Mass Spectrometry in Drug Metabolism and Disposition E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Veröffentlichungsjahr: 2011

Beschreibung

This book examines the background, industrial context, process, analytical methodology, and technology of metabolite identification. It emphasizes the applications of metabolite identification in drug research. While primarily a textbook, the book also functions as a comprehensive reference to those in the industry. The authors have worked closely together and combine complementary backgrounds to bring technical and cultural awareness to this very important endeavor while serving to address needs within academia and industry It also contains a variety of problem sets following specific sections in the text.

Leseprobe

Contents

Foreword

Preface

Contributors

Part I : Basic Concepts of Drug Metabolism and Disposition

Chapter 1 : Progression of Drug Metabolism

1.1 Introduction

1.2 Historical Phases of Drug Metabolism

1.3 Next Step in the Progression of DM

1.4 Perspective on the Magnitude of the Challenge

1.5 Are there more Sensitive Alternatives to MS?

1.6 Summary

References

Chapter 2 : Common Biotransformation Reactions

2.1 Introduction

2.2 Oxidative Reactions

2.3 Reductive Reactions

2.4 Hydrolytic Reactions

2.5 Glucuronidation Reactions

2.6 Sulfation Reactions

2.7 Acylation Reactions

2.8 Methylation Reactions

2.9 Glutathione Conjugation Reactions

2.10 Conclusions

References

Chapter 3 : Metabolic Activation of Organic Functional Groups Utilized in Medicinal Chemistry

3.1 Introduction

3.2 Bioactivation of Drugs

3.3 Experimental Strategies to Detect Reactive Metabolites

3.4 Functional Group Metabolism to Reactive Intermediates

3.5 Structural Alerts and Drug Design

3.6 Reactive Metabolite Trapping and Covalent Binding Studies as Predictors of Idiosyncratic Drug Toxicity

3.7 Dose As An Important Mitigating Factor for IADR

3.8 Concluding Remarks

References

Chapter 4 : Drug-Metabolizing Enzymes, Transporters, and Drug–Drug Interactions

4.1 Introduction

4.2 Drug-Metabolizing Enzymes

4.3 Metabolism-Based DDIS

4.4 CYP Conclusion

4.5 Drug Transporters

4.6 Tools of The Transporter Trade

4.7 Uptake and Efflux Transporter Tools

4.8 Sample Analysis

4.9 Automation

4.10 In Vitro DDI Assays

4.11 In Vitro–in Vivo Correlations

4.12 Kinetic Models

4.13 Transporter Conclusion

Acknowledgment

References

Chapter 5 : Experimental Models of Drug Metabolism and Disposition

5.1 Introduction

5.2 ADME Study Strategy in Drug Discovery

5.3 ADME Experimental Models

5.4 Data Interpretation

5.5 Summary

Acknowledgments

References

Chapter 6 : Principles of Pharmacokinetics: Predicting Human Pharmacokinetics in Drug Discovery

6.1 Introduction

6.2 Physiological Pharmacokinetics

6.3 Prediction of Absorption

6.4 Distribution

6.5 Metabolism and Excretion

6.6 Drug–Drug Interactions

6.7 Practical Issues That Need To Be Considered

Abbreviations and Notations

References

Chapter 7 : Drug Metabolism Research as Integral Part of Drug Discovery and Development Processes

7.1 Introduction

7.2 Metabolic Clearance

7.3 Metabolite Profiling/Mass Balance Studies

7.4 Safety Testing of Drug Metabolites

7.5 Reaction Phenotyping

7.6 Assessment of Potential Toxicology of Metabolites

7.7 Assessment of Potential for Active Metabolites

7.8 Summary

References

Part II : Mass Spectrometry in Drug Metabolism: Principles and Common Practice

Chapter 8 : Theory and Instrumentation of Mass Spectrometry

8.1 Basic Concepts and Theory of Mass Spectrometry

8.2 Major Components of A Mass Spectrometer

8.3 ION Sources

8.4 Mass Analyzers

References

Chapter 9 : Common Liquid Chromatography–Mass Spectrometry (LC–MS) Methodology for Metabolite Identification

9.1 Introduction

9.2 Strategies for Metabolite Identification

9.3 In Silico Tools

9.4 Conclusions and Future Trends

Acknowledgment

References

Chapter 10 : Mass Spectral Interpretation

10.1 Molecular Weight and Empirical Formula Determination

10.2 Common Fragmentation Reactions

10.3 Practical Applications

10.4 Conclusion

Acknowledgment

References

Chapter 11 : Techniques to Facilitate the Performance of Mass Spectrometry: Sample Preparation, Liquid Chromatography, and Non-Mass-Spectrometric Detection

11.1 Introduction

11.2 Sample Preparation for Bioanalysis

11.3 Sample Preparation for Metabolite Profiling and Identification

11.4 Liquid Chromatographic Separation in Bioanalysis

11.5 Liquid Chromatographic Separation Technologies in Metabolite Profiling

11.6 Liquid Chromatographic Detection

References

Part III : Applications of New LC–MS Techniques in Drug Metabolism and Disposition

Chapter 12 : Quantitative In Vitro ADME Assays Using LC–MS as a Part of Early Drug Metabolism Screening

12.1 Introduction

12.2 Metabolic Stability Assays

12.3 Drug Absorption and Permeability Assays

12.4 Cytochrome P450 (CYP) Assays

12.5 New Technology for High-Throughput Assays

12.6 Conclusions

References

Chapter 13 : High-Resolution Mass Spectrometry and Drug Metabolite Identification

13.1 Introduction

13.2 Challenges Presented by Different Samples

13.3 Fundamental Advantage of High-Resolution Mass Spectrometry: Specificity/Selectivity in A Single Generic Method

13.4 High-Resolution Mass Spectrometry: Important Concepts

13.5 High-Resolution Instrumentation

13.6 Advantages of High-Resolution MS: The Concept of Mass Defect Filtration

13.7 Postprocessing Strategies for Identifying Metabolites in Complex High-Resolution Data Sets

13.8 Control Comparison

13.9 Background Subtraction

13.10 Isotope Filtration

13.11 “All-In-One” Data Analysis

13.12 Rationalization of Novel Metabolites

13.13 Assigning Product ION Spectra Using The Power of Accurate Mass

13.14 Localization: The Final Frontier

13.15 Quantitative And Qualitative In Vivo Pharmacokinetic Data From A Single Injection Per Sample

13.16 Future Opportunities

References

Chapter 14 : Distribution Studies of Drugs and Metabolites in Tissue by Mass Spectrometric Imaging

14.1 Introduction

14.2 Tissue Imaging Techniques

14.3 Mass Spectrometric Imaging Background

14.4 MSI Methodology

14.5 Applications of MSI For Detection of Drug Metabolites in Tissue

14.6 Conclusions

Acknowledgments

References

Chapter 15 : Use of Triple Quadrupole–Linear Ion Trap Mass Spectrometry as a Single LC–MS Platform in Drug Metabolism and Pharmacokinetics Studies

15.1 Introduction

15.2 Instrumentation and Scan Functions

15.3 In Vitro and In Vivo Metabolite Profiling and Identification

15.4 Reactive Metabolite Screening and Characterization

15.5 In Vitro Drug Interaction Studies

15.6 Quantification and Screening of Drugs and Small Molecules

15.7 Summary

References

Chapter 16 : Quantitative Drug Metabolism with Accelerator Mass Spectrometry

16.1 Relevance of AMS to Drug Metabolism

16.2 Introduction to AMS

16.3 Fundamentals of AMS Instruments

16.4 Sample Definition and Interfaces

16.5 AMS Quantitation

16.6 LC–AMS Analysis of Drug Metabolites

16.7 Comparative Resolution of Fraction LC Measurements

16.8 Quantitative Extraction and Recovery

16.9 LC–AMS Background and Sensitivity

16.10 Clinical Aspects of AMS Metabolite Studies

16.11 AMS Analysis of Reactive Metabolites

16.12 Species Metabolite Comparison

16.13 New Metabolic Studies Enabled By AMS

16.14 Conclusions

References

Chapter 17 : Standard-Free Estimation of Metabolite Levels Using Nanospray Mass Spectrometry: Current Statutes and Future Directions

17.1 Introduction

17.2 Current Approaches for Metabolite Quantitation in The Absence of Synthetic Standards

17.3 Use of Nanospray for Standard-Free Metabolite Quantitation

17.4 Future Directions

References

Chapter 18 : Profiling and Characterization of Herbal Medicine and Its Metabolites Using LC–MS

18.1 Introduction

18.2 Characterization of Chemical Constituents in Chinese Herbal Medicine

18.3 Profiling The Integral Metabolism of Herbal Medicine

18.4 Conclusions

Acknowledgment

References

Chapter 19 : Liquid Chromatography Mass Spectrometry Bioanalysis of Protein Therapeutics and Biomarkers in Biological Matrices

19.1 General Introduction

19.2 Protein Quantitation By LC–MS/MS

19.3 Protein Quantitation Using Intact Proteins By LC–MS/MS

19.4 Protein Quantitation Using Representative Peptides By LC–MS/MS

19.5 Consideration of Internal Standard for Protein Quantitation

19.6 Matrix Effect, Matrix Suppression/Enhancement, and Recovery

19.7 Sensitivity Enhancement VIA Immunocapture/Purification

19.8 Sensitivity Enhancement VIA Depletion of Abundant Proteins

19.9 Practical Aspects of LC–MS Assay for Proteins in Drug Development

19.10 Conclusions

Acknowledgments

References

Chapter 20 : Mass Spectrometry in the Analysis of DNA, Protein, Peptide, and Lipid Biomarkers of Oxidative Stress

20.1 Introduction

20.2 DNA Biomarkers of Oxidative Stress

20.3 Protein and Peptide Biomarkers of Oxidative Stress

20.4 Lipid Biomarkers of Oxidative Stress

20.5 Creatinine: The Common Denominator

20.6 Summary and Conclusions

Acknowledgments

References

Chapter 21 : LC–MS in Endogenous Metabolite Profiling and Small-Molecule Biomarker Discovery

21.1 Introduction

21.2 Measuring The Metabolome

21.3 Analytical Approaches

21.4 Experimental Design

21.5 Data Processing and Analysis

21.6 Conclusion

References

Appendix

Index

WILEY SERIES ON PHARMACEUTICAL SCIENCE AND BIOTECHNOLOGY: PRACTICES, APPLICATIONS AND METHODS

Series Editor:

Mike S. Lee

Milestone Development Services

Mike S. Lee • Integrated Strategies for Drug Discovery Using Mass Spectrometry

Birendra Pramanik, Mike S. Lee, and Guodong Chen • Characterization of Impurities and Degradants Using Mass Spectrometry

Mike S. Lee and Mingshe Zhu • Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and Applications

Copyright © 2011 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Mass spectrometry in drug metabolism and disposition: basic principles and applications / edited by Mike S. Lee, Mingshe Zhu.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-0-470-40196-5 (cloth) 1. Drugs—Metabolism—Analysis. 2. Metabolites—Spectra. 3. Mass spectrometry. 4. Mass spectrometry. I. Lee, Mike S., 1960- II. Zhu, Mingshe.

[DNLM: 1. Pharmaceutical Preparations—metabolism. 2. Biopharmaceutics—methods. 3. Drug Design. 4. Mass Spectrometry—methods. 5. Pharmacokinetics. QV 38]

RM301.55.M367 2011

615′.7—dc22

2010028341

FOREWORD

Studies in the areas of drug metabolism and pharmacokinetics have assumed progressively greater importance in pharmaceutical research over the past two decades, reflecting an increased awareness of the critical impact on successful drug development of the absorption, metabolism, distribution, elimination, and toxicity (ADMET) properties of candidate therapeutic agents. Indeed, the role of drug metabolism studies in the pharmaceutical industry, formerly limited to later phases of the development process, now spans the continuum from early discovery efforts through lead optimization, preclinical development, clinical trials, and postmarketing surveillance. Information on the identities and exposure levels of drug metabolites, first in animals and subsequently in human subjects, represents an essential component of preclinical and clinical safety assessment programs and, in those cases where circulating metabolites are pharmacologically active, provides the basis for assessing their pharmacokinetic/pharmacodynamic (PK/PD) relationships and contribution to the effects of the parent drug. Chemically reactive drug metabolites, which can be detected and characterized through specialized in vitro “trapping” techniques, generally are viewed as risk factors in drug development in light of their association with several forms of drug-induced toxicities, and early information on their identities is key to the design of optimized new chemical entities that lack this potential liability.

The detection, structural characterization, and quantitative analysis of drug metabolites in complex biological matrices often is a challenging endeavor, given the low levels that derive from highly potent parent compounds that are administered at doses of a few milligrams per day or less. As a result, stringent demands are placed on the analytical methodology employed for drug metabolism studies conducted either in vitro or in vivo, in terms of sensitivity and specificity of detection, and of wide dynamic range. In this regard, mass spectrometry, which always has been an important technique in drug metabolism studies, rapidly became the dominant technology in the field following the introduction, in the early 1990s, of the first commercial LC–MS/MS systems. Over the past decade, remarkable technical advances have been made in ion source design and ionization methods, rapid scanning and highly sensitive mass analyzers, efficient methods for inducing fragmentation of parent ions, rapid-response detectors with wide dynamic range, and powerful data acquisition and processing systems with sophisticated software packages and expert systems designed specifically for investigations in drug metabolism. The evolution of hybrid mass analyzers for MS/MS studies, and ancillary techniques such as ion mobility spectrometry, have added new dimensions to the mass spectrometry experiment, while the advent of high mass resolution capabilities on an LC time scale (even with “fast” chromatography) is having a truly revolutionary impact on the utility of LC–MS/MS in this field.

Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and Applications addresses each of these areas through a series of chapters authored by eminent scientists well versed in the application of contemporary mass spectrometry techniques to problems in drug metabolism and pharmacokinetics, with an emphasis on issues in drug discovery and development. The reader cannot help but be impressed by the capabilities of the current generation of LC–MS/MS instruments, which provide a combination of sensitivity, specificity, versatility, and speed of analysis that was difficult to envisage only a few years ago, and which have transformed the way drug metabolism studies are conducted. One can only wonder what lies in the years ahead!

Thomas A. Baillie

Seattle, WA

August, 2010

PREFACE

Two decades ago, drug metabolism research in the pharmaceutical industry was limited to radiolabeled in vivo drug disposition studies conducted in late stages of development. Drug metabolite identification was accomplished via a long and tedious process: metabolite separation and isolation followed by mass spectrometric and nuclear magnetic resonance analysis. Now, drug metabolism plays a critical role in the drug discovery and development process from lead optimization to clinical drug–drug interaction studies. Commercialized liquid chromatography/mass spectrometry (LC/MS) platforms have become the dominant analytical instrument employed in drug metabolism and pharmacokinetics (DMPK) studies and revolutionized the productivity of drug metabolism research. Certainly, the need for fast, sensitive and accurate measurements of drugs and metabolites in complex biological matrices has driven the continued development of novel LC/MS technology. Drug metabolism science and mass spectrometry technology have been integrated into an inseparable arena in drug discovery and development as well in related academic research activities. This book provides a unique thesis on mass spectrometry in drug metabolism with specific emphasis on both principles and applications in drug design and development. We believe that this integration will provide a unique contribution to the field that details both drug metabolism and analytical perspectives. Therefore, this book can be used as a teaching and/or reference tool to delineate and understand the “why” and “how” with regard to the many creative uses of mass spectrometry in drug metabolism and disposition studies. This work, authored by internationally renowned researchers, represents a combination of complementary backgrounds to bring technical and cultural awareness to this very important endeavor while serving to address needs within academia and industry.

The book is organized into three parts. Part I provides the reader with the basic concepts of drug metabolism and disposition. These concepts are intended to build a unique foundation of knowledge for drug metabolism and the subsequent studies performed during drug discovery and drug development endeavors. The book begins with an elegant perspective on drug metabolism. This review or perhaps “state of the union” provides unique insight into where we are, how we got there, and where we appear to be headed. Next we delve into the details of drug metabolism with a chapter on common biotransformation reactions. Further detail is provided in Chapter 3 from a medicinal chemistry perspective as the metabolic activation of organic functional groups is described along with considerations on how to address the reactive metabolite issues in drug design. Chapter 4 provides an overview on metabolizing enzymes, transporters, and their involvement with drug–drug interactions. In vitro experimental approaches to assess and predict drug–drug interactions in humans are elaborated. Chapter 5 starts with DMPK strategies in a drug discovery setting followed by a comprehensive overview of various experimental models applied in drug metabolism and disposition studies. Selection and data interpretation of the appropriate model are also discussed. Prediction of human pharmacokinetics is the focus of Chapter 6. Basic concepts and principles are discussed along with the use of mathematical models to predict pharmacokinetics. The actual use of drug metabolism information within the pharmaceutical industry is described in Chapter 7. In this chapter, the reader will obtain insight into the strategies used to design experiments for characterizing drug metabolism properties and addressing drug metabolism related issues from drug discovery to regulatory registrations.

Part II of the book highlights the principles and common practices of mass spectrometry in drug metabolism. The basic concepts and theory of mass spectrometry are presented in Chapter 8. In this chapter the reader will be able to obtain an updated thesis on the major components of this enabling instrumentation as well as the various mass analyzer platforms in use today. Some of the most common LC/MS-based methods used for metabolite identification is described in Chapter 9. Strategies for identification are reviewed and include a variety of mass spectrometry formats. Chapter 10 provides a review of common fragmentation reactions in the gas phase that are the foundation for mass spectral interpretation. Detailed examples provide the reader with the necessary tools for metabolite structure eludication. We dedicate an entire chapter to techniques that facilitate the performance of mass spectrometry during metabolite studies. And so, Chapter 11 provides concise background and industrial use of liquid chromatographic techniques as well as other detection techniques that are used to enhance the analytical performance of the mass spectrometer.

Part III of the book focuses on LC/MS techniques in drug metabolism and disposition. The application of quantitative LC/MS in drug metabolism and disposition is highlighted in Chapter 12. Critical studies that are routinely performed in drug discovery that involve metabolic stability, enzyme kinetics, metabolizing enzyme inhibition and induction, permeability and absorption, and in vitro transporter experiments are described. Chapter 13 provides both background and advantages of modern high-resolution mass spectrometry along with the use of newly developed data-mining tools for in vitro and in vivo drug metabolite identification. The understanding of the tissue distribution of a drug and the corresponding metabolites is illustrated in Chapter 14. The recent applications of imaging mass spectrometry for these studies are described. Novel instrumentation and mass spectrometry scan functions of hybrid triple quadrupole–linear ion trap mass spectrometry are discussed in Chapter 15. The applications for both bioanalysis and metabolite identification are highlighted. Quantitative drug metabolism studies using accelerator mass spectrometry are introduced and described in Chapter 16. The utility of this powerful technique for microdosing and DMPK studies in early clinical studies is described. Chapter 17 provides a provocative description of novel approaches, typically used in the field of protein identification, to obtain standard-free estimation of metabolite levels using nanospray mass spectrometry. A unique perspective on the profiling and characterization of Chinese herbal medicine and their metabolites using LC/MS is provided in Chapter 18. The approaches to determine the chemical composition of these medicines and their subsequent metabolites are discussed. The emerging area of bioanalysis of protein therapeutics in biological matrices is discussed in Chapter 19. Unique perspectives on digestion efficiency, internal standards, and biomarker validation are discussed in detail. Biomarker analysis is an exciting and emerging area of interest. Chapter 20 provides the reader with real-world application of mass spectrometry for the analysis of DNA, protein, peptide, and lipid biomarkers of oxidative stress. Part III of the book concludes with an updated perspective on endogenous metabolite profiling and small-molecule biomarker discovery (Chapter 21). In this chapter, the relationship between a perturbation and effected biochemical pathways is described within the context of biomarker discovery.

So, what criteria will emerge as the most desirable analytical figure of merit for high-performance LC/MS analysis in drug metabolism? It is our sincere hope that this book will provide an updated perspective on mass spectrometry in drug metabolism and disposition with recent applications, novel technologies, and innovative workflows.

Finally, the authors wish to acknowledge the contributions of many who transformed this book from idea to passion to reality. Specifically, the contributions from the authors and their respective collaborators, the editorial staff at John Wiley & Sons, and, of course, the families of the editors.

Mike S. Lee

Mingshe Zhu

CONTRIBUTORS

Zeper Abliz

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Maria D. Bacolod

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

Tom Baillie

School of Pharmacy

University of Washington

Seattle, Washington

Kevin P. Bateman

Drug Metabolism and Pharmacokinetics

Merck Frosst Canada Ltd.

Quebec, H9H 3L1 Canada

Ian A. Blair

Centers for Cancer Pharmacology and Excellence in Environmental Toxicology

University of Pennsylvania

Philadelphia, Pennsylvania

Manuel Cajina

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

Dongmei Dai,

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Xinxin Ding

Wadsworth Center,

New York State Department of Health

Albany, New York

Stephen R. Dueker

Vitalea Science

Davis, California

Timothy J. Garrett

Department of Medicine

University of Florida

Gainesville, Florida

Stacy L. Gelhaus

Centers for Cancer Pharmacology and Excellence in Environmental Toxicology

University of Pennsylvania

Philadelphia, Pennsylvania

Ping Geng

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Qing Ping Han

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

Mark Hayward

Analytical, Automation, and Formulation Laboratories

Department of Chemistry

Lundbeck Research USA

Paramus, New Jersey

Jiuming He

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Akihiro Hisaka

Department of Pharmacy

The University of Tokyo Hospital

Faculty of Medicine

The University of Tokyo

Tokyo, 113-8655 Japan

Serhiy Hnatyshyn

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Princeton, New Jersey

Gérard Hopfgartner

Life Sciences Mass Spectrometry

School of Pharmaceutical Sciences

University of Geneva

University of Lausanne

Geneva, Switzerland

W. Griffith Humphreys

Department of Biotransformation

Bristol-Myers Squibb Research and Development

Princeton, New Jersey

Qin C. Ji

Bioanalytical Sciences, Analytical Research & Development

Bristol-Myers Squibb

Princeton, New Jersey

Wenying Jian

BA/DMPK

Pharmaceutical Research and Development

Johnson & Johnson

Raritan, New Jersey

Elliott B. Jones

Applied Biosystems Inc.

Foster City, California

Amit S. Kalgutkar

Pharmacokinetics, Dynamics, and Metabolism Department,

Pfizer Global Research and Development

Eastern Point Road, Groton, Conneticut

Brad Keck

Vitalea Science

Davis, California

Lewis J. Klunk

Department of Drug Metabolism and Pharmacokinetics

Biogen Idec

Cambridge, Massachusetts

Walter Korfmacher

Exploratory Drug Metabolism

Merck Research Laboratories

Kenilworth, New Jersey

Mike Lee

Milestone Development Services

Newtown, PA

Fumin Li

Bioanalytical Department

Covance Laboratories

Madison, Wisconsin

Jian Liu

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Peter Lohstroh

Vitalea Science

Davis, California

Steven W. Louie

Department of Pharmacokinetics and Drug Metabolism

Amgen, Inc.

Thousand Oaks, California

Chuang Lu

Drug Metabolism and Pharmacokinetics

Millennium Pharmaceuticals, Inc

Cambridge, Massachusetts

Gang Luo

Covance Laboratories

Madison, Wisconsin

Daniel P. Magparangalan

Department of Chemistry

University of Florida

Gainesville, Florida

Russell J. Mortishire-Smith

Janssen Pharmaceutical Companies of Johnson & Johnson

B-2340 Beerse, Belgium

Sidney D. Nelson

Department of Medicinal Chemistry, School of Pharmacy

University of Washington

Seattle, Washington

Chandra Prakash

Department of Drug Metabolism and Pharmacokinetics

Biogen Idec

Cambridge, Massachusetts

Birendra Pramanik

Chemical Research

Merck Research Laboratories

Kenilworth, New Jersey

Richard F. Reich

Department of Chemistry

University of Florida

Gainesville, Florida

Michael D. Reily

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Princeton, New Jersey

Petia Shipkova

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Pennington, New Jersey

Magang Shou

Department of Pharmacokinetics and Drug Metabolism

Amgen, Inc.

Thousand Oaks, California

Hiroshi Suzuki

Department of Pharmacy

The University of Tokyo Hospital

Faculty of Medicine

The University of Tokyo

Tokyo, 113-8655 Japan

John S. Vogel

Vitalea Science

Davis, California

Bo Wen

Department of Drug Metabolism and Pharmacokinetics

Hoffmann-La Roche

Nutley, New Jersey

Ronald E. White

White Global Pharma Consultants

Cranbury, New Jersey

Jing-Tao Wu

Drug Metabolism and Pharmacokinetics

Millennium Pharmaceuticals, Inc.

Cambridge, MA

Lin Xu

Department of Drug Metabolism and Pharmacokinetics

Biogen Idec

Cambridge, Massachusetts

Ming Yao

Department of Biotransformation

Bristol-Myers Squibb Research and Development

Princeton, New Jersey

Takehito Yamamoto

Department of Pharmacy,

The University of Tokyo Hospital

Faculty of Medicine

The University of Tokyo

Tokyo, 113-8655 Japan

Richard A. Yost

Department of Chemistry

University of Florida

Gainesville, Florida

Donglu Zhang

Department of Biotransformation

Bristol-Myers Squibb

Princeton, New Jersey

Haiying Zhang

Department of Biotransformation

Bristol-Myers Squibb Research & Development

Pennington, New Jersey

Li-Kang Zhang

Global Analytical Chemistry

Merck Research Laboratoriess

Kenilworth, New Jersey

Ruiping Zhang

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing, 100050 China

Mingshe Zhu

Department of Biotransformation

Bristol-Myers Squibb Research and Development

Princeton, New Jersey

Zack Zou

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

PART I

Basic Concepts of Drug Metabolism and Disposition

Chapter 1

Progression of Drug Metabolism

RONALD E. WHITE

White Global Pharma Consultants, Cranbury, New Jersey

1.1 Introduction1.2 Historical Phases of Drug Metabolism 1.2.1 The “Chemistry” Phase (1950–1980)1.2.2 The “Biochemistry” Phase (1975–Present)1.2.3 The “Genetics” Phase (1990–Present)1.2.4 The “Biology” Phase (2010 and Beyond)1.3 Next Step in the Progression of DM 1.3.1 New Regulatory Expectation1.3.2 New Challenges for Technology1.4 Perspective on the Magnitude of the Challenge 1.4.1 Ultimate Limits on Metabolite Quantitation1.4.2 Practical Limits on Metabolite Quantitation1.4.3 Natural Limit Due to Dose Size1.5 Are There More Sensitive Alternatives to MS?1.6 SummaryReferences

1.1 INTRODUCTION

In a certain sense, the field of drug metabolism (DM) is standing still. More specifically, the basic experiment of drug metabolism (i.e., administering a new drug to an animal or human and determining the structures, amounts, and disposition of the metabolites) has changed very little over a period of decades. Remarkably, the experimental design and resulting data set from a typical absorption, distribution, metabolism, and excretion (ADME) study conducted today would be instantly recognized and understood by DM scientists from 50 years ago. This is not the case with most other disciplines in the life sciences. For instance, 20 years ago protein sequencing was based on peptide chemistry, and the basic experiment was the assay of amino acids released from tryptic peptides, a process that required months or years to complete. Now, protein sequencing is based on nucleotide chemistry, and the basic experiment is the automated assay of oligonucleotides from partial hydrolysis of complementary deoxyribonucleic acid (cDNA) a process that takes about a day. The reason that ADME experiments have not evolved much is that we have never devised a surrogate for the whole human body for metabolism studies. All systems tried up to this point (e.g., animals, transgenic animals, perfused organs, in vitro incubations, three-dimensional (3D) microfluidic cell culture devices, in silico calculations) fail to reliably predict the actual metabolic fate of NCEs. To be sure, the technology we use for the ADME experiment has advanced greatly, but even with the newest methods, DM is still essentially a chemical exercise at its core, and the backbone technology remains mass spectrometry (MS).

However, if we consider the more complete picture, DM has expanded enormously in scope and level of understanding in those 50 years. While the chemistry-based core remains intact and actively growing, several other kinds of DM studies have layered over the core, all coexisting and relevant in contemporary DM science. Thus, in addition to the purely chemical description of the structures of metabolites and probable chemical mechanisms of their formation, we now have a very good biochemical understanding of the various enzymes that catalyze these biotransformation processes, as well as a cellular and genetic understanding of the expression and regulation of those enzymes. We are even making progress toward reliable prediction of the fates of xenobiotic substances in human beings (Anderson et al., 2009), although this goal remains out of reach for the present. The current level of understanding of DM is presented by several experts in Part I of this book.

1.2 HISTORICAL PHASES OF DRUG METABOLISM

Near the end of the twentieth century, I suggested that there had been four overlapping phases of DM in industrial drug discovery and development (White, 1998). These can be summarized as follows.

1.2.1 The “Chemistry” Phase (1950–1980)

During this period, only a descriptive account of the disposition of a new chemical entity (NCE) was provided, largely consisting of chemical information. Major urinary and fecal metabolites were isolated and identified by classic chemical techniques including column and thin-layer chromatography, crystallization, and derivatization. Eventually, spectroscopy was used for the structural elucidation, including mass spectroscopy, infrared, and nuclear magnetic resonance (NMR). These techniques required much smaller quantities to be isolated and allowed high-performance liquid chromatography (HPLC) to replace column chromatography. Interestingly, early in this period R.T. Williams published his monograph Detoxication Mechanisms, which can be considered the first identification of DM as a discrete field of study (Murphy, 2008). The publication of that book also showed that academic researchers were beginning to think about the biological basis and implications of DM, although this way of thinking took some time to make an impact in the industrial world.

1.2.2 The “Biochemistry” Phase (1975–Present)

Starting in the mid-1970s, we began to determine the underlying biochemical processes responsible for the disposition of xenobiotics (e.g., which enzymes were involved). Illustrating the indistinct separation of these phases, the pioneers in this phase often came from a chemical background, and they sought to describe the enzymes in chemical terms. The proteins were isolated so that they could be treated as discreet chemical reagents, describable in classical chemical terms of composition, reaction stoichiometry, thermodynamics, and reaction mechanism. However, after about a decade or so, the chemical approach to studying the enzymes transitioned to a biochemical and cell biology approach in which enzyme kinetics and protein–protein and membrane–protein interactions became the “hottest” topics. All of the important DM enzymes were characterized, named, and even made commercially available to industrial researchers. The latest advance in the biochemistry phase was the realization that even the exposure of drugs to the drug-metabolizing enzymes was a biochemical event, mediated by physical enzymes called transporters (Wu and Benet, 2005). Advances in our understanding of the biochemistry of DM in academic laboratories were reflected in a greater expectation by regulatory agencies that a biochemical description be provided in addition to the purely chemical description of DM of an NCE.

1.2.3 The “Genetics” Phase (1990–Present)

In this phase, we began to account for individual variations in the pharmacokinetic rates and molecular sites of metabolism by genotyping human test subjects with respect to an ever-growing list of genetically polymorphic drug-metabolizing enzymes. Equally important, regulation of DM enzymes was recognized to occur mainly at the gene expression level, whether resulting from heredity, disease processes, or environment. This pharmacogenetic characterization has become a routine expectation for the registration packages of NCEs and continues to expand. As before, the new genetic information did not replace any previous requirements for DM information but instead added an additional dimension to that information package.

1.2.4 The “Biology” Phase (2010 and Beyond)

We are beginning to view drug metabolism in terms of systems biology. This involves taking a holistic view of the simultaneous interaction of a xenobiotic molecule with all the enzymes and receptors in the human body. Some of these receptors are the pharmacological targets that lead to therapeutic benefits, some are unintended targets that generate adverse events and toxicities, and some are the enzymes and nuclear receptors of DM. In our overall description of the disposition of the compound, interactions of the compound with these DM targets are especially complex to relate to safety and efficacy. When designing a practical clinical medicine, we need to establish a balance between too rapid metabolism, leading to reduced efficacy, and too sluggish metabolism, leading to accumulation and possible toxicity. In the clinic, we need to determine whether metabolism decreases or increases the desired pharmacological effect (i.e., active metabolites). And, finally, in this holistic biological view, we need to assess how the metabolites interact with all the off-target human enzymes and receptors, especially the phenomenon of reactive metabolites covalently binding to proteins and nucleic acids, leading to toxic sequelae (Baillie, 2009).

These four phases are graphically depicted in Figure 1.1. They are layered in the figure because we continue to do all the activities of each preceding phase as we proceed through the evolution of industrial DM. Thus, the total amount of DM characterization work for a new drug has increased dramatically over the years. The meaning of the step increase in work at around 2010 in the figure will be discussed in the next section.

FIGURE 1.1 Progression of amounts of DM information required for regulatory filing of a new chemical entity. The vertical axis is the total amount of DM information in the registration application on a relative scale. The information classes are segregated as discussed in the text. The chemistry component (C, black) continually increases with time but abruptly increases about 2010 due to enhanced regulatory surveillance of metabolites. Starting around 1975, biochemistry (B, dark gray) begins to be included in the DM characterization and slowly increases with time. Genetics information (G, light gray) continues to increase, mainly in clinical trials. The apparent jump in B and G work around 2010 is due to the abrupt increase in C. Actual B and G work would not increase. Systems biology (S, white) is nearly zero in 2010 but is expected to increase subsequently.

1.3 NEXT STEP IN THE PROGRESSION OF DM

The first three of these historical phases of industrial DM serve to summarize and rationalize the scientific questions of yesteryear and today. The questions of tomorrow are described by the biology phase. However, now we can also discern the beginning of an additional new trend that could be called the “regulatory” phase. This phase is not primarily concerned with the physiological process of DM, as are the other phases. Instead, the regulatory phase is concerned with the human safety of the metabolites, once they are formed. But even though the focus of this phase is safety, it may well produce the greatest increment of additional DM work to be done in the future.

1.3.1 New Regulatory Expectation

Regulatory interest in metabolites has developed into a formal Guidance for Industry (Safety Testing of Drug Metabolites) issued by the U.S. Food and Drug Administration (FDA), which instructs sponsors on the qualitative and quantitative characterization of metabolites in both clinical and preclinical toxicological settings (U.S. FDA, 2008). A similar concern about metabolite safety is expressed in a Guidance from the International Conference on Harmonisation (ICH), currently in draft stage (ICH, 2009). We may succinctly state the requirement as follows: Human circulating metabolites that exceed 10% of the total exposure of all drug-related materials in circulation at pharmacokinetic steady state require safety assessment before large-scale clinical trials can proceed. These Guidances have implications for bioanalysis and safety assessment, but here we wish to focus on the implications for biotransformation studies. Development of these Guidances, though initiated by an industry-sponsored group (Baillie et al., 2002), has resulted in an inevitable regulatory emphasis on metabolite characterization much earlier than traditionally carried out. Importantly, the relative levels of parent drug and metabolites at pharmacokinetic steady state have been little studied previously. Consequently, we can expect surprises, possibly even new phenomena, as we watch the time evolution of drug metabolism during the approach to steady state. The traditional approach to definitive metabolite characterization (i.e., single-dose 14C-labeled clinical ADME studies performed during Phase II or III) is inadequate for the new regulatory demands and either a new approach to 14C studies or new nonradiolabel-based methodology is required.

1.3.2 New Challenges for Technology

Distressingly, both the qualitative and quantitative requirements of the new paradigm potentially push the existing technology past present limits. Qualitatively, technology is now needed for metabolite detection in early clinical development. Up to this time, MS was only required to elucidate the chemical structures of metabolites, augmented as necessary by NMR and synthesis of authentic standards. However, detection of the presence of metabolites in a sample relied on the observation of nonparent radioactive peaks in the chromatogram. Now we are asking the mass spectroscopist to also detect the metabolites, without the benefit of radiolabel or prior knowledge of the structures. This requirement for MS-based detection as well as characterization has necessitated the development and validation of new algorithms of data acquisition and processing. In fact, as will be seen in later chapters, reliable nonradiometric MS-based methods of metabolite detection are now a reality.

Quantitatively, technology limits are pushed in two ways. First, the mass spectroscopist is asked to estimate the concentration of each metabolite in a plasma profile and categorize it as more or less than 10% of the total. This is problematic because the structures may not be completely known and authentic standards may not be available. Second, for those metabolites at levels more than 10%, a validated assay will be required, pushing the sensitivity limits that may be required in some cases. As the typical drug candidate becomes ever more potent, with ever smaller administered doses, the accurate estimation of analyte exposures that are as much as an order of magnitude less than that of the parent could become a challenge. Experts will discuss technological advances in MS related to the problems of detection and estimation of amounts of metabolites in Part II of this book and the experimental application of this technology to these problems in Part III. However, we can put some perspective here on the magnitude of the challenge.

1.4 PERSPECTIVE ON THE MAGNITUDE OF THE CHALLENGE

1.4.1 Ultimate Limits on Metabolite Quantitation

Let us start by considering whether there is any amount of metabolite that is too little to be meaningful. Since the present regulatory criterion for the amount of metabolite requiring assay is expressed as a percentage of a variable quantity (dose), then as new, more potent drugs are introduced, there is no regulatory lower limit on the absolute quantity of a metabolite that might need to be detected and assayed. So a literal reading of the Guidances is that no amount of metabolite is too little to be of regulatory interest. This approach to metabolite safety assessment has been questioned on the basis of the likelihood of metabolite-driven toxicity from minute body burdens (Smith and Obach, 2009), and it seems unlikely that regulatory authorities would apply the 10% rule to very low dose drugs (ICH, 2009). However, for this examination of limits, let us assume the worst case, that the 10% rule was always applied. Is there any other limitation of how much metabolite might need to be detected and quantitated? Actually, a moment’s practical consideration of the question of lower limit reminds us that there is a fundamental limit imposed by nature (i.e., one molecule). Obviously, in this hypothetical limiting case, one molecule in a whole human body could only be assayed by exsanguinating the subject, so the stipulation that the subject must survive the assay clearly requires many more molecules than one in the body for detectability. To estimate how many more, in the next section we will use the “one-molecule” concept in the opposite direction (i.e., from the detector’s point of view).

1.4.2 Practical Limits on Metabolite Quantitation

At very low concentrations, the discrete nature of molecules means that a detector signal no longer appears to be continuous. At the ultimate limit, either zero or one molecule will enter the inlet of the mass spectrometer; one cannot analyze half a molecule. Thus, one molecule entering the mass spectrometer inlet during a single duty cycle is the natural ultimate limit of sensitivity. Working backward from this limit and assuming that about 5% of the injected mass is actually sampled in a single duty cycle, we see that at least 20 molecules would have to be injected, on average, to get one into the inlet during a single duty cycle. Given a 10-μL injection from a 100-μL reconstituted extract of a 100-μL plasma aliquot of an original 1-mL plasma sample, we can estimate that plasma from a human subject would have to contain at least 2000 molecules per milliliter to be measurable in a practical sense by current liquid chromatography (LC)/MS laboratory methods. Although one could propose taking a larger plasma sample from a subject, reconstituting the extract in a smaller volume, and injecting a larger fraction onto the instrument, there are natural limitations for these volumes as well. For instance, a full pharmacokinetic profile for a subject that required 10 mL of plasma (i.e., about 20 mL of blood) for each of 10 time points would surely be close to the limit that physicians would ever accept under any circumstances, and even larger samples would clearly be out of the question. Thus, for this thought experiment, let us accept that a realistically and routinely measurable concentration could not be much less than 1000 molecules per milliliter of plasma based on the natural limit of one molecule interacting with a mass spectral detector. Application of Avogadro’s number to this ensemble of 1000 molecules allows us to state that plasma concentrations less than about 1 attomolar (1 aM, 10−18 molar) will never be accessible in a practical sense.

1.4.3 Natural Limit Due to Dose Size

Now let us translate the natural-limit concept back to the real-world quantity of dose. If a midrange volume of distribution of 50 L is assumed for a typical drug, then a meaningful detection limit of 1 aM for a metabolite implies a concentration of 10 aM for the parent drug (i.e., metabolite is 10% of parent), which is equivalent to a total dose of 500 attomol. If the drug has a molecular weight (MW) of 400, then, the dose would be 0.2 pg. For comparison, what is the lowest dose of drug for which we are ever likely to be expected to characterize circulating metabolites? The most potent substances administered therapeutically are hormones, and their doses are exceedingly low. For instance, the labor-inducing hormone oxytocin is effective at a vanishingly low dose of about 40 ng, while the calcitriol dose is only about 4 μg. These examples may represent the lowest human doses that will ever be used for any drug. Amazingly, some actual drugs already approach these dose levels. For instance, the inhaled β-agonist formoterol is given at 12 μg, and the inhaled glucocorticoid beclomethasone is delivered at 40 μg. Circulating levels of these two drugs and their metabolites are almost unmeasurable (Cmax in each case is ca. 10 pg/mL). The most potent oral drugs are also in the same range, with the dose of clonidine being about 100 μg. If, as suggested above, future drug candidates will not be more potent than the most potent drugs in use today, then we can say that levels of metabolites requiring detection and characterization will likely never be less than about 1 pg/mL. This is good news because, although they are not routine, MS-based assays with picogram/milliliter sensitivity are already in use. Thus, we can see that there is a natural limit to the ultimate sensitivity required to detect and characterize circulating metabolites, and it is not far from what our current MS technology already allows us to do. Most new drugs in development are dosed in the 1 to 1000-mg range, and metabolite levels for these drugs are well within contemporary MS sensitivity.

1.5 ARE THERE MORE SENSITIVE ALTERNATIVES TO MS?

An interesting extension of the concept of natural limitation is to remove the assumption that one molecule gives one quantum of signal (i.e., destructive analysis). Spectroscopic methods such as NMR and fluorescence can time integrate numerous signal pulses from a single molecule, resulting in no theoretical limit to how much signal could be accumulated with unlimited acquisition time. In fact, quantitative NMR spectroscopy has been proposed as a readily available solution to the main problems of implementing the Guidances, namely recognition and quantitation of metabolites without radiolabel at steady state (Espina et al., 2009; Vishwanathan et al., 2009). However, both NMR and fluorescence still face the indivisibility of individual molecules when applied to ex vivo samples such as plasma, so that a practical lower limit of concentration would still exist. Moreover, given the insensitivity of NMR relative to MS, it is unlikely that NMR could ever access concentrations that MS could not, even with the advantage of signal accumulation. Conversely, fluorescence might conceivably achieve the requisite sensitivity, but unlike MS and NMR, fluorescence is not generally applicable to all drug candidates. Clearly, then, the only currently available technology for low-level metabolite characterization is MS, which combines sensitivity, structural information, and general applicability.

1.6 SUMMARY

In summary, DM is a traditional yet dynamic discipline, comprising a constant core overlaid with evolving successive layers of related activities. The core activity of detecting and determining the chemical structure of human metabolites has changed little in decades and, in fact, is the starting point for all the other activities in areas such as enzymology, regulation, and genetics. For example, it is meaningless to inquire which cytochrome P450 (CYP) enzyme is principally responsible for the metabolism of a new drug until the chemical structure of the major metabolite shows that it was likely formed by a CYP enzyme. Today, structural elucidation of a metabolite almost always begins with MS, followed by complementary methods such as NMR, as necessary. Conversely, detection of metabolites has traditionally been accomplished by radiochromatography. However, in response to evolving regulatory expectations, it is likely that detection will become the job of MS also. Thus, MS is the most important single technique in DM and is likely to remain so going forward into the future. This conclusion explains the need for continued advancement of DM applications of MS technology, as described in the remainder of this book.

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Chapter 2

Common Biotransformation Reactions

BO WEN

Department of Drug Metabolism and Pharmacokinetics, Hoffmann-La Roche, Nutley, New Jersey