DRUG METABOLIZING ENZYMES Cytochrome P450 and Other Enzymes in Drug Discovery and Development © 2003 by Taylor & Francis Group, LLC DRUG METABOLIZING ENZYMES Cytochrome P450 and Other Enzymes in Drug Discovery and Development Edited by Jae S.Lee, R.Scott Obach and Michael B.Fisher © 2003 by Taylor & Francis Group, LLC Published in 2003 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487–2742 © 2003 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group This book is a joint publication of FontisMedia S.A. and Taylor & Francis Group, LLC No claim to original U.S. Government works International Standard Book Number-10:0-8247-4293-1 (Print Edition) (Hardcover) International Standard Book Number-13:978-0-8247-4293-5 (Print Edition) (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com © 2003 by Taylor & Francis Group, LLC DEDICATION This book is dedicated to Michael R.Rowley (1969–2002), who passed away January 19, 2002, after a long battle with cancer. Mike, you were a good friend, and you are sorely missed. M.B.Fisher © 2003 by Taylor & Francis Group, LLC vii Contents Editors’ Preface xiii Foreword xv Anthony Y.H.Lu Chapter 1 DIOXYGEN ACTIVATION BY CYTOCHROMES P450: A ROLE FOR MULTIPLE OXIDANTS IN THE OXIDATION OF SUBSTRATES 1 Alfin D.N.Vaz 1 INTRODUCTION 1 1.1 Ligands to heme in CYP enzymes 2 1.2. Reaction cycle of CYP enzymes 3 2 ACTIVE OXIDANT(S) IN CYP REACTIONS 6 2.1 Heme-oxene as oxidant 6 2.2 Heme-peroxo as an oxidant 11 2.3 Multiple Oxidant Hypothesis in CYP-catalyzed oxidations 17 3 HEME-HYDROPEROXO AS AN OXIDANT 19 4 HEME-SUPEROXO AS ACTIVE OXIDANT 24 5 THE TWO-STATE THEORY 25 CONCLUSIONS 27 REFERENCES 27 Chapter 2 APPLICATION OF LC/MS, LC/NMR, NMR AND STABLE ISOTOPES IN IDENTIFYING AND CHARACTERIZING METABOLITES 33 A.E.Mutlib and John P.Shockcor 1 INTRODUCTION 33 2 SEPARATION OF METABOLITES FROM ENDOGENOUS COMPONENTS 36 2.1 Sample Clean-up on Solid Phase Cartridges 37 3 LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY 39 © 2003 by Taylor & Francis Group, LLC viii 4 NUCLEAR MAGNETIC RESONANCE (NMR) 46 4.1 Continuous-Flow LC-NMR 46 4.2 Time-Slice LC-NMR 47 4.3 Stop-Flow LC-NMR 47 4.4 Loop-Storage 49 4.5 LC-NMR-MS 49 4.6 Recent Advances in NMR 50 5 SPECIFIC EXAMPLES 51 5.1 Metabolism of Efavirenz (DPC 266) and Renal Toxicity 51 5.2 Characterization of Unusual Metabolites 54 5.3 Pharmacologically Active Metabolites 80 6 CONCLUSIONS 81 REFERENCES 83 Chapter 3 BIOACTIVATION 87 Jack Uetrecht 1 INTRODUCTION 87 2 INVOLVEMENT OF REACTIVE METABOLITES IN ADVERSE REACTIONS 88 2.1 Types of Adverse Drug Reactions 88 2.2 The Hapten Hypothesis 89 2.3 The Danger Hypothesis 91 2.4 Immune Response in the Absence of Covalent Binding 92 3 ENZYME SYSTEMS RESPONSIBLE FOR METABOLIC ACTIVATION 93 4 TYPES OF REACTIVE METABOLITES 94 4.1 General Types of Reactive Metabolites 94 4.2 Alkyl Halides 96 4.3 Primary Aryl Amines 97 4.4 Aromatic Nitro Drugs 101 4.5 Hydrazines 104 4.6 Other Nitrogen-Containing Aromatic Compounds 106 4.7 Acyl Glucuronides and Co-A Esters 107 4.8 Quinone-type Reactive Metabolites 110 4.10 Metabolites, such as Epoxides, that are Reactive because of Ring Strain 121 4.11 Furans and Thiophenes 125 4.12 Sulfhydryl-Containing Drugs 126 4.13 Thiono Sulfur Compounds 127 4.14 Isocyanates 129 4.15 Acetylenes 131 © 2003 by Taylor & Francis Group, LLC ix 4.16 Methylenedioxyphenyl compounds 131 4.17 Free Radicals 132 5 THE USE OF BIOACTIVATION SCREENS FOR LEAD DRUG SELECTION 135 6 SUMMARY AND CONCLUSIONS 138 REFERENCES 139 Chapter 4 CHEMICALLY REACTIVE METABOLITES IN DRUGDISCOVERY AND DEVELOPMENT 147 Thomas A.Baillie REFERENCES 154 Chapter 5 CYTOCHROME P450 AND ITS PLACE IN DRUG DISCOVERY AND DEVELOPMENT 155 Dennis Smith INTRODUCTION 155 1 FROM SINGLE ENZYME TO SUPERFAMILY OF HUMAN ISOFORMS 155 2 INHIBITION OF HUMAN ISOFORMS 160 3 INDUCTION OF HUMAN ISOFORMS 163 4 DIVERSITY OF HUMAN ISOFORMS 166 5 RESPONSE TO P450 METABOLISM IN DRUG DESIGN 168 6 P450 AS A DRUG TARGET 173 7 SUMMARY 176 REFERENCES 176 Chapter 6 CYTOCHROME P450 IN LABORATORY ANIMAL SPECIES 179 Margit Spatzenegger, Stephanie L.Born and James R.Halpert 1 INTRODUCTION 179 2 LABORATORY ANIMAL MODELS AS PREDICTORS OF HUMAN METABOLISM 179 2.1 Basic Approaches 180 2.2 Interspecies Scaling 182 3 FROM ANIMAL TO MAN: COMPARISON OF CATALYTIC SELECTIVITY BY ONE SUBFAMILY 185 © 2003 by Taylor & Francis Group, LLC x 3.1 CYP1A 185 3.2 CYP2A 186 3.3 CYP2B 187 3.4 CYP2C 188 3.5 CYP2D 189 3.6 CYP2E 189 3.7 CYP3A 190 3.8 CYP4A 191 4 REGULATION OF LABORATORY ANIMAL CYTOCHROME P450 192 4.1 Regulation of CYP1 genes 192 4.2 Regulation of CYP2B genes 193 4.3 Regulation of CYP3 A genes 195 4.4 Regulation of CYP4A genes 196 5 NON-NATIVE ANIMAL MODELS 197 5.1 The Gene Knockout Mouse Model 197 5.2 The Humanized Mouse Model 200 6 CONCLUSIONS 201 REFERENCES 202 Chapter 7 TYPICAL AND ATYPICAL ENZYME KINETICS 211 J.Brian Houston, Kathryn E.Kenworthy and Aleksandra Galetin 1 INTRODUCTION 212 1.1 General considerations of In Vitro Experimentation 213 1.2 Scope of this chapter 215 2 THE MICHAELIS-MENTEN APPROACH FOR ANALYSIS OF ENZYME KINETIC DATA 215 2.1 Enzyme Kinetic Studies in Drug Metabolism 216 2.2 Assumptions in Enzyme Kinetics 216 2.3 Methodological Considerations 217 2.4 Analysis of Substrate Kinetic Data 218 2.5 Analysis of Enzyme Inhibition Data 223 3 MULTISITE APPROACH FOR ANALYSIS OF A TYPICAL KINETIC DATA 227 3.1 Homotropic Effects 229 3.2 Heterotropic Effects 235 3.3 Criteria for Selection of an Appropriate Multisite Kinetic Model in Prediction of Drug Interactions 246 4 IN VIVO RELEVANCE OF IN VITRO ATYPICAL KINETICS AND CURRENT PERSPECTIVE 248 REFERENCES 251 © 2003 by Taylor & Francis Group, LLC xi Chapter 8 CYTOCHROME P450 REACTION PHENOTYPING 255 Larry C.Wienkers and Jeffrey C.Stevens 1 INTRODUCTION 256 2 EFFECT OF INCUBATION CONDITIONS ON REACTION PHENOTYPING 262 3 ENZYME KINETICS AND REACTION PHENOTYPING 266 4 CORRELATION ANALYSIS 274 5 EXPRESSED P450S AND REACTION PHENOTYPING 281 6 CHEMICAL INHIBITORS AND REACTION PHENOTYPING 285 7 ANTIBODIES 294 8 CONCLUDING REMARKS 299 9 ACKNOWLEDGEMENTS 300 REFERENCES 300 Chapter 9 DRUG-DRUG INTERACTIONS AND THE CYTOCHROMES P450 311 Kenneth A.Bachmann, Barbara J.Ring and Steven A.Wrighton 1 INTRODUCTION 311 1.1 The Numerology of Drug-Drug Interactions 311 1.2 The Centrality of the Cytochromes P450 in Drug-Drug Interactions 312 1.3 Clinical Ramifications of CYP-Based Drug-Drug Interactions 314 1.4 Electronic Sources of Information on CYP-Based Drug-Drug Interactions 315 2 IN VITRO DRUG-DRUG INTERACTIONS: DETERMINING THE POTENTIAL OF AN NCE TO INHIBIT THE CYPS 317 2.1 In Vitro to In Vivo Extrapolations 319 2.2 Irreversible or Quasi-Irreversible Inhibition 321 3 IN VIVO DRUG-DRUG INTERACTION STUDIES 322 3.1 CYP-Based Interactions with Nutraceuticals and Food 327 3.2 Transporter-Mediated Drug-Drug Interactions Masquerading as CYP-Based Interactions 330 3.3 Pharmacogenetics and CYP-Based Drug-Drug Interactions 331 4 CONCLUSION 332 REFERENCES 332 © 2003 by Taylor & Francis Group, LLC xii Chapter 10 CYP GENE INDUCTION BY XENOBIOTICS AND DRUGS 337 Academic authors: Jean-Marc Pascussi, Sabine Gerbal-Chaloin, Martine Daujat, Lionel Drocourt, Lydiane Pichard-Garcia, Marie-José Vilarem and Patrick Maurel Drug Industry authors: Sylvie Klieber, François Torreilles, Martine Bourrié, François Guillou and Gérard Fabre 1 INTRODUCTION 337 2 CLINICAL AND PHARMACOLOGICAL CONSEQUENCES OF CYP1-3 GENE INDUCTION IN MAN 338 3 MECHANISMS OF CYP GENE INDUCTION 339 3.1 Induction of the CYP1A family 339 3.2 Cross-talks between the AhR pathway and other transcriptional factors and signal transduction pathways 340 3.3 Induction of CYP2 and CYP3 families 344 3.4 Cross-talk between PXR and CAR 349 3.5 Cross-talk between PXR and CAR and other nuclear receptors 350 4 IN VITRO SYSTEMS TO SCREEN CYP INDUCERS 354 4.1 Direct receptor-ligand binding assays 355 4.2 Enhanced green fluorescent protein (EGFP)-based recombinant cell bioassay 356 4.3 Ligand-induced receptor-coactivator interaction assays 356 4.4 Cell lines for the direct screening of CYP1A inducers 357 4.5 Cell line co-transfections: gene reporter assays 358 4.6 Primary human hepatocytes for the direct screening of CYP inducers 359 5 IN VIVO EVALUATION OF CYP INDUCTION IN MAN 360 5.1 Induction of CYP1A2 isoform 361 5.2 Induction of CYP3A4 isoform 362 6 CONCLUSION 364 REFERENCES 365 Chapter 11 CYTOCHROME P450 PHARMACOGENETICS 375 Robert L.Haining and Aiming Yu 1 INTRODUCTION 375 1.1 Chapter Introduction 375 1.2 Brief History of Pharmacogenetics 375 1.3 Fundamentals of Pharmacogenetic Mutations 376 1.4 In Vivo versus In Vitro Considerations 380 1.5 Key principles of pharmacogenetics 380 © 2003 by Taylor & Francis Group, LLC