362 Electrolytes, drug-induced disturbances, 9-10 Elimination, drugs, 8-10 Encephalomyelitis, experimental allergic, ciclosporin therapy, 140-141 Endothelial cells, ciclosporin effects, 178-1 79 Endothelin ciclosporin-induced acute nephrotoxicity and hypertension, 201-202 smooth muscle proliferation and contraction, 200-201 Eosinophils, ciclosporin effects, 175-176 Epithelial cells ciclosporin effects, 178-179 prostatic, normal and neoplastic, kinetic parameters, 284-286 Erythema, 34 Erythrocytes, turkey ADP-ribosyltransferases, 250-252 transferase A, in vitro substrates, 252-253 Escherichia coli heat-labile enterotoxin Arg7,264-265 GlullO and Glu112,267-268 mutants, as host, 329 Estrogen-replacement therapy, as cholesterol-lowering agent, 96-98 Ethanol drug interactions, 11-14 sites, 12-13 effect on degradation of drugs, 12-13 metabolism, ethnic differences, 13-14 Ethnic differences ethanol metabolism, 13-14 nutrient-drug interactions, 17, 20

Index

Eukaryotic cells, mono-ADP-ribosylation, 248-249 Excretion, drugs, 8-10 Exotoxin A, Pseudomonas aeruginosa, 262 His440,263 Experimental allergic encephalomyelitis, ciclosporin therapy, 140-141

Familial Atherosclerosis Treatment Study, 85-86, 309 Fibric-acid derivatives, 101-104 efficacy, 102 mechanism of action, 101-102 side effects and drug interactions, 102-103 Fish oil supplementation, graft arteriosclerosis, 199 as triglyceride-lowering agent, 104-105 Fluvastatin, 88 Folate, drugs affecting activity, 15 Food, see also Nutrient-drug interactions genetically engineered, 2 Formylpeptide receptor antagonism, selective, ciclosporin and, 188-1 89

Gastrointestinal absorption, factors affecting, 3-8 Gastrointestinal inflammation, nitric oxide in, 57-58 Gemfibrozil, 101- 104 Gene therapy, cancer, 336-343 bone marrow protection, 341-343 herpes thymidine kinase, 336-342 Genetic selection, random, for biologically active proteins, 324-325 Geriatrics, nutrient-drug interactions, 20-22 a-Glucosidases, inhibition, 23 G protein, mono-ADP-ribosylation, 248 Graft arteriosclerosis, ciclosporin effects, 195-196, 198-199,204 Graft-versus-host disease, 129-130 ciclosporin-induced, 167 nitric oxide in, 56-57 syngeneic, 167-169 Growth factor p, T cell inhibition, 141-142

Hair follicles, ciclosporin effects, 179 Heidelberg Exercise-Diet Study, 316-317

389

Helsinki Heart Study, fibric-acid derivatives, 103-104 Herpes thymidine kinase, gene therapy in cancer, 336-342 changing substrate specificity, 341-342 functionally active substitutions, 339-340 oligonucleotide insertion, 338-339 protocol, 337 thermostability, 340-341 HIV-1, replication, inhibition by ciclosporin, 189- 190 Host, choosing, biopharmaceutics, 329 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, 87-94 Canadian Coronary Atherosclerosis Intervention Trial, 92 clinical trials, 90-94 efficacy, 88 mechanism of action, 88 Monitored Atherosclerosis Regression Study, 91 Multicenter Anti-Atheroma Study, 93 Pravastatin Limitation of Atherosclerosis in the Coronary Arteries, 92 Scandinavian Simvastatin Survival Study, 93-94 side effects and drug interactions, 89-90 Hyperlipidemia, 79- 107 agents predominantly lowering cholesterol, 80-98 bile-acid sequestrants, 81-87 estrogen-replacement therapy, 96-98 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, 87-94 probucol, 94-96 agents predominantly lowering triglyceride, 98-105 fibric-acid derivatives, 101-104 fish oil, 104-105 nicotinic acid, 99-101 combination-drug therapy, 105 drug mechanisms and effects, 79-80 future developments, 105-107 lipid-regulating agents, clinical trials, 81 Hypersensitivity, delayed-type precursors, 134 T cell suppression by ciclosporin, 133-137 Hypertension, ciclosporin-induced, 202 Hypoglycemics, nutrient-drug interactions, 22-23

390

Index

Immunosuppressants, see Ciclosporin Inflammation, 27-61 acute, mechanisms, nitric oxide in, 51-53 arginine metabolism, 48-50 chronic ciclosporin, 154 mechanisms, nitric oxide in, 53-55 prostanoids, 37-38 classic signs, 27 complement- and cell-mediated models, 52-53 cyclo-oxygenase,40-4 1 inhibition by ciclosporin, 116 neurogenic, 53 nitric oxide, cellular production and activity, 47-48 nitric oxide synthase, 45-51 pain, NSAIDs, 38-39 prostanoids and, 33-37 dnterferon, effects on morphine withdrawal syndrome, 157 Interleukin-2, inhibition by ciclosporin, 135-136 Interleukin-2 receptor, expression, induction and ciclosporin, 136-137 Keratinocyte, proliferation, ciclosporin effects, 183-185 P-Lactamase, mutant enzyme production, 334-335 Lactose intolerance, 17, 20 Langerhans cells, epidermal, ciclosporin effects, 183 Learning animal models, linopirdine effects, 358-360 lesioned and aged rodents, linopirdine effects, 359-361 Leiden Intervention Trial, 315-316 Lifestyle Heart Trial, 316 Linopirdine, 349-380 Alzheimer’s disease, rationale behind use, 351-352 anticholinesterase activity, 363 behavioral effects, 358-361 animal models of learning and memory, 358-360 conclusions, 361 learning in lesioned and aged rodents, 359-361

effect on neuritic branching, 362-363 electroencephalographic effects, 361-362 electrophysiological studies, 37 1-3 80 effect on M-current, 374-378 selectivity of M-current effect, 378-380 spike frequency adaptation reduction, 371-374 evoked neurotransmitter release enhancement, 353-357 characteristics, 353-355 endogenous neurotransmitters, 356-357 in vivo, 357 K+ channel blockers, 354-355 multiple neurotransmitter systems, 35 5-35 6 metabolic effects, 362 neurochemical studies, 364-371 binding site identification, 364-367 CaZ+involvement in release enhancement, 369-370 interactions with other pharmacological agents, 367-369 K+ concentration and release enhancement, 370-371 neuromuscular effects, 363-364 structure, 351 Lipid-regulating agents, clinical trials, 81 Lipid Research Clinics Coronary Primary Prevention Trial, bile-acid sequestrants, 83-84 Local graft-versus-hostreaction, 129-130 Local lymph node weight assay, 129 Lovastatin, 88, 90-92 coronary angiographic trials, 309-314 Lymphocytes, ciclosporin effects in asthma, 176 Lymphokine, release from cytotoxic T cells, 130 Macrophages ciclosporin effects, 180-1 83 nitric oxide production, 50 Mammalian ADP-ribosylargininehydrolase, 270-271 Mast cells, ciclosporin effects in asthma, 176-1 77 M-current, linopirdine effect, 374-378 selectivity, 378-380 Memory, animal models, linopirdine effects, 358-360 Metabolic effects, linopirdine, 362

Index

Metabolism, bioavailability and, 8 Minerals, drug interactions, 15-16, 18-19 Mixed function oxidase system, 6, 8 Molecular evolution, unnatural nucleotide sequences, 323-324 Monitored Atherosclerosis Regression Study, 91,310-311 Mono-ADP-ribosylation, 247-272; see also ADP-ribosyltransferases cysteine, 249-250 eukaryotic cells, 248-249 Monocytes, ciclosporin effects, 181-1 82 Morphine, withdrawal syndrome, ciclosporin and a-interferon effects, 157 Multicentre Anti-Atheroma Study, 93, 314 Multiple sclerosis, nitric oxide in, 56 Muscle ADP-ribosyltransferase, 256-258 Mutagenesis, site-specific, unnatural nucleotide sequences, 322-323 Myositis, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, 89

NAD:arginine ADP-ribosyltransferase, activity, 248 National Heart, Lung, and Blood Institute Type I1 Coronary Intervention Study, 84-85, 308 Nephropathies, drug-induced or chemicalinduced, 9, 11 Nephrotoxicity ,ciclosporin-induced, 1 17, 201-202 Neuritic branching, linopirdine effect, 362-363 Neuromuscular effects, linopirdine, 363-364 Neutrophils ciclosporin effects, 178 nitric oxide synthesis, 47-48 NHLBI Type I1 Coronary Intervention Study, 84-85, 308 Niacin, coronary angiographic trials, 308-310, 314 Nicotinic acid, as triglyceride-lowering agent, 99-101 Nitric oxide action on prostaglandin production, 60-61 acute inflammation mechanisms, 51-53 in angiogenesis, 54-55

39 I

cellular production and activity at inflammatory sites, 47-48 chronic inflammation mechanisms, 53-55 cytotoxicity and tissue damage, 50-51 in gastrointestinal inflammation, 57-58 in graft-versus-host reaction, 56-57 induction, human macrophages, 50 in inflammatory pain, 59 in multiple sclerosis, 56 in renal inflammation, 57 in rheumatoid arthritis, 55-56 Nitric oxide synthase, see also Inflammation complement- and cell-mediated inflammatory models, 52-53 induction, 46-47 cell types, 50 inflammation, 45-51 inhibition, 46-47 interaction with cyclo-oxygenase pathway, 59-61 isoforms, 46 Non-functional inserts, unnatural nucleotide sequences, 330 Nonsteroidal anti-inflammatory drugs inflammatory pain, 38-39 inhibitory capacity on cyclo-oxygenase, 41-43 Nuclear factor of activated T cells, 127 Nucleic acids, libraries, unnatural nucleotide sequences, 326-327 Nucleotides, unnatural sequences, 321-344 combinatorial chemical libraries, 328 molecular evolution and its consequences, 323-324 mutant enzyme production, 334-336 p-lactamase, 334-335 related studies, 335-336 nucleic acid libraries, 325-327 peptide libraries for modeling peptide hormones and drugs, 325-326 phage display libraries for binding proteins, 324-326 random genetic selection for biologically active proteins, 324-325 random sequence selection, 328-333 applications, 333 combinatorial consideration, 332-333 non-functional inserts, 330 oligonucleotide inserts containing random nucleotide sequences, 330 plasmid vector and host choice, 329 protocol, 328-329

392

Index

Nucleotides (continued) randomization of oligonucleotides, 331 regulatory DNA sequences and binding proteins, 333-334 site-specific mutagenesis and rational drug design, 322-323 status, summary, and future prospects, 343-344 Nutrient-drug interactions, 1-24 antimicrobials, 10-12 ethanol, 11-14 factors affecting gastrointestinal absorption, 3-8 genetic differences, 17, 20 geriatrics, 20-22 hypoglycemics, 22-23 mechanism in elderly, 21 mineral interactions, 15-16, 18-19 Phase I reactions, 5-8 physiological interactions, 3-4, 7 risk factors, 2-3 total parenteral nutrition, 23 vitamin interactions. 14-15

Oligonucleotides inserts containing random nucleotide sequences, 330 randomization, 33 1

Pain inflammatory, nitric oxide in, 59 prostaglandins and, 38-39 Paracetamol, 39 Parenteral nutrition, nutrient-drug interactions, 23 Peptides, hormones and rugs, peptide libraries for modeling, 325-326 Pertussis toxin, His35, 267 Pharmacologic agents interactions with linopirdine, 367-369 transethnic responsiveness, 17, 20 Plants, genetic engineering, 2 Plasmid vector, choosing, biopharmaceutics, 329 Poly(ADP-ribose) polymerase, activity, 261-262 Postmenopausal Estroged’rogestin Interventions, 97-98

Potassium, concentration and release enhancement due to linopirdine, 370-371 Potassium channel blockers, as Ach release enhancers, 354-355 Potassium currents, see also M-current linopirdine effect, 378 Pravastatin, 88, 90, 92 Pravastatin Limitation of Atherosclerosis in the Coronary Arteries, 92 Probucol, 94-96 Probucol Quantitative Regression Swedish Trial, 96 Programmed cell death, see Apoptosis Program on the Surgical Control of the Hyperlipidemias, 3 17 Prolactin antagonism, ciclosporin, 154- 156 Prostacyclin, 36 Prostaglandin D2, 37 Prostaglandin El, 33-36 immunomodulatory and antiinflammatory effects, 35-36 pro-inflammatory effects, 33-34 Prostaglandin F,, 37 Prostaglandins pain and, 38-39 production, nitric oxide action on, 60-61 Prostanoids, 31, 33-38 chronic inflammation, 37-38 inhibition of formation, 28 Prostate, cell cycle, redefining, 295 Prostate cancer, 281-302 androgen ablation-induced apoptosis p S 3 expression, 294-295 recruitment into, perturbed cell cycle, 293-294 androgen-independent cells, proliferationindependent therapeutic approaches, 285,287 androgen sensitivity, 282-283 apoptosis induced by castration, cell proliferation role, 291-293 therapeutic implication, 295-296 cell kinetics during progression, 283-286 incidence, 281-282 normal and neoplastic cells, kinetic parameters, 284-286 thapsigargin as therapy, 300-302

index

Prostate gene, expression during apoptosis, 290-291 Prostate specific antigen, apoptosis, 301-302 Prostatic glandular cells, normal, apoptosis following androgen ablation, 287-289 Proteins binding phage display libraries, 324-326 regulatory DNA sequences, 333-334 biologically active, random genetic selection, 324-325 Pseudomonas aeruginosa exotoxin A, 262 His440, 263 Psoriasis, ciclosporin therapeutic effects, 172-175 Renal allografts, histoincompatible, ciclosporin effects, 197-198 Renal inflammation, nitric oxide in, 57 Renal transport mechanisms, organic acid and base, 8-9 Rhabdomyolysis, 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors, 89-90 Rheumatoid arthritis, nitric oxide in, 55-56 Sandimmun, clinical pharmacokinetics, 204-207 Sandimmun Neoral, 207-208 Scandinavian Simvastatin Survival Study, 3hydroxy-3-methylglutarylcoenzyme A reduaase inhibitors, 93-94 Simonsen test, 129 Simvastatin, 88, 93-94 Smooth muscle cells, stimulation, ciclosporin effect, 200 Smooth muscles, contraction, ciclosporin and, 186-187 Sodium salicylate, 39 Spike frequency adaptation, reduction by linopirdine, 371-374 Stanford Coronary Risk Intervention Project, 314-315 Stockholm Ischaemic Heart Disease Secondary Prevention Study, nicotinic acid, 101

393

St. Thomas’ Atherosclerosis Regression Study, 85, 315 Suppressor T cells cell-mediated function, ciclosporin and, 137-143 development and ciclosporin, 165 Systemic graft-versus-host reaction, 129 Tacrolimus, 127 T cells activated, nuclear factor, 127 autoreactive, ciclosporin treatment, 168-169 cytotoxic, see Cytotoxic T cells functions for help, memory, and delayedtyped hypersensitivity, suppression by ciclosporin, 133-137 mammalian mono-ADPribosyltransferases, 260 mediating suppression, functional significance, 138-139 signaling pathways, 11 8 suppressor, see Suppressor T cells Thapsigargin apoptosis activation, 297-300 structure, 297 as therapy for prostate cancer, 300-302 T-helper cells, ciclosporin and, 134-135 Thromboxane A2, 36-37 Thymus, effect of ciclosporin treatment, 166 Tissue damage, nitric oxide, 50-51 Tolerance, see Antigen-specific functional unresponsiveness Transducin, ADP-ribosylation, 253 Transferase A and B, turkey erythrocyte, 250-252 Transplanted patients, early posttransplant period, 123 Triglycerides, agents lowering, 98-105 fibric-acid derivatives, 101-104 fish oil, 104-105 nicotinic acid, 99-101 Tubulin, ADP-ribosylation, 253 Turkey ADP-ribosylarginine hydrolase, 269-270 Turkey erythrocytes ADP-ribosyltransferases, 250-252

3 94

Index

Turkey erythrocytes (continued) transferase A, in vitro substrates, 252-253

Veto cell, mediation of cytotoxic T cell suppression, 141 Vitamin, drug interactions, 14-15

Ulcerative colitis, nitric oxide in, 58 University of California, San Francisco, Specialized Center of Research Intervention Trial, 309-310

World Health Organization Cooperative Trial, fibric-acid derivatives, 103

Contents of Previous Volumes

Volume 26 Cyclic GMP: Synthesis, Metabolism, and Function Edited by Ferid Murad Introduction and Some Historical Comments Ferid Murad

Cloning of Guanylyl Cyclase Isoforms Masaki Nakane and Ferid Murad

Regulation of Cytosolic Guanylyl Cyclase by Nitric Oxide: The NO-Cyclic GMP Signal Transduction System Ferid Murad

Regulation of Cytosolic Guanylyl Cyclase by Porphyrins and Metalloporphyrins Louis J. lgnarro

Regulation of Articulate Guanylate Cyclase by Natriuretic Peptides and Escherichiu coli Heat-Stable Enterotoxin Dale C. Leitman, Scott A. Waldman, and Ferid Murad

Cyclic GMP and Regulation of Cyclic Nucleotide Hydrolysis William K. Sonnenberg and Joseph A. Beavo

Progress in Understanding the Mechanism and Function of Cyclic GMP-dependent Protein Kinase Sharron H. Francis and Jackie D. Corbin

395

396

Contents of Previous Volumes

Effects of Cyclic GMP in Smooth Muscle Relaxation Timothy D. Warner, Jane A. Mitchell, Hong Sheng, and Ferid Murad

Interrelationships of Cyclic GMP, Inositol Phosphates, and Calcium Masato Hirata and Ferid Murad

Cyclic GMP Regulation of Calcium Slow Channels in Cardiac Muscle and Vascular Smooth Muscle Cells Nicholas Sperelakis, Noritsugu Tohse, Yusuke Ohya, and Hiroshi Masuda

Effect of Cyclic GMP on Intestinal Transport Arie B. Vaandrager and Hugo R. De Jonge

Cyclic GMP in Lower Forms Joachim E. Schultz and Susanne Klumpp

Clinical Relationships of Cyclic GMP Jean R. Cusson, Johanne Tremblay, Pierre Larochelle, Ernest0 L. Schiffrein, Jolanta Gutkowska, and Pave1 Hamet

Future Directions Ferid Murad

Volume 27 Conjugation-Dependent Carcinogenicity and Toxicity of Foreign Compounds Edited by M. W. Anders and Wolfgang Dekant

Historical Perspectives on Conjugation-Dependent Bioactivation of Foreign Compounds James A. Miller and Young-Joon Surb

Part I: Glutathione-Dependent Toxicity Enzymology of Microsomal Glutathione S-Transferase Claes Andersson. Erifili Mosialou, Rolf Weinander, and Ralf Morgenstern

Enzymology of Cytosolic Glutathione S-Transferases Brain Keterer and Lucia G. Christodoulides

Enzymology of Cysteine S-Conjugate b-Lyases Arthur J. L. Cooper

Contents of Previous Volumes

397

Formation and Fate of Nephrotoxic and Cytotoxic Glutathione S-Conjugates: Cysteine Conjugates: Cysteine Conjugate b-Lyase Pathway Wolfgang Dekant, Spyridon Vamvakas, and M. W. Anders

Reversibility in Glutathione-Conjugate Formation Thomas A. Baillie and Kelem Kassahun

Glutathione Conjugation as a Mechanisms for the Transport of Reactive Metabolites Terrence J. Monks and Serrine S. Lau

Metabolism and Genotoxicity of Dihaloalkenes F. Peter Guengerich

Bioactivation of Thiols by One-Electron Oxidation Rex Munday

Glutathione Mercaptides as Transport Forms of Metals N. Ballatori

Part 11: Sulfate Conjugate-Dependent Toxicity Biochemistry of Cytosolic Sulfotransferases Involved in Bioactivation Charles N. Falany and Teresa W. Wilborn

Carcinogen Activation by Sulfate Conjugate Formation Christopher J. Michejda and Marily B. Kroeger-Koepke

Part 111: Glucuronide Conjugate-Dependent Toxicity UDP-Glucuronosyltransferase and Their Role in Metabolism and Disposition of Carcinogens Karl Water Bock

Bioactivation by Glucuronide-Conjugate Formation Parnian Zia-Amirhosseini. Hildegard Spahn-Langguth, and Leslie Z. Benet

Part IV:Bioactivation and Bioconversion N-Actelytransferases, 0-Acetyltransferases, and N,OAcetyltransferases: Enzymology and Bioactivation Patrick E. Hanna

Aminocylases M. W. Anders and Wolfgang Dekant

398

Contents of Previous Volumes

Bioactivation by S-Adenosylation, S-Methylation, or N-Methylation jerald L. Hoffman

Bioconversion of Prodrugs by Conjugate-Processing Enzymes Spyridon Vamvakas and M. W. Anders

Volume 28

Regulation of Endothelial Cell Adhesion Molecular Expression with Antisense Oligonucleotides C. Frank Bennett and Stanley T. Crooke

The Role of the I-Arginine: Nitric Oxide Pathway in Circulatory Shock Christoph Thiemermann

Platelet-Activating Factor Antagonists: Scientific Background and Possible Clinical Applications Matyas Koltai, Philippe Guinot, David Hosford, and Pierre G. Broquet

Therapeutic Implications of Delivery and Expression of Foreign Genes in Hepatocytes Adam W. Grasso and George Y. W u

Recombinant Toxins Robert J. Kreitman and Ira Paston

Therapeutic Potential of the Lazaroids (21-Aminosteroids) in Acute Central Nervous System Trauma, Ischemia, and Subarachnoid Hemorrhage Edward D. Hull, John M. McCall and Eugene D. Means

Angiotensin I1 Receptor Pharmacology Kathy K. Griendling, Bernard Lasskgue. Thomas 1. Murphy, and R. Wayne Alexander

New Developments in Macrolides: Structures and Antibacterial and Prokinetic Activities P. A. Lartey, H. Nellans, and S. K. Tanaka Volume 29A DNA Topoisomerases: Molecular Targets for Chemotherapy of Cancer and Infectious Diseases Edited by Leroy F. Liu

Contents of Previous Volumes

399

ADNA Topoisomerases as Targets of Therapeutics: An Overview James C. Wang

Biochemistry of Bacterial Type 1-DNA Topoisomerases Yuk-Ching and Tse-Dinh

The Biochemistry and Biology of DNA Gyrase Rolf Menzel and Martin Gellert

Mechanism of Catalysis of Eukaryotic DNA Topoisomerase I JamesJ. Champoux

The DNA Binding, Cleavage, and Religation Reactions of Eukaryotic Topoisomerases I and I1 Anni H. Andersen, Jesper Q. Svejstrup, and Ole Westergaard

Roles of DNA Topoisomerases in Chromosomal Replication and Segregation Jon Nitiss

Roles of DNA Topoisomerases in Transcription Marc Drolet, Hai-Young Wu, and Leroy F. Liu

DNA Topoisomerase-Mediated Illegitimate Recombination Hideo lkeda

Cellular Regulation of Mammalian DNA Topoisomerases jualang Hwang and Ching-Long Hwong

Structure of Eukaryotic Type I DNA Topoisomerase Tao-shih Hsieh, Maxwell P. Lee, and Sheryl D. Brown

4-Quinolones and the Physiology of DNA Gyrase Karl Drlica and Barry Kreiswirth

Molecular Mechanisms of DNA Gyrase Inhibition by Quinolone Antibacterials Linus L. Shen

Volume 29B Topoisomerase-Targeting Antitumor Drugs Edited by Leroy F. Liu Clinical Development of Topoisomerase-Interactive Drugs Franco M. Muggia and Howard A. Burris

400

Contents of Previous Volumes

Topoisomerases in Human Leukemia David Peereboom, Martin Charron. and Scott H. Kaufmann

Preclinical and Clinical Development of Camptothecins Dan Costin and Milan Potmesil

Mechanisms of Topoisomerase I Inhibition by Anticancer Drugs Yves Pommier, Akihiko Taninwa, and Kurt W. Kohn

Drug Resistance Mechanisms of Topoisomerase I Drugs Toshiwa Andoh and Kosuke Okada

Mechanism of Action of Topoisomerase 11-Targeted Antineoplastic Drugs Neil Osheroff, Anita H. Corbett, and Megan J. Robinson

Determinants of Sensitivity to Topoisomerase-Targeting Antitumor Drugs Peter DArpa

Resistance of Mammalian Tumor Cells in Inhibitors of DNA Topoisomerase I1 William T. Beck, Mary K. Dank, Judith S. Wolverton, Mei Chen, Bernd Granzen, Ryungsa Kim, and D. Parker Suttle

A Bacteriophage Model System for Studying Topoisomerase Inhibitors Kenneth N. Kreuzer

Drugs Affecting Trypanosome Topoisomerases Theresa A. Shapiro

Yeast as a Genetic Model System for Studying Topoisomerase Inhibitors John L. Nitiss

DNA Topoisomerase Inhibitors as Antifungal Agents Linus L. Shen and Jennifer M. Fostel

Design of Topoisomerase Inhibitors to Overcome MDR1Mediated Drug Resistance Allan Y. Chen and Leroy F. Liu

Appendix I. An Introduction to DNA Supercoiling and DNA Topoisomerase-Catalyzed Linking Number Changes of Supercoiled DNA James C. Wang

Contents of Previous Volumes

40 I

Appendix 11. Alignment of Primary Sequences of DNA Topoisomerases Paul R. Caron and James C. Wang

Volume 30

Neuroprotective Actions of Excitatory Amino Acid Receptor Antagonists V. L. Woodburn and G. N. Woodruff

Pharmacologic Therapy of Obsessive Compulsive Disorders Joseph DeVeaugh-Geiss

Mechanism of Action of Antibiotics in Chronic Pulmonary Pseudomonas Infection Niels Holby, Birgit Giwercman. Elsebeth Tvenstrup Jensen, Svend Stenvang Pedersen, Chritian Koch. and Arsalan Kharazmi

Quinolinic Acid in Neurological Disease: Opportunities for Novel Drug Discovery John F. Reinhard, Jr.,Joel B. Erickson, and Ellen M. Flanagan

Pharmacologic Management of Shock-Induced Renal Dysfunction Anupam Agarwal, Gunnar Westberg, and Leopoldo Raij

Autoantibodies against Cytochromes P450: Role in Human Diseases Philippe Beaune, Dominique Pessayre, Patrick Dansette, Daniel Mansuy, and Michael Manns

Activation and inactivation of Gene Expression Using RNA Sequences Boro Dropulic, Stephen M. Smith, and Kuan-Teh Jeang

Therapy of Cancer Metastasis by Systemic Activation of Macrophages Isaiah J. Fidler

5-Hydroxytryptomine Receptor Subtypes: Molecular and Functional Diversity Frederic Saudou and Rene Hen

Volume 31 Anesthesia and Cardiovascular Disease Edited by Zeljko J. Bosnjak and John P. Kampine

402

Contents of Previous Volumes

Regulation of the Calcium Slow Channels of the Heart by Cylic Nucleotides and Effects of Ischemia Nicholas Sperelakis

Functional Adaptation to Myocardial Ischemia: Interaction with Volatile Anesthetics in Chronically Instrumented Dogs Patrick F. Wouters, Hugo Van Aken, Marc Van de Velde, Marco A. E. Marcus, and Willem Flameng

Excitation-Contraction Uncoupling and Vasodilators for Long-Term Cold Preservation of Isolated Hearts David F. Stowe

Troponin T as a Marker of Perioperative Myocardial Cell Damage H. Machler, H. Gombotz, K. Sabin, and H. Metzler

Silent Myocardial Ischemia: Pathophysiology and Perioperative Management Anders G. Hedman

Effect of Halothane on Sarcolemmal Calcium Channels during Myocardial Ischemia and Reperfusion Benjamin Drenger, Yehuda Ginosar, and Yacov Gozal

Myocardial Ishemic Preconditioning Donna M. Van Winkle, Grace L. Chien, and Richard F. Davis

Effects Hypoxiahleoxygenation on Intracellular Calcium Ion Homeostasis in Ventricular Myocytes during Halothane Exposure Paul R. Knight, Mitchell D. Smith, and Bruce A. Davidson

Mechanical Consequences of Calcium Channel Modulation during Volatile Anesthetic-Induced Left Ventricular Systolic and Diastolic Dysfunction Paul S. Pagel and David C. Warltier Anesthetic Actions on Calcium Uptake and CalciumDependent Adenosine Triphophatase Activity of Cardiac Sarcoplasmic Reticulum Ning Miao, Martha J. Frazer, and Carl Lynch 111

Interaction of Anesthetics and Catecholamines on Conduction in the Canine His-Purkinje System L. A. Turner, S. Vodanovic, and Z. J. Bosnjak

Contents of Previous Volumes

403

Anesthetics, Catecholamines, and Ouabain on Automaticity of Primary and Secondary Pacemakers John L. Atlee 111, Martin N. Vincenzi, Harvey J. Woehlck, and Zelijko J. Bosnjak

The Role of L-Type Voltage-Dependent Calcium Channels in Anesthetic Depression of Contractility T. J. J. Blanck, D. L. Lee, S. Yasukochi, C. Hollmann, and J. Zhang Effects of Inhibition of Transsarcolemmal Calcium Influx on Content and Releasability of Calcium Stored in Sarcoplasmic Reticulum of Intact Myocardium Hirochika Komai and Ben F. Rusy

Arrhythmogenic Effect of Inhalation Anesthetics: Biochemical Heterogeneity between Conduction and Contractile Systems and Protein Unfolding lssaku Ueda and JanShing Chiou

Potassium Channel Current and Coronary Vasodilatation by Volatile Anesthetics Nediijka Buljubasic, lure Mariijic, and Zelijko J. Bosnjak

Potassium Channel Opening and Coronary Vasodilation by Halothane D. R. Larach, H. G. Schuler, K. A. Zangari, and R. L. McCann

Volatile Anesthetics and Coronary Collateral Circulation Judy R. Kersten, Craig Hartman, Paul S. Pagel, and David C. Warltier

Myocardial Oxygen Supply-Demand Relations during Isovolemic Hermodilution Geroge J. Crystal

Plasma Membrane Ca2+-ATPaseas a Target for Volatile Anesthetics Danuta Kosk-Kosicka

Enhancement of Halothane Action at the Ryanodine Receptor by Unsaturated Fatty Acids Jeffrey E. Fletcher and Vincent E. Welter

Adrenergic Receptors: Unique Localization in Human Tissues Debra A. Schwinn

404

Contents of Previous Volumes

Volatile Anesthetic Effects on Inositol Triphosphate-Gated Intracellular Calcium Stores in GH3 Cells Alex S. Evers and M. Delawar Hossain Differential Control of Blood Pressure by Two Subtypes of Carotid Baroreceptors Jeanne L. Seagard

Sympathetic Activation with Desflurane in Humans Thomas J. Ebert and M. Muzi

Randomized, Prospective Comparison of Halothane, Isoflurane, and Enflurane on Baroreflex Control of Heart Rate in Humans Michael Muzi and Thomas J. Ebert

Baroreflex Modulation by Isoflurane Anesthesia in Normotensive and Chronically Hypertensive Rabbits Leonard B. Bell

Effects of Isoflurane on Regulation of Capacitance Vessels in the Normotensive and Chronically Hypertensive Condition Thomas A. Stekiel, Leonard B. Bell, Zelijko, J. Bosnjak, and john P. Kampine

Effect of Volatile Anesthetics on Baroreflex Control of Mesenteric Venous Capacitance J. Bruce McCallum, Thomas A. Stekiel, Anna Stadnicka, Zelijko j. Bosnjak, and john P. Kampine

Effect of General Anesthesia on Modulation of Sympathetic Nervous System Function Margaret Wood

Inhibition of Nitric Oxide-Dependent Vasodilaton by Halogenated Anesthesia Ming Jing, Jayne L. Hart. Saiid Bina, and Sheila M. Muldoon

Effects of Epidural Anesthesia on Splanchnic Capacitance Quinn H. Hogan, Anna Stadnicka, and john P. Kampine

Anesthetic Modulation of Pulmonary Vascular Regulation Paul A. Murray

Pulmonary Mechanics Changes Associated with Cardiac Surgery Ron Dueck

Contents of Previous Volumes

405

Inhaled Nitric Oxide in Adult Respiratory Distress Syndrome and Other Lung Diseases Warren M. Zap01 and William E. Hurford

First Pass Uptake in the Human Lung of Drugs Used during Anesthesia David L. Roerig, Susan B. Ahlf, Christopher A. Dawson, John H. Linehan, and John P. Kampine

Lactic Acidosis and pH on the Cardiovascular System C.Wong

Yuguang Huang, James B. Yee, Wang-Hin Yip, and K.

Role of Oxygen Free Radicals and Lipid Peroxidation in Cerebral Reperfusion Injury Richard J. Traystman and Daniel Nyhan

Effects of Volatile Anesthetics on Cerbrocortical Laser Doppler Flow: Hyperemia, Autoregulation, Carbon Dioxide Response, Flow Oscilliations, and Role of Nitric Oxide Antal G. Hudetz, Joseph G. Lee, Jeremy J. Smith, Zelijko J. Bosnjak, and John P. Kampine

Cerebral Blood Flow during Isovolemic Hemodilution: Mechanistic Observations Michael M. Todd

Cerebral Physiology during Cardiopulmonary Bypass: Pulsatile versus Nonpulsatile Flow Brad Hindman

Anesthetic Actions of Cardiovascular Control Mechanisms in the Central Nervous System William T. Schmeling and Neil E. Farber

Volume 32

Signal Sorting by G-Protein-Linked Receptors Graeme Milligan

Regulation of Phospholipase A2 Enzymes: Selective Inhibitors and Their Pharmacological Potential Keith B. Glaser

Platelet Activating Factor Antagonists James B. Summers and Daniel H. Albert

406

Contents of Previous Volumes

Pharmacological Management of Acute and Chronic Bronchial Asthma Michael K. Gould and Thomas A. Raffin

Anti-Human Immunodeficiency Virus Immunoconjugates Seth H. Pincus and Vladimir V. Tolstikov

Recent Advances in the Treatment of Human Immunodeficiency Virus Infections with Interferons and Other Biological Response Modifiers Orjan Stranneg’drd

Advances in Cancer Gene Therapy Wei-Wei Zhang, Toshiyoshi Fujiwara, Elizabeth A. Grimm, and Jack A. Roth

Melanoma and Melanocytes; Pigmentation, Tumor Progression, and the Immune Response to Cancer Setaluri Vijayasaradhi and Alan N. Houghton

High-Density Lipoprotein Cholesterol, Plasma Triglyceride, and Coronary Heart Disease: Pathophysiology and Management Wolfgang Patsch and Antonio M. Gotto, Jr.

Neurotransmitter-like Actions of I-DOPA Yoshimi Misu, Hiroshi Ueda, and Yoshio Goshima

New Approaches to the Drug Treatment of Schizophrenia Gavin P. Reynolds and Carole Czudek

Membrane Trafficking in Nerve Terminals Flavia Valtorta and Fabio Benfenati

Volume 33

Endothelin Receptor Antagonism Terry J. Opgenorth

The Ryanodine Receptor Family of Intracellular Calcium Release Channels Vincenzo Sorrentino

Design and Pharmacology of Peptide Mimetics Graham J. Moore, Julian R. Smith, Barry W. Baylis, and John M. Mauoukas

Contents of Previous Volumes

407

Alternative Approaches for the Application of Ribozymes as Gene Therapies for Retroviral Infections Thomas B. Campbell and Bruce A. Sullenger

Inducible Cyclooxygenase and Nitric Oxide Synthase Kenneth K. W u

Regulation of Airway Wall Remodeling: Propsects for the Development of Novel Antiasthma Drugs Alastair G. Stewart, Paul R. Tomlinson, and John W. Wilson

Advances in Selective Immunosuppression Lucian0 Adorini, Jean-Charles Guery, and Sylvie Trembleau

Monoclonal Antibody Therapy of Leukemia and Lymphoma Joseph G. Jurcic, Philip C. Caron, and David A. Scheinberg

4-Hydroxyphenylretinamide in the Chemoprevention of Cancer Harmesh R. Naik, Gregory Kalemkerian, and Kenneth J. Pienta

Immunoconjugates and Immunotoxins for Therapy of Carcinomas lngegerd Hellstrom, Karl Erik Hellstrom. Clay B. Siegall, and Pamela A. Trail

Discovery and in Vitro Development of AIDS Antiviral Drugs as Biopharmaceuticals William G. Rice and John P. Bader

Volume 34 Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications Edited by Louis lgnarro and Ferid Murad

Chemistry of Nitric Oxide: Biologically Relevant Aspects Jon M. Fukuto

Reactions between Nitric Oxide, Superoxide, and Peroxynitrite: Footprints of Peroxynitrite in Vivo John P. Crow and Joseph S. Beckman

Oxygen Radical-Nitric Oxide Reactions in Vascular Diseases Bruce A. Freeman, Roger White, Hector Gutierrez, Andres Paler-Martinez, Margaret Tarpey, and Homero Rubbo

Nitric Oxide Synthases: Gene Structure and Regulation Yang Wang and Philip A. Marsden

408

Contents of Previous Volumes

Transcription of the Human Neuronal Nitric Oxide Synthase Gene in the Central Nervous System Is Mediated by Multiple Promoters Anthony P. Young, Ferid Murad, Harald Vaessin, JinlingXie, and Terrie K. Rife

Regulation of the Expression of the Inducible Isoform of Nitric Oxide Synthase Csaba Szabo and Christoph Thiernerrnann

Regulation and Function of Inducible Nitric Oxide Synthase during Sepsis and Acute Inflammation James W. Wong and Timothy R. Billiar

Expression and Expressional Control of Nitric Oxide Synthases in Various Cell Types Ulrich Forsterrnann, Hartmut Kleinert, lngolf Gath, Petra Schwarz. Ellen 1. Closs, and Nae J. Dun

Control and Consequences of Endothelial Nitric Oxide Formation Ingrid Fleming and Rudi Busse

Control of Electron Transfer in Neuronal Nitric Oxide Synthase by Calmodulin, Substrate, Substrate Analogs, and Nitric Oxide Dennis J. Stuehr, Husarn M. Abu-Soud, Denis L. Rousseau, Paul L. Feldrnan, and JianlingWang

Negative Modulation of Nitric Oxide Synthase by Nitric Oxide and Nitroso Compounds Jeanette M. Griscavage, Adrian J. Hobbs, and Louis J. lgnarro

Regulation of Nitric Oxide Synthase: Role of Oxygen Radicals and Cations in Nitric Oxide Formation Chandra K. Mittal and Chander S. Mehta

Why Tetrahydrobiopterin? Bernd Mayer and Ernst R. Werner

Nitric Oxide and cGMP Signaling Lee J. McDonald and Ferid Murad

Nitric Oxide and Intracellular Heme Young-Myeong Kim, Hector A. Bergonia, Claudia Mi", W. David Watkins, and Jack R. Lancaster, Jr.

Bruce R. Pitt,

High-Level Expression of Biologically Active Soluble Guanylate Cyclase Using the Baculovirus System Is Strongly Heme-Dependent Wolfgang A. Buechler, Sujay Singh, Janet Aktas, Stefan Mullet-, Ferid Murad, and Rupert Gerzer

Contents of Previous Volumes

409

cGMP Signaling through CAMP-and cGMP-Dependent Protein Kinases Thomas M. Lincoln, Padmini Komalavilas, Nancy J. Boerth, Lee Ann MacMillan-Crow, and Trudy L. Cornwell

Physiological and Toxicological Actions of Nitric Oxide in the Central Nervous System Valina L. Dawson and Ted M. Dawson

S-Nitrosothiols: Chemistry, Biochemistry, and Biological Actions Gilbert R. Upchurch, Jr., George N. Welch, and Joseph Loscalzo

Glyceraldehyde-3-Phosphate Dehydrogenase: A Target for Nitric Oxide Signaling Bernhard Brune and Eduardo G. Lapetina

Nitric Oxide Donors: Biochemical Pharmacology and Therapeutics John Anthony Bauer, Brian P. Booth, and Ho-Leung Fung

Nitric Oxide Donors: A Continuing Opportunity in Drug Design Stephen R. Hanson, Thomas C. Hutsell, Larry K. Keefer, Daniel L. Mooradian, and Daniel 1. Smith

Nitric Oxide and Peripheral Adrenergic Neuromodulation Roberto Levi, Kwan Ha Park, Michiaki Imamura, Nahid Seyedi, and Harry M. Lander

A Study on Tumor Necrosis Factor, Tumor Necrosis Factor Receptors, and Nitric Oxide in Human Fetal Glial Cultures Barbara A. St. Pierre, Douglas A. Granger, Joyce L. Wong, and Jean E. Merrill

Inhaled Nitric Oxide, Clinical Rationale and Applications Claes G. Frostell and Warren M. Zap01

Inhaled Nitric Oxide Therapy of Pulmonary Hypertension and Respiratory Failure in Premature and Term Neonates Steven H. Abman and John P. Kinsella

Clinical Applications of Inhaled Nitric Oxide in Children with Pulmonary Hypertension David L. Wessel and Ian Adatia

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