MASS SPECTROMETRY IN DRUG METABOLISM AND DISPOSITION docx

Louie and Magang Shou 5 Experimental Models of Drug Metabolism and Disposition 151 Gang Luo, Chuang Lu, Xinxin Ding, and Donglu Zhang 6 Principles of Pharmacokinetics: Predicting Human T

MASS SPECTROMETRY IN DRUG METABOLISM AND DISPOSITION

AND BIOTECHNOLOGY: PRACTICES, APPLICATIONS

AND METHODS

Series Editor:

Mike S Lee

Milestone Development Services

Mike S Lee Integrated Strategies for Drug Discovery Using Mass SpectrometryBirendra Pramanik, Mike S Lee, and Guodong Chen Characterization of Impuritiesand Degradants Using Mass Spectrometry

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

MASS SPECTROMETRY

IN DRUG METABOLISM AND DISPOSITION

Basic Principles and Applications

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 u.7—dc22

2010028341 Printed in Singapore

Bo Wen and Sidney D Nelson

3 Metabolic Activation of Organic Functional Groups Utilized

Amit S Kalgutkar

4 Drug-Metabolizing Enzymes, Transporters, and

Steven W Louie and Magang Shou

5 Experimental Models of Drug Metabolism and Disposition 151

Gang Luo, Chuang Lu, Xinxin Ding, and Donglu Zhang

6 Principles of Pharmacokinetics: Predicting Human

Takehito Yamamoto, Akihiro Hisaka, and Hiroshi Suzuki

7 Drug Metabolism Research as Integral Part of Drug Discovery

W Griffith Humphreys

v

PART II MASS SPECTROMETRY IN DRUG METABOLISM:

Ge´rard Hopfgartner

9 Common Liquid Chromatography–Mass Spectrometry

(LC–MS) Methodology for Metabolite Identification 291

Lin Xu, Lewis J Klunk, and Chandra Prakash

Li-Kang Zhang and Birendra N Pramanik

11 Techniques to Facilitate the Performance of Mass

Spectrometry: Sample Preparation, Liquid Chromatography,

Mark Hayward, Maria D Bacolod, Qing Ping Han, Manuel Cajina,

and Zack Zou

12 Quantitative In Vitro ADME Assays Using LC–MS as a

Walter Korfmacher

13 High-Resolution Mass Spectrometry and Drug Metabolite

Russell J Mortishire-Smith, Haiying Zhang, and Kevin P Bateman

14 Distribution Studies of Drugs and Metabolites in Tissue

Richard F Reich, Daniel P Magparangalan, Timothy J Garrett,

and Richard A Yost

15 Use of Triple Quadrupole–Linear Ion Trap Mass

Spectrometry as a Single LC–MS Platform in Drug

Wenying Jian, Ming Yao, Bo Wen, Elliott B Jones, and Mingshe Zhu

16 Quantitative Drug Metabolism with Accelerator Mass

John S Vogel, Peter Lohstroh, Brad Keck, and Stephen R Dueker

17 Standard-Free Estimation of Metabolite Levels Using

Nanospray Mass Spectrometry: Current Statutes and

Jing-Tao Wu

18 Profiling and Characterization of Herbal Medicine and

Zeper Abliz, Ruiping Zhang, Ping Geng, Dongmei Dai,

Jiuming He, and Jian Liu

19 Liquid Chromatography Mass Spectrometry Bioanalysis of

Protein Therapeutics and Biomarkers in Biological Matrices 613

Fumin Li and Qin C Ji

20 Mass Spectrometry in the Analysis of DNA, Protein, Peptide,

Stacy L Gelhaus and Ian A Blair

21 LC–MS in Endogenous Metabolite Profiling and Small-Molecule

Studies in the areas of drug metabolism and pharmacokinetics have assumedprogressively greater importance in pharmaceutical research over the past twodecades, reflecting an increased awareness of the critical impact on successfuldrug development of the absorption, metabolism, distribution, elimination, andtoxicity (ADMET) properties of candidate therapeutic agents Indeed, the role

of drug metabolism studies in the pharmaceutical industry, formerly limited tolater phases of the development process, now spans the continuum from earlydiscovery efforts through lead optimization, preclinical development, clinicaltrials, and postmarketing surveillance Information on the identities andexposure levels of drug metabolites, first in animals and subsequently in humansubjects, represents an essential component of preclinical and clinical safetyassessment programs and, in those cases where circulating metabolites arepharmacologically active, provides the basis for assessing their pharmacoki-netic/pharmacodynamic (PK/PD) relationships and contribution to the effects

of the parent drug Chemically reactive drug metabolites, which can be detectedand characterized through specialized in vitro “trapping” techniques, generallyare viewed as risk factors in drug development in light of their associationwith several forms of drug-induced toxicities, and early information on theiridentities is key to the design of optimized new chemical entities that lack thispotential liability

The detection, structural characterization, and quantitative analysis of drugmetabolites in complex biological matrices often is a challenging endeavor,given the low levels that derive from highly potent parent compounds that areadministered at doses of a few milligrams per day or less As a result, stringentdemands are placed on the analytical methodology employed for drugmetabolism studies conducted either in vitro or in vivo, in terms of sensitivityand specificity of detection, and of wide dynamic range In this regard, massspectrometry, which always has been an important technique in drug metabo-lism studies, rapidly became the dominant technology in the field following theintroduction, in the early 1990s, of the first commercial LC MS/MS systems.Over the past decade, remarkable technical advances have been made in ionsource design and ionization methods, rapid scanning and highly sensitive massanalyzers, efficient methods for inducing fragmentation of parent ions, rapid-response detectors with wide dynamic range, and powerful data acquisition andprocessing systems with sophisticated software packages and expert systemsdesigned specifically for investigations in drug metabolism The evolution of

ix

hybrid mass analyzers for MS/MS studies, and ancillary techniques such as ionmobility spectrometry, have added new dimensions to the mass spectrometryexperiment, while the advent of high mass resolution capabilities on an LC timescale (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 andApplicationsaddresses each of these areas through a series of chapters authored

by eminent scientists well versed in the application of contemporary massspectrometry techniques to problems in drug metabolism and pharmacoki-netics, with an emphasis on issues in drug discovery and development Thereader cannot help but be impressed by the capabilities of the currentgeneration of LC MS/MS instruments, which provide a combination ofsensitivity, specificity, versatility, and speed of analysis that was difficult toenvisage only a few years ago, and which have transformed the way drugmetabolism studies are conducted One can only wonder what lies in the yearsahead!

Thomas A Baillie

Seattle, WA

August, 2010

Two decades ago, drug metabolism research in the pharmaceutical industrywas limited to radiolabeled in vivo drug disposition studies conducted in latestages of development Drug metabolite identification was accomplished via along and tedious process: metabolite separation and isolation followed by massspectrometric and nuclear magnetic resonance analysis Now, drug metabolismplays a critical role in the drug discovery and development process from leadoptimization to clinical drug–drug interaction studies Commercialized liquidchromatography/mass spectrometry (LC/MS) platforms have become thedominant analytical instrument employed in drug metabolism and pharmaco-kinetics (DMPK) studies and revolutionized the productivity of drug metabo-lism research Certainly, the need for fast, sensitive and accurate measurements

of drugs and metabolites in complex biological matrices has driven the tinued development of novel LC/MS technology Drug metabolism science andmass spectrometry technology have been integrated into an inseparable arena

con-in drug discovery and development as well con-in related academic researchactivities This book provides a unique thesis on mass spectrometry in drugmetabolism with specific emphasis on both principles and applications indrug design and development We believe that this integration will provide aunique contribution to the field that details both drug metabolism andanalytical perspectives Therefore, this book can be used as a teaching and/orreference tool to delineate and understand the “why” and “how” with regard tothe many creative uses of mass spectrometry in drug metabolism and disposi-tion studies This work, authored by internationally renowned researchers,represents a combination of complementary backgrounds to bring technicaland cultural awareness to this very important endeavor while serving to addressneeds within academia and industry

The book is organized into three parts Part I provides the reader with thebasic concepts of drug metabolism and disposition These concepts areintended to build a unique foundation of knowledge for drug metabolismand the subsequent studies performed during drug discovery and drug devel-opment endeavors The book begins with an elegant perspective on drugmetabolism This review or perhaps “state of the union” provides uniqueinsight 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 commonbiotransformation reactions Further detail is provided in Chapter 3 from amedicinal chemistry perspective as the metabolic activation of organic

xi

functional groups is described along with considerations on how to addressthe reactive metabolite issues in drug design Chapter 4 provides an overview

on metabolizing enzymes, transporters, and their involvement with drug–druginteractions In vitro experimental approaches to assess and predict drug–drug interactions in humans are elaborated Chapter 5 starts with DMPKstrategies in a drug discovery setting followed by a comprehensive overview ofvarious experimental models applied in drug metabolism and dispositionstudies Selection and data interpretation of the appropriate model are alsodiscussed Prediction of human pharmacokinetics is the focus of Chapter 6.Basic concepts and principles are discussed along with the use of mathematicalmodels to predict pharmacokinetics The actual use of drug metabolisminformation within the pharmaceutical industry is described in Chapter 7 Inthis chapter, the reader will obtain insight into the strategies used to designexperiments for characterizing drug metabolism properties and addressing drugmetabolism related issues from drug discovery to regulatory registrations.Part II of the book highlights the principles and common practices ofmass spectrometry in drug metabolism The basic concepts and theory ofmass spectrometry are presented in Chapter 8 In this chapter the reader will beable to obtain an updated thesis on the major components of this enablinginstrumentation as well as the various mass analyzer platforms in use today.Some of the most common LC/MS-based methods used for metaboliteidentification is described in Chapter 9 Strategies for identification are reviewedand include a variety of mass spectrometry formats Chapter 10 provides

a review of common fragmentation reactions in the gas phase that are thefoundation for mass spectral interpretation Detailed examples providethe reader with the necessary tools for metabolite structure eludication

We dedicate an entire chapter to techniques that facilitate the performance ofmass spectrometry during metabolite studies And so, Chapter 11 providesconcise background and industrial use of liquid chromatographic techniques aswell as other detection techniques that are used to enhance the analyticalperformance of the mass spectrometer

Part III of the book focuses on LC/MS techniques in drug metabolism anddisposition The application of quantitative LC/MS in drug metabolismand disposition is highlighted in Chapter 12 Critical studies that are routinelyperformed 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 bothbackground and advantages of modern high-resolution mass spectrometryalong with the use of newly developed data-mining tools for in vitro and invivo drug metabolite identification The understanding of the tissue distribution

of a drug and the corresponding metabolites is illustrated in Chapter 14 Therecent applications of imaging mass spectrometry for these studies aredescribed Novel instrumentation and mass spectrometry scan functions ofhybrid triple quadrupole–linear ion trap mass spectrometry are discussed inChapter 15 The applications for both bioanalysis and metabolite identification

are highlighted Quantitative drug metabolism studies using accelerator massspectrometry are introduced and described in Chapter 16 The utility ofthis powerful technique for microdosing and DMPK studies in early clinicalstudies is described Chapter 17 provides a provocative description of novelapproaches, typically used in the field of protein identification, to obtainstandard-free estimation of metabolite levels using nanospray mass spectro-metry A unique perspective on the profiling and characterization of Chineseherbal medicine and their metabolites using LC/MS is provided in Chapter 18.The approaches to determine the chemical composition of these medicines andtheir subsequent metabolites are discussed The emerging area of bioanalysis ofprotein therapeutics in biological matrices is discussed in Chapter 19 Uniqueperspectives on digestion efficiency, internal standards, and biomarker valida-tion are discussed in detail Biomarker analysis is an exciting and emerging area

of interest Chapter 20 provides the reader with real-world application of massspectrometry for the analysis of DNA, protein, peptide, and lipid biomarkers ofoxidative stress Part III of the book concludes with an updated perspective onendogenous metabolite profiling and small-molecule biomarker discovery(Chapter 21) In this chapter, the relationship between a perturbation andeffected biochemical pathways is described within the context of biomarkerdiscovery

So, what criteria will emerge as the most desirable analytical figure of meritfor high-performance LC/MS analysis in drug metabolism? It is our sincerehope that this book will provide an updated perspective on mass spectrometry

in drug metabolism and disposition with recent applications, novel gies, and innovative workflows

technolo-Finally, the authors wish to acknowledge the contributions of many whotransformed this book from idea to passion to reality Specifically, thecontributions from the authors and their respective collaborators, the editorialstaff at John Wiley & Sons, and, of course, the families of the editors

Mike S LeeMingshe Zhu

ZEPERABLIZ

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

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

Drug Metabolism and Pharmacokinetics

Merck Frosst Canada Ltd

Lundbeck Research USA,

Paramus, New Jersey

DONGMEIDAI,

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

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

xv

Wadsworth Center,

New York State Department of Health

Albany, New York

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

QINGPINGHAN

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

Analytical, Automation, and Formulation Laboratories

Department of Chemistry

Lundbeck Research USA

Paramus, New Jersey

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

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Princeton, New Jersey

Life Sciences Mass Spectrometry

School of Pharmaceutical Sciences

Bristol-Myers Squibb Research and Development

Princeton, New Jersey

Pharmaceutical Research and Development

Johnson & Johnson

Raritan, New Jersey

ELLIOTTB JONES

Applied Biosystems Inc

Foster City, California

Pharmacokinetics, Dynamics, and Metabolism Department,

Pfizer Global Research and Development

Eastern Point Road, Groton, Conneticut

Exploratory Drug Metabolism

Merck Research Laboratories

Kenilworth, New Jersey

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

Drug Metabolism and Pharmacokinetics

Millennium Pharmaceuticals, Inc

Cambridge, Massachusetts

Janssen Pharmaceutical Companies of Johnson & Johnson

Merck Research Laboratories

Kenilworth, New Jersey

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Princeton, New Jersey

Bioanalytical and Discovery Analytical Sciences

Applied and Investigational Metabonomics

Bristol-Myers Squibb

Pennington, New Jersey

White Global Pharma Consultants

Cranbury, New Jersey

JING-TAOWU

Drug Metabolism and Pharmacokinetics

Millennium Pharmaceuticals, Inc

Bristol-Myers Squibb Research & Development

Pennington, New Jersey

LI-KANGZHANG

Global Analytical Chemistry

Merck Research Laboratoriess

Kenilworth, New Jersey

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

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing, 100050 China

Department of Biotransformation

Bristol-Myers Squibb Research and Development

Princeton, New Jersey

ZACKZOU

Department of Chemistry

Lundbeck Research USA,

Paramus, New Jersey

PART I

Basic Concepts of Drug Metabolism and Disposition

1 Progression of Drug Metabolism

RONALD E WHITE

White Global Pharma Consultants, Cranbury, New Jersey

1.1 Introduction

1.2 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 Expectation

1.3.2 New Challenges for Technology

1.4 Perspective on the Magnitude of the Challenge

1.4.1 Ultimate Limits on Metabolite Quantitation

1.4.2 Practical Limits on Metabolite Quantitation

1.4.3 Natural Limit Due to Dose Size

1.5 Are There More Sensitive Alternatives to MS?

Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and Applications, First Edition Edited by Mike S Lee and Mingshe Zhu.

r 2011 John Wiley & Sons, Inc Published 2011 by John Wiley & Sons, Inc.

3

For instance, 20 years ago protein sequencing was based on peptide chemistry,and the basic experiment was the assay of amino acids released from trypticpeptides, a process that required months or years to complete Now, proteinsequencing is based on nucleotide chemistry, and the basic experiment is theautomated assay of oligonucleotides from partial hydrolysis of complementarydeoxyribonucleic acid (cDNA) a process that takes about a day The reasonthat ADME experiments have not evolved much is that we have never devised asurrogate for the whole human body for metabolism studies All systems tried

up to this point (e.g., animals, transgenic animals, perfused organs, in vitroincubations, three-dimensional (3D) microfluidic cell culture devices, in silicocalculations) fail to reliably predict the actual metabolic fate of NCEs To besure, the technology we use for the ADME experiment has advanced greatly,but even with the newest methods, DM is still essentially a chemical exercise atits core, and the backbone technology remains mass spectrometry (MS).However, if we consider the more complete picture, DM has expandedenormously in scope and level of understanding in those 50 years While thechemistry-based core remains intact and actively growing, several other kinds

of DM studies have layered over the core, all coexisting and relevant incontemporary DM science Thus, in addition to the purely chemical description

of the structures of metabolites and probable chemical mechanisms of theirformation, we now have a very good biochemical understanding of the variousenzymes that catalyze these biotransformation processes, as well as a cellularand genetic understanding of the expression and regulation of those enzymes

We are even making progress toward reliable prediction of the fates ofxenobiotic substances in human beings (Anderson et al., 2009), although thisgoal 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

Near the end of the twentieth century, I suggested that there had been fouroverlapping 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 newchemical entity (NCE) was provided, largely consisting of chemical information.Major urinary and fecal metabolites were isolated and identified by classicchemical techniques including column and thin-layer chromatography, crystal-lization, and derivatization Eventually, spectroscopy was used for the structuralelucidation, including mass spectroscopy, infrared, and nuclear magnetic reso-nance (NMR) These techniques required much smaller quantities to be isolatedand allowed high-performance liquid chromatography (HPLC) to replace

column chromatography Interestingly, early in this period R.T Williams lished his monograph Detoxication Mechanisms, which can be considered thefirst identification of DM as a discrete field of study (Murphy, 2008) Thepublication of that book also showed that academic researchers were beginning

pub-to think about the biological basis and implications of DM, although this way ofthinking 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 biochemicalprocesses responsible for the disposition of xenobiotics (e.g., which enzymes wereinvolved) Illustrating the indistinct separation of these phases, the pioneers in thisphase often came from a chemical background, and they sought to describethe enzymes in chemical terms The proteins were isolated so that they could

be treated as discreet chemical reagents, describable in classical chemical terms ofcomposition, reaction stoichiometry, thermodynamics, and reaction mechanism.However, after about a decade or so, the chemical approach to studyingthe enzymes transitioned to a biochemical and cell biology approach in whichenzyme kinetics and protein protein and membrane protein interactionsbecame the “hottest” topics All of the important DM enzymes were character-ized, named, and even made commercially available to industrial researchers Thelatest advance in the biochemistry phase was the realization that even the exposure

of drugs to the drug-metabolizing enzymes was a biochemical event, mediated byphysical enzymes called transporters (Wu and Benet, 2005) Advances in ourunderstanding of the biochemistry of DM in academic laboratories were reflected

in a greater expectation by regulatory agencies that a biochemical description beprovided 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 kinetic rates and molecular sites of metabolism by genotyping human testsubjects with respect to an ever-growing list of genetically polymorphic drug-metabolizing enzymes Equally important, regulation of DM enzymes wasrecognized to occur mainly at the gene expression level, whether resulting fromheredity, disease processes, or environment This pharmacogenetic character-ization has become a routine expectation for the registration packages of NCEsand continues to expand As before, the new genetic information did notreplace any previous requirements for DM information but instead added anadditional dimension to that information package

pharmaco-1.2.4 The “Biology” Phase (2010 and Beyond)

We are beginning to view drug metabolism in terms of systems biology Thisinvolves taking a holistic view of the simultaneous interaction of a xenobiotic

molecule with all the enzymes and receptors in the human body Some ofthese receptors are the pharmacological targets that lead to therapeutic benefits,some are unintended targets that generate adverse events and toxicities, and someare the enzymes and nuclear receptors of DM In our overall description of thedisposition of the compound, interactions of the compound with these DMtargets are especially complex to relate to safety and efficacy When designing apractical clinical medicine, we need to establish a balance between too rapidmetabolism, leading to reduced efficacy, and too sluggish metabolism, leading toaccumulation and possible toxicity In the clinic, we need to determine whethermetabolism decreases or increases the desired pharmacological effect (i.e., activemetabolites) And, finally, in this holistic biological view, we need to assess howthe metabolites interact with all the off-target human enzymes and receptors,especially the phenomenon of reactive metabolites covalently binding to proteinsand nucleic acids, leading to toxic sequelae (Baillie, 2009).

These four phases are graphically depicted in Figure 1.1 They are layered inthe 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

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 withtime but abruptly increases about 2010 due to enhanced regulatory surveillance ofmetabolites Starting around 1975, biochemistry (B, dark gray) begins to be included inthe DM characterization and slowly increases with time Genetics information (G, lightgray) continues to increase, mainly in clinical trials The apparent jump in B and G workaround 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

years The meaning of the step increase in work at around 2010 in the figure will

be discussed in the next section

The first three of these historical phases of industrial DM serve to summarizeand rationalize the scientific questions of yesteryear and today The questions

of tomorrow are described by the biology phase However, now we can alsodiscern the beginning of an additional new trend that could be called the

“regulatory” phase This phase is not primarily concerned with the gical process of DM, as are the other phases Instead, the regulatory phase isconcerned with the human safety of the metabolites, once they are formed Buteven though the focus of this phase is safety, it may well produce the greatestincrement of additional DM work to be done in the future

physiolo-1.3.1 New Regulatory Expectation

Regulatory interest in metabolites has developed into a formal Guidance forIndustry (Safety Testing of Drug Metabolites) issued by the U.S Food and DrugAdministration (FDA), which instructs sponsors on the qualitative and quantita-tive characterization of metabolites in both clinical and preclinical toxicologicalsettings (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 asfollows: Human circulating metabolites that exceed 10% of the total exposure ofall drug-related materials in circulation at pharmacokinetic steady state requiresafety assessment before large-scale clinical trials can proceed These Guidanceshave implications for bioanalysis and safety assessment, but here we wish to focus

on the implications for biotransformation studies Development of these dances, though initiated by an industry-sponsored group (Baillie et al., 2002), hasresulted in an inevitable regulatory emphasis on metabolite characterization muchearlier than traditionally carried out Importantly, the relative levels of parent drugand metabolites at pharmacokinetic steady state have been little studied pre-viously Consequently, we can expect surprises, possibly even new phenomena, as

Gui-we watch the time evolution of drug metabolism during the approach to steadystate The traditional approach to definitive metabolite characterization (i.e.,single-dose14C-labeled clinical ADME studies performed during Phase II or III)

is inadequate for the new regulatory demands and either a new approach to14Cstudies or new nonradiolabel-based methodology is required

1.3.2 New Challenges for Technology

Distressingly, both the qualitative and quantitative requirements of thenew paradigm potentially push the existing technology past present limits

Qualitatively, technology is now needed for metabolite detection in earlyclinical development Up to this time, MS was only required to elucidate thechemical structures of metabolites, augmented as necessary by NMR andsynthesis of authentic standards However, detection of the presence ofmetabolites in a sample relied on the observation of nonparent radioactivepeaks in the chromatogram Now we are asking the mass spectroscopist to alsodetect the metabolites, without the benefit of radiolabel or prior knowledge ofthe structures This requirement for MS-based detection as well as character-ization has necessitated the development and validation of new algorithms ofdata acquisition and processing In fact, as will be seen in later chapters, reliablenonradiometric MS-based methods of metabolite detection are now a reality.Quantitatively, technology limits are pushed in two ways First, the massspectroscopist is asked to estimate the concentration of each metabolite in aplasma profile and categorize it as more or less than 10% of the total This isproblematic because the structures may not be completely known and authenticstandards may not be available Second, for those metabolites at levels morethan 10%, a validated assay will be required, pushing the sensitivity limits thatmay be required in some cases As the typical drug candidate becomes evermore potent, with ever smaller administered doses, the accurate estimation

of analyte exposures that are as much as an order of magnitude less than that ofthe parent could become a challenge Experts will discuss technologicaladvances 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 thistechnology to these problems in Part III However, we can put some perspectivehere 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 istoo 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 regula-tory lower limit on the absolute quantity of a metabolite that might need to bedetected 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 tometabolite 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 oflimits, let us assume the worst case, that the 10% rule was always applied Isthere any other limitation of how much metabolite might need to be detectedand 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, sothe stipulation that the subject must survive the assay clearly requires manymore molecules than one in the body for detectability To estimate how manymore, in the next section we will use the “one-molecule” concept in the oppositedirection (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 adetector signal no longer appears to be continuous At the ultimate limit, eitherzero or one molecule will enter the inlet of the mass spectrometer; one cannotanalyze half a molecule Thus, one molecule entering the mass spectrometerinlet during a single duty cycle is the natural ultimate limit of sensitivity.Working backward from this limit and assuming that about 5% of the injectedmass is actually sampled in a single duty cycle, we see that at least 20 moleculeswould have to be injected, on average, to get one into the inlet during a singleduty cycle Given a 10-μL injection from a 100-μL reconstituted extract of a100-μL plasma aliquot of an original 1-mL plasma sample, we can estimate thatplasma from a human subject would have to contain at least 2000 molecules permilliliter to be measurable in a practical sense by current liquid chromato-graphy (LC)/MS laboratory methods Although one could propose taking alarger plasma sample from a subject, reconstituting the extract in a smallervolume, and injecting a larger fraction onto the instrument, there are naturallimitations for these volumes as well For instance, a full pharmacokineticprofile 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 physicianswould ever accept under any circumstances, and even larger samples wouldclearly be out of the question Thus, for this thought experiment, let us acceptthat a realistically and routinely measurable concentration could not be muchless than 1000 molecules per milliliter of plasma based on the natural limit ofone molecule interacting with a mass spectral detector Application of Avoga-dro’s number to this ensemble of 1000 molecules allows us to state that plasmaconcentrations less than about 1 attomolar (1 aM, 10218molar) will never beaccessible 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 typicaldrug, then a meaningful detection limit of 1 aM for a metabolite implies aconcentration 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 molecularweight (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 tocharacterize circulating metabolites? The most potent substances administeredtherapeutically are hormones, and their doses are exceedingly low Forinstance, the labor-inducing hormone oxytocin is effective at a vanishinglylow dose of about 40 ng, while the calcitriol dose is only about 4 μg Theseexamples may represent the lowest human doses that will ever be used for anydrug Amazingly, some actual drugs already approach these dose levels Forinstance, the inhaled β-agonist formoterol is given at 12 μg, and the inhaledglucocorticoid beclomethasone is delivered at 40μg Circulating levels of thesetwo drugs and their metabolites are almost unmeasurable (Cmaxin each case is

ca 10 pg/mL) The most potent oral drugs are also in the same range, with thedose of clonidine being about 100 μg If, as suggested above, future drugcandidates will not be more potent than the most potent drugs in use today,then we can say that levels of metabolites requiring detection and characteriza-tion 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/millilitersensitivity are already in use Thus, we can see that there is a natural limit tothe ultimate sensitivity required to detect and characterize circulating metabo-lites, 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, andmetabolite levels for these drugs are well within contemporary MS sensitivity

An interesting extension of the concept of natural limitation is to remove theassumption that one molecule gives one quantum of signal (i.e., destructiveanalysis) Spectroscopic methods such as NMR and fluorescence can timeintegrate numerous signal pulses from a single molecule, resulting in notheoretical limit to how much signal could be accumulated with unlimitedacquisition time In fact, quantitative NMR spectroscopy has been proposed

as a readily available solution to the main problems of implementing theGuidances, namely recognition and quantitation of metabolites without radio-label at steady state (Espina et al., 2009; Vishwanathan et al., 2009) However,both NMR and fluorescence still face the indivisibility of individual moleculeswhen applied to ex vivo samples such as plasma, so that a practical lower limit

of concentration would still exist Moreover, given the insensitivity of NMRrelative 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 MSand NMR, fluorescence is not generally applicable to all drug candidates.Clearly, then, the only currently available technology for low-level metabolitecharacterization is MS, which combines sensitivity, structural information, andgeneral applicability

1.6 SUMMARY

In summary, DM is a traditional yet dynamic discipline, comprising a constantcore overlaid with evolving successive layers of related activities The coreactivity of detecting and determining the chemical structure of human meta-bolites has changed little in decades and, in fact, is the starting point for all theother activities in areas such as enzymology, regulation, and genetics Forexample, it is meaningless to inquire which cytochrome P450 (CYP) enzyme isprincipally responsible for the metabolism of a new drug until the chemicalstructure of the major metabolite shows that it was likely formed by a CYPenzyme Today, structural elucidation of a metabolite almost always beginswith MS, followed by complementary methods such as NMR, as necessary.Conversely, detection of metabolites has traditionally been accomplished byradiochromatography However, in response to evolving regulatory expecta-tions, it is likely that detection will become the job of MS also Thus, MS is themost important single technique in DM and is likely to remain so goingforward into the future This conclusion explains the need for continuedadvancement of DM applications of MS technology, as described in theremainder of this book

Espina R, Yu L, Wang J, Tong Z, Vashishtha S, Talaat R, Scatina J, Mutlib A Nuclearmagnetic resonance spectroscopy as a quantitative tool to determine the concentra-tions of biologically produced metabolites: Implications in metabolites in safetytesting Chem Res Toxicol 2009;22:299 310

ICH (International Conference on Harmonisation of Technical Requirements forRegistration of Pharmaceuticals for Human Use) Guidance on Nonclinical SafetyStudies for the Conduct of Human Clinical Trials and Marketing Authorization forPharmaceuticals, M3(R2); Step 4 version, 11, 2009, June available: www.ich.org/cache/compo/276-254-1.html

Murphy PJ The development of drug metabolism research as expressed in thepublications of ASPET: Part 1, 1909 1958 Drug Metab Dispos 2008;36:1 5.Smith DA, Obach RS Metabolites in safety testing (MIST): Considerations ofmechanisms of toxicity with dose, abundance, and duration of treatment ChemRes Toxicol 2009;22:267 279

U.S Food and Drug Administration Guidance for Industry: Safety Testing of DrugMetabolites Center for Drug Evaluation and Research (CDER), Rockville, MD,

2008, available: www.fda.gov/cder/guidance/6897fnl.pdf

Vishwanathan K, Babalola K, Wang J, Espina R, Yu L, Adedoyin A, Talaat R, Mutlib

A, Scatina J Obtaining exposures of metabolites in preclinical species throughplasma pooling and quantitative NMR: Addressing metabolites in safety testing(MIST) guidance without using radiolabeled compounds and chemically synthesizedmetabolite standards Chem Res Toxicol 2009;22:311 322

White RE Short and long-term projections about the involvement of drug metabolism indrug discovery and development Drug Metab and Dispos 1998;26:1213 1216

Wu CY, Benet LZ Predicting Drug Disposition via Application of BCS: Transport/Absorption/Elimination Interplay and Development of a Biopharmaceutics DrugDisposition Classification System Pharm Res 2005;22:11 23

2.2.1 Cytochrome P450 Oxidative Reactions

2.2.2 Oxidations by Flavin Monooxygenases

2.2.3 Oxidations by Monoamine Oxidases

2.2.4 Oxidations by Molybdenum Hydroxylases

2.2.5 Oxidations by Alcohol and Aldehyde Dehydrogenases

2.2.6 Oxidations by Peroxidases

2.3 Reductive Reactions

2.3.1 Reductions by Cytochrome P450s

2.3.2 Reductions by Molybdenum-Containing Enzymes

2.3.3 Reductions by Alcohol Dehydrogenases and Carbonyl Reductases2.3.4 Reductions by Cytochrome P450 Reductase and

Quinone Oxidoreductase

2.3.5 Reductions by Intestinal Microflora

2.4 Hydrolytic Reactions

2.4.1 Hydrolysis by Epoxide Hydrolases

2.4.2 Hydrolysis of Esters, Amides, and Related Structures

2.5 Glucuronidation Reactions

2.5.1 Glucuronidation of Hydroxy Groups

2.5.2 Glucuronidation of Amines and Amides

2.5.3 Glucuronidation of Thiols and Thiocarbonyl Compounds

2.5.4 Glucuronidation of Relatively Acidic Carbon Atoms

Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and Applications, First Edition Edited by Mike S Lee and Mingshe Zhu.

r 2011 John Wiley & Sons, Inc Published 2011 by John Wiley & Sons, Inc.

13

2.6 Sulfation Reactions

2.6.1 Sulfation of Alcohols

2.6.2 Sulfation of Hydroxylamines and Hydroxyamides

2.6.3 Sulfation of Amines and Amides

2.7 Acylation Reactions

2.7.1 Acetylation of Primary Amines and Hydrazines

2.7.2 Amino Acid Conjugation of Carboxylic Acids

a few cases, the metabolite or its sequential products may cause adversereactions that can lead to toxic effects The biotransformation reactions thatlead to the various products or metabolites are governed by basic physico-chemical principles, and most can be described with standard one- or two-electron chemical reactions This chapter will survey the most commonbiotransformation reactions and is not intended to provide mechanistic details.For a more complete description of the chemical and enzymatic mechanisticaspects of drug metabolism, written for nonchemists and chemists, the reader isreferred to a recent textbook on this topic (Uetrecht and Trager, 2007) Foradditional information on drug metabolism in general, the reader is referred to

a recent handbook (Pearson and Wienkers, 2008)

2.2 OXIDATIVE REACTIONS

Oxidations are the most common biotransformation reactions that occur withmost drugs There are several classes of enzymes that carry out these reactions:cytochrome P450s, flavin monooxygenases, monoamine oxidases, xanthineoxidase, aldehyde oxidases, aldehyde dehydrogenases, and peroxidases Typicalreactions and substrate substructures for each of these classes of enzymes will

be described

2.2.1 Cytochrome P450 Oxidative Reactions

Cytochrome P450s are a superfamily of hemoproteins that exhibit a visibleabsorption band at approximately 450 nm when carbon monoxide is bound tothe reduced (ferrous) protein (Ortiz de Montellano, 2005; Guengerich, 2008;P450 Homepage: http://drnelson.utmem.edu/CytochromeP450.html) Cyto-chrome P450s are ubiquitous in nature with over 8000 genes found as of

2008 and utilize reduced nicotinamide adenine dinucleotide phosphate(NADPH) as the cofactor Although 115 cytochrome P450 genes have beenidentified in humans, only 57 are known to be functional, of which about halfare known to metabolize drugs Some of these enzymes, particularly those thatoxidize physiological substrates, have high substrate selectivities, whereas manythat metabolize drugs have broad and overlapping substrate selectivities Theseenzymes catalyze a broad range of oxidative reactions that is usually driven bythe reaction of an electron-deficient hypervalent iron-oxo species Thus, for themost part, the reactions feature a one-electron radical abstraction/recombina-tion that, depending on the particular drug substrate substructure, yieldsseveral different kinds of products as categorized below In a few cases, theregiochemistry of the products may be dictated by electron-rich and radicalstabilizing elements of particular substructures However, in most cases,interactions of the substrate with specific active site residues are favored anddictate both the regio and stereochemistry of the products

2.2.1.1 Aliphatic Hydrocarbon Hydroxylation These oxidations occur onallylic and benzylic sites (or similar aliphatic groups on heteroaromaticstructures), as well as theω and ω-1 positions on alkyl chains:

2.2.1.2 Aromatic Hydroxylation Phenols and phenol-like compounds (ortheir tautomers) are major metabolites of most benzenoid substructures,including heteroaromatic substructures The hydroxylation may or may notproceed through an arene oxide, but the physicochemical/enzymic parametersthat dictate which pathway will prevail are still largely unknown Only a fewaromatic epoxides have been stable enough to characterize:

O

N C

Carbamazepine Epoxide O

2.2.1.4 Oxidative N, O, and S Dealkylation Oxidative dealkylation is one

of the most common biotransformation reactions observed with drugs.Essentially, all drugs that have an alkyl amine or amide, alkyl ether or ester,

or alkyl thioether or thioester substructure that contains an α-carbon atomwith at least one hydrogen atom, will be oxidized by cytochrome P450s at thatcarbon atom to form an intermediate carbinol (carbinolamine in the case ofamines) Most of these semistable intermediates will spontaneously dealkylate

to yield the corresponding heteroatom-containing product that has lost thealkyl group that was hydroxylated, and which forms an aldehyde or ketone

in the process However, several stable hydroxyamides, or similar structuresthat contain nonbasic nitrogens, do not spontaneously dealkylate (Note thatoxidative deamination is just another case of oxidative N-dealkylation in whichthe amine is a primary amine; thus, ammonia is lost.)

undergo a similar reaction to that described for oxidative dealkylation, withintermediate formation of a halohydrin that spontaneously dehalogenates Thebest leaving groups are iodide bromide chloride fluoride:

Ar N H R

where Ar = aromatic or heteroaromatic ring.

a

Ar N O

R H

O H H

R1 HC O H

R2

R1 C R2O

R

H2C

H2C

2.2.2 Oxidations by Flavin Monooxygenases

Flavin monooxygenases (FMOs) are a family of enzymes that catalyze themonooxygenation of soft nucleophilic groups (N, S, P, Se) through the formation

of an enzyme-bound hydroperoxyflavin that is a stable, but relatively weak,oxidant (Krueger and Williams, 2005; Testa and Kramer, 2007; Strolin-Benedetti

et al., 2006) Primary substructures are tertiary amines that are oxidized to oxides FMOs also will metabolize primary alkylamines sequentially to hydro-xylamines and oximes and secondary amines to N-hydroxy and nitrone products.Aromatic amines and amides are not substrates Thioethers can be oxidized tosulfoxides; and thiols, thioamides, and thiocarbamates can be oxidized by bothflavin monooxygenases and cytochrome P450s to reactive sulfenic acids, sulfines,and sulfenes:

2.2.3 Oxidations by Monoamine Oxidases

Whereas cytochrome P450s and flavin monooxygenases are mostly microsomalenzymes, monoamine oxidases (MAOs) are located in mitochondria and arepresent in particularly high concentrations in nerve terminals (Testa andKramer, 2007; Strolin-Benedetti et al., 2006; Youdim et al., 2006) Only twoforms (MAO-A and MAO-B) have been characterized Their substruc-ture substrates are most commonly primary amines, though MAOs canoxidize some secondary and tertiary amines, such as the drug sumatriptan andthe neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) MAOsare flavin-containing enzymes, like FMOs, but they do not form a peroxy-flavin oxidant The MAOs apparently react via a radical abstraction mechanismthat forms imines that are hydrolyzed to the amines and aldehydes with retention

of oxygen from water rather than oxygen in the aldehyde product:

N H

N

C S

H N

H N

C O H

2.2.4 Oxidations by Molybdenum Hydroxylases

Xanthine oxidase (XO) and aldehyde oxidase (AO) are molybdenum-containingcytosolic enzymes whose normal substrate substructures are iminelike sp2-hybridized carbon atoms (Strolin-Benedetti et al., 2006; Garattini et al., 2008;Kitamura et al., 2006) The product amides contain oxygen that comes fromwater These enzymes, particularly AO, which is present in relatively highconcentrations in human liver, appear to play a greater role in the metabolism

of new drugs that often contain nitrogen heterocycles:

2.2.5 Oxidations by Alcohol and Aldehyde Dehydrogenases

There are several classes of alcohol dehydrogenases that catalyze the reversibleoxidation/reduction reaction of alcohols to aldehydes (Testa and Kramer, 2007;