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Library of Congress Cataloging-in-Publication Data
Names: Pearson, Paul G. (Paul Gerard), 1960- editor. | Wienkers, Larry C., editor.
Title: Handbook of drug metabolism.
Description: Third edition / [edited by] Paul G. Pearson, Larry C. Wienkers. | Boca Raton, Florida : CRC Press, 2019. | Series: Drugs and the pharmaceutical sciences | Includes bibliographical references and index.
Identifiers: LCCN 2018057997| ISBN 9781482262032 (hardback : alk. paper) | ISBN 9780429190315 (ebook)
Subjects: LCSH: Drugs--Metabolism--Handbooks, manuals, etc.
LC record available at https://lccn.loc.gov/2018057997
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com
and the CRC Press Web site at http://www.crcpress.com
This book is dedicated to Professors William F. Trager and Sidney D. Nelson who trained and inspired a generation of drug metabolism scientists.
Contributors xv
Section I Fundamental Aspects of Drug Metabolism
1. The Evolution of Drug Metabolism Research ............................................................................... 3 Patrick J. Murphy
2. Pharmacokinetics of Drug Metabolites ........................................................................................17 Philip C. Smith
3. The Cytochrome P450 Oxidative System ..................................................................................... 57
Paul R. Ortiz de Montellano
4. Aldehyde Oxidases an Emerging Group of Enzymes Involved in Xenobiotic Metabolism: Evolution, Structure, and Function 83
Enrico Garattini and Mineko Terao
5. UDP-Glucuronosyltransferases 109
Robert S. Foti and Upendra A. Argikar
Section II Factors W hich Affect Drug Metabolism
6. Non-CYP Drug Metabolizing Enzymes and Their Reactions 163
Shuguang Ma, Ryan H. Takahashi, Yong Ma, Sudheer Bobba, Donglu Zhang, and S. Cyrus Khojasteh
7. The Genetic Basis of Variation in Drug Metabolism and Toxicity ......................................... 205 Tore Bjerregaard Stage and Deanna L. Kroetz
8. Inhibition of Drug Metabolizing Enzymes ................................................................................ 223 F. Peter Guengerich
9. Quantitative Approaches to Human Clearance Projection in Drug Research and Development 253 Nigel J. Waters
10. Sites of Extra Hepatic Metabolism, Part I: The Airways and Lung 279
John G. Lamb and Christopher A. Reilly
11. Sites of Extra Hepatic Metabolism, Part II: Gut 315
Dan-Dan Tian, Emily J. Cox, and Mary F. Paine
12. Sites of Extra Hepatic Metabolism, Part III: Kidney ...............................................................
Lawrence H. Lash
Section III Technologies to Study Drug Metabolism
Bo Wen
14. The Role of NMR as a Qualitative and Quantitative Analytical Technique in Biotransformation Studies ..........................................................................................................
Gregory S. Walker, Raman Sharma, and Shuai Wang
15. In Vitro Metabolism: Subcellular Fractions ...............................................................................
Michael A. Mohutsy
16.
R. Scott Obach and Kimberly Lapham 17.
Joshua G. Dekeyser and Jan L. Wahlstrom
18. Transporters in Drug Discovery and Development
Yaofeng Cheng and Yurong Lai
19. Experimental Characterization of Cytochrome P450 Mechanism-Based Inhibition
Dan Rock, Michael Schrag, and Larry C. Wienkers
Section IV Applications of Metabolism Studies in Drug Discovery and Development
Kirk R. Henne, George R. Tonn, and Bradley K. Wong
21.
Sanjeev Kumar, Kaushik Mitra, and Thomas A. Baillie
22. Kinetic Differences between Generated and Preformed Metabolites: A Dilemma in Risk Assessment
Thomayant Prueksaritanont and Jiunn H. Lin
23. Numerical Approaches to Drug Metabolism Kinetics and Pharmacokinetics
Ken Korzekwa and Swati Nagar
24.
Sylvie E. Kandel and Jed N. Lampe
25. ADME of Antibody Drug Conjugates ........................................................................................
Jiajie Yu, Cinthia Pastuskovas, and Brooke M. Rock
Amit
S. Kalgutkar
27. Applications of 14C Accelerator Mass Spectrometry in Drug Development
Raju Subramanian and Mark Seymour
Preface
It is almost a decade since the second edition of the Handbook of Drug Metabolism was published. Since its inception, the goal of the Handbook was to provide a comprehensive text to serve as a graduate course in Drug Metabolism, a useful reference for academic and industrial drug metabolism scientists, but also as an important reference tool for those pursuing a career in drug discovery and development. The third edition of the Handbook of Drug Metabolism has been markedly updated to capture a decade of advances in our understanding of factors that impact the pharmacokinetics and metabolism of therapeutic agents in humans. Moreover, we have sought to include new chapters that reflect significant advances that have occurred in major areas viz., the role transporters in drug disposition, active metabolites in drug development, predicting clinical pharmacokinetics, nonP450 biotransformation reactions, pharmacogenetics in drug metabolism and toxicity, drug interactions, and antibody drug conjugates.
The third edition of the Handbook of Drug Metabolism is organized into four sections. The first three sections capture scientific and experimental concepts around drug metabolism. Section I reviews fundamental aspects of drug metabolism, including a history of drug metabolism, a review of oxidative and non-oxidative biotransformation mechanisms, a review of liver structure, and function and pharmacokinetics of drugs metabolites. Section II details factors that impact drug metabolism, including pharmacogenetics, drug-drug interactions, and the role of extra-hepatic organs in drug biotransformation. Section III provides in depth insights into analytical technologies and methodologies to study drug metabolism at the molecular, subcellular, and cellular levels, and considerations of factors, viz. enzyme inhibition and induction that influence drug metabolism and therapeutic response. Section IV has been expanded substantially from the second edition to illustrate the highly integrated role of drug metabolism in drug discovery and drug development. In this regard, Section IV focuses on clinical and preclinical drug metabolism studies, safety considerations for drug metabolites (chemically-reactive and non-reactive metabolites) in the selection and development of promising therapeutic candidates and highlights the increased focus of regulatory agencies on safety considerations of drug metabolites.
The discipline of drug metabolism is now a highly integrated component of contemporary drug discovery and development programs—the results of these efforts have lead to only a small number of clinical development candidates that fail in clinical development for unacceptable pharmacokinetic and drug metabolism properties.
This book is dedicated to two groups of exceptional individuals. First, we thank the distinguished academic and industrial leaders (many of whom have contributed to this book) who have trained a generation, or more, of exceptional drug metabolism scientists. Second, we thank the many graduate students, post-doctoral fellows, and industrial colleagues who have challenged us and enriched our lives over the last two decades. We thank all of you for advancing the field of drug metabolism; your efforts have enabled our discipline to advance promising therapeutic agents with increased probability of success in finding medicines to treat serious illness.
Lastly, it has been a privilege to interact with this collection of expert authors, and we would like to express our sincere gratitude to them for their contributions to the third edition to the Handbook of Drug Metabolism.
Paul G. Pearson
Larry C. Wienkers
Editors
Paul G. Pearson is the president and CEO of Pearson Pharma Partners, Westlake Village, California, United States. Dr. Pearson received his PhD in Pharmaceutical Sciences from Aston University, Birmingham, UK. He has had an extensive and successful career in pharmaceutical science, previously serving as vice president of Pharmacokinetics and Drug Metabolism at Amgen, Inc. and executive director of Preclinical Drug Metabolism at Merck & Co, Inc. He is an active member of several international and national professional organizations. Dr. Pearson has contributed to numerous peer-reviewed publications and has been an honored invited lecturer at conferences, society meetings, and symposia on drug development, drug metabolism, and drug discovery. He is actively engaged in the discovery and development of new human therapeutics in the fields of oncology and neuroscience to make a dramatic difference to the lives of patients.
Larry C. Wienkers is a principal scientist, Wienkers Consulting, Bainbridge Island Washington, United States. Prior to consulting, Dr. Wienkers served as vice president of Pharmacokinetics and Drug Metabolism at Amgen, Inc. and senior director of Pharmacokinetics, Dynamics, and Metabolism at Pfizer. He obtained his PhD in Medicinal Chemistry from the University of Washington, Seattle, Washington, United States and in 2014 he was awarded the University of Washington, School of Pharmacy Distinguished Alumni Award in Pharmaceutical Science and Research. Larry is a member of several professional societies, where he has served as Chair of American Society of Pharmacology and Experimental Therapeutics Division of Drug Metabolism and in 2013 he was elected as a fellow in the American Association of Pharmaceutical Scientists. Dr. Wienkers has published over 80 peer-reviewed publications and book chapters, has been an invited lecturer at conferences and symposia on large- and small-molecule drug metabolism and drug discovery.
Contributors
Upendra A. Argikar
Pharmacokinetic Sciences Department
Novartis Institutes for BioMedical Research, Inc.
Cambridge, Massachusetts
Thomas A. Baillie
Department of Medicinal Chemistry School of Pharmacy
University of Washington Seattle, Washington
Sudheer Bobba
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc.
South San Francisco, California
Yaofeng Cheng
Drug Metabolism
Gilead Sciences, Inc.
Foster City, California
Joshua G. Dekeyser
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, California
Robert S. Foti
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
Cambridge, Massachusetts
Enrico Garattini
Laboratory of Molecular Biology
IRCCS-Istituto di Ricerche Farmacologiche
“Mario Negri” Milano, Italy
F. Peter Guengerich
Department of Biochemistry
Vanderbilt University School of Medicine Nashville, Tennessee
Kirk R. Henne
DMPK, Denali Therapeutics
South San Francisco, California
Emily J. Cox
Providence Medical Research Center
Providence Health & Services Spokane, Washington
Amit S. Kalgutkar
Medicinal Sciences Department
Pfizer Worldwide Research and Development
Cambridge, Massachusetts
Sylvie E. Kandel
Department of Pharmacology, Toxicology and Therapeutics
The University of Kansas Medical Center Kansas City, Kansas
S. Cyrus Khojasteh
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc. South San Francisco, California
Ken Korzekwa
Temple University School of Pharmacy Philadelphia, Pennsylvania
Deanna L. Kroetz
Department of Bioengineering and Therapeutic Sciences
University of California San Francisco, California
Sanjeev Kumar
Department of Drug Metabolism and Pharmacokinetics
Department of Pharmacology and Toxicology College of Pharmacy University of Utah Salt Lake City, Utah
Jed N. Lampe
Department of Pharmacology, Toxicology and Therapeutics
The University of Kansas Medical Center Kansas City, Kansas
Kimberly Lapham
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
Pfizer Global Research and Development Groton, Connecticut
Lawrence H. Lash
Department of Pharmacology
Wayne State University School of Medicine Detroit, Michigan
Jiunn H. Lin
Department of Drug Metabolism and Pharmacokinetics
Merck Research Laboratories West Point, Pennsylvania
Shuguang Ma
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc.
South San Francisco, California
Yong Ma
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc.
South San Francisco, California
Kaushik Mitra
Department of Safety Assessment and Laboratory Animal Resources
Merck Research Laboratories (MRL)
Merck & Co., Inc. West Point, Pennsylvania
Michael A. Mohutsy
Lilly Research Laboratories
Eli Lilly & Company Indianapolis, Indiana
Patrick J. Murphy
Consultant, Drug Metabolism and Disposition Carmel, Indiana
Swati Nagar
Temple University School of Pharmacy Philadelphia, Pennsylvania
R. Scott Obach
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
Pfizer Global Research and Development Groton, Connecticut
Paul R. Ortiz de Montellano
Department of Pharmaceutical Chemistry
School of Pharmacy
University of California San Francisco, California
Mary F. Paine
Department of Pharmaceutical Sciences
College of Pharmacy and Pharmaceutical Sciences
Washington State University Spokane, Washington
Cinthia Pastuskovas
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
South San Francisco, California
Thomayant Prueksaritanont
Department of Drug Metabolism and Pharmacokinetics
Merck Research Laboratories West Point, Pennsylvania
Christopher A. Reilly
Department of Pharmacology and Toxicology
College of Pharmacy
University of Utah
Salt Lake City, Utah
Brooke M. Rock
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
South San Francisco, California
Dan Rock
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
Seattle, Washington
Michael Schrag
Department of Drug Metabolism
Array BioPharma Inc. Boulder, Colorado
Mark Seymour
Department of Metabolism, Covance Laboratories Ltd., Harrogate, United Kingdom
Raman Sharma Pfizer, Inc.
Groton, Connecticut
Philip C. Smith
School of Pharmacy
University of North Carolina at Chapel Hill Chapel Hill, North Carolina
Tore Bjerregaard Stage Clinical Pharmacology and Pharmacy
Department of Public Health
University of Southern Denmark Odense, Denmark and
Department of Bioengineering and Therapeutic Sciences
University of California San Francisco, California
Raju Subramanian
Department of Drug Metabolism and Pharmacokinetics
Gilead Sciences, Inc. Foster City, California
Ryan H. Takahashi
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc.
South San Francisco, California
Mineko Terao
Laboratory of Molecular Biology
IRCCS-Istituto di Ricerche Farmacologiche
“Mario Negri” Milano, Italy
Dan-Dan Tian
Department of Pharmaceutical Sciences College of Pharmacy and Pharmaceutical Sciences
Washington State University Spokane, Washington
George R. Tonn DMPK, Denali Therapeutics South San Francisco, California
Jan L. Wahlstrom
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
One Amgen Center Drive Thousand Oaks, California
Gregory S. Walker Pfizer, Inc. Groton, Connecticut
Shuai Wang
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
Cambridge, Massachusetts
Nigel J. Waters
Nonclinical Development
Relay Therapeutics
Cambridge, Massachusetts
Bo Wen
Department of Drug Metabolism and Pharmacokinetics
GlaxoSmithKline
Collegeville, Pennsylvania
Larry C. Wienkers
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
Seattle, Washington
Bradley K. Wong
Wong DMPK Consulting, LLC
Redwood City, California
Jiajie Yu
Department of Pharmacokinetics and Drug Metabolism
Amgen Inc.
South San Francisco, California
Donglu Zhang
Department of Drug Metabolism and Pharmacokinetics
Genentech Inc.
South San Francisco, California
Section I Fundamental Aspects of Drug Metabolism
1
The Evolution of Drug Metabolism Research
Patrick J. Murphy
Introduction
Drug metabolism research has grown from a desire to understand the workings of the human body in chemical terms to a major force in the effort to develop drugs tailored to the individual. This essay will trace the beginnings of what are now major branches of drug metabolism to provide some background to the current state of the art represented in the many chapters of this book.
Chemistry—Major Metabolic Routes
In 1828, the laboratory of Friedrich Woehler was abuzz with the synthesis of urea, the first “organic” synthetic achievement. Woehler then turned his attention to potential chemical transformations in the body. He had been interested in compounds found in urine since his undergraduate days, and when Liebig identified hippuric acid as a normal urinary product, Woehler suggested that it might be formed from benzoic acid and glycine in the body. His initial experiments in dogs, however, were inconclusive [1]. Alexander
a
for
heard about
idea and reasoned that if
of urea due to the use of nitrogen in the glycine conjugate. Ure took benzoic acid and isolated hippuric acid from his urine [2]. Woehler had his associate, Keller, repeat the experiment and confirmed that ingested benzoic acid was indeed excreted in the urine as hippuric acid [3].
These studies initiated a period lasting to the end of the nineteenth century when scientists and their collaborators subjected themselves to interesting molecules to “see what would happen.” Many of the studies followed logical extensions of the earlier work.
Erdmann and Marchand administered cinnamic acid to volunteers and isolated a product tentatively identified as hippuric acid [4,5]. They proposed that the cinnamic acid was oxidized to benzoic acid and then conjugated with glycine. Woehler and Friederick Frerichs confirmed this transformation in dogs [6]. They also showed that benzaldehyde was converted to hippuric acid in dogs and rabbits.
The oxidation of benzene to phenol was discovered by the clinician, Bernhard Naunyn, during the course of experimental treatment of stomach “fermentation” with benzene. He was surprised to find that phenol was excreted following the administration of benzene. Naunyn then collaborated with the chemist, Schultzen, to study the fate of a number of hydrocarbons, including toluene, xylene, and larger molecules [7]. Aromatic hydroxylation, which had proven difficult for the chemists of the day, was readily accomplished in humans.
Studies on aromatic hydroxylation led Stadeler to discover conjugated phenols in human and animal urine [8]. Munk, after ingesting varying amounts of benzene, monitored the excretion of a “ phenol-forming substance” in his urine by hydrolyzing the urine with acid and measuring the released phenol [9].
Baumann, using the color of indigo as his guide, purified an indigo-forming substance from urine and showed that upon hydrolysis both indigo and sulfate were released [10]. Baumann made many pioneering studies on sulfates formed from a variety of compounds, including catechol, bromobenzene, indole, and aniline.
The surprising ability of the body to methylate compounds was discovered by His in 1887 when he was able to isolate and identify N-methyl pyridinium hydroxide from the urine of dogs dosed with pyridine [11]. Over 60 years later, MacLagan and Wilkinson discovered the more significant O -methylation pathway using the phenol butyl-4-hydroxy 3,5-diiodobenzoate [12]. This is the pathway that led Axelrod to his Nobel Prize related to the methylation of catecholamines.
N-Acetylation was first described by Cohn in his studies on the fate of m-nitrobenzaldehyde. The oxidized, reduced compound is acetylated and conjugated with glycine to yield the hippurate of N-acetylm-aminobenzoic as a major metabolite [13].
Mercapturic acids were initially isolated in the laboratories of Baumann and Preuss studying the fate of bromobenzene and by Jaffe looking at chloro and iodobenzene [14,15]. The actual structure of these acetylcysteine conjugates was determined by Baumann in 1884 [16]. The nature of the cofactors in the conjugation reactions would not be known until the twentieth century.
A unique, primarily human, conjugation of glutamine with aryl acetic acids was discovered by Thierfelder and Sherwin in 1914 [17].
Active Metabolites
By the early part of the twentieth century, the major drug-metabolizing reactions had been identified. A unifying theory on the role of metabolism was developed by John Paxson Sherwin. Sherwin was one of the most prominent Americans in the field of metabolism [18]. A native of Bristol, Indiana, he was educated in Indiana and Illinois and then spent two years in Tubingen, Germany, before returning to the Midwest. He formulated the “chemical defense” theory elaborated in his reviews on drug metabolism in 1922, 1933, and 1935 [19 –21]. The latter two reviews carried the title “Detoxication Mechanisms.” This same title was used by R.T. Williams in his groundbreaking summaries of drug metabolism in 1947 and 1959 [22]. Although Williams was troubled with the general classification of all metabolic reactions as “detoxication,” he accepted it as the most practical appellation. A mind-set that metabolism led to detoxication was so logical and had so many examples that when a compound was actually made more
active, it engendered disbelief. But, even at this early stage of drug development, the examples of activation began to accumulate.
The world’s first major drug, arsphenamine ( Figure 1.1), an arsenical used for the treatment of syphilis, was ineffective in vitro [23]. Twelve years after its launch, studies showing that the drug worked through an oxidation product were published by Voegtlin and coworkers [24]. This compound, which evolved from the “magic bullet” concept of Ehrlich, set the stage for future worldwide “blockbusters.”
A more dramatic impact of metabolism occurred with the launch of prontosil ( Figure 1.1), the first major antibacterial agent. Prontosil was discovered in the early 1930s in the laboratories of I.G. Farben,
FIGURE 1.1 Examples of drug activation.
the world’s largest chemical company. G. Domagk and coworkers used Ehrlich’s concept, wherein compounds that could be shown to bind to tissues may lead to specific antagonists of infectious agents. The early work, therefore, concentrated on derivatives of azo dyes. After numerous failures, they came across the compound prontosil, an azo dye containing a sulfonamide moiety [25]. This molecule had striking activity and was an instant success. Launched initially in Europe, it quickly stormed the United States when President Roosevelt’s son was cured by its administration [26]. Domagk was awarded the Nobel Prize in 1939 for his work.
But, like arsphenamine, prontosil had very low activity in vitro. This puzzled workers in the laboratories of Trefouel in France. They proceeded to test both prontosil and the sulfonamide breakdown product and came to the conclusion that it was the metabolite formed by the azo reduction that was the true antibacterial [27]. This was confirmed by studies in England that showed the presence of aminobenzenesulfonamide in the plasma and urine of patients treated with prontosil [28]. Once it became clear that any derivative that would release the active sulfonamide in vivo could represent effective therapy, chemical companies around the world began making variations that would spawn the birth of the modern pharmaceutical industry.
Acetanilide ( Figure 1.1) provides a bridge from active to toxic metabolites. Brodie and Axelrod found that acetanilide was converted to aniline, which explained the methemoglobinemia, which had been observed at high doses, and to acetaminophen, a superior analgesic [29]. This study launched the illustrious career of Julius Axelrod in the field of metabolism.
There are numerous examples of prodrugs, either by fortune or design, that must be activated for full pharmacological effect. Esters such as enalapril or clofibrate have to be hydrolyzed for activity. Methyldopa is decarboxylated and hydroxylated for activation, and cyclophosphamide is hydroxylated for activation. There are also many other examples where the metabolites have some or all of the activity designated for the parent. One of the most striking and significant of these is the antihistamine terfenadine ( Figure 1.1). The parent is oxidized to the active carboxylic acid and other metabolites by cytochrome P450 enzymes. When the P450s are inhibited, parent terfenadine reaches higher than normal levels [30,31]. This interaction led to cardiovascular problems in patients taking terfenadine and ketoconazole or erythromycin. Because of this interaction, terfenadine was taken off the market and new regulatory guidelines were put in place by the Food and Drug Administration (FDA) to alert drug developers of the need for interaction studies before approval.
Toxic and Reactive Metabolites
The products of metabolism are determined by the reaction mechanism of the enzymes involved and by the chemical structure of the reactant. Whether a metabolic product is more or less active is independent of these two interacting forces. Humans have evolved over the years, whereby we have a certain capacity to handle whatever the environment and our diets present. We learn to avoid certain toxins, which cannot be handled metabolically. The time frame of evolution does not permit the type of adaptation necessary to dispose of every new compound in a safe and beneficial manner. It is impossible to estimate what percentage of new molecular entities are converted to active or toxic metabolites, but it is clear that it has to be a higher percentage than we see just looking at marketed drugs. The fact is that if a toxic metabolite is produced during drug development, the candidate is usually eliminated from consideration. Therefore, the number of compounds actually activated by metabolism is necessarily higher than the overall documented occurrences. With any new compound, there is a significant chance that metabolism will yield pharmacologically active derivatives.
Reactive Intermediates
The formation of reactive intermediates is of particular concern in the development of new agents. Guroff and coworkers found that during the course of aromatic hydroxylation the hydrogen on the position to be hydroxylated could shift to the adjacent position [32]. This was termed the “NIH” shift and
was subsequently explained by the formation of a reactive epoxide intermediate. The formation of “green pigments” during administration of 2-allyl-2-isopropylacetamide was shown by de Matteis to be due to destruction of P450 [33]. Ethylene and other olefins had similar destructive properties [34]. The most significant chemical moieties giving rise to reactive molecules and/or P450 inhibition have been reviewed [35]. Many of these compounds will react with glutathione in an inactivation step. Excretion of mercapturic acids is often taken as a sign of the formation of the reactive species. In the absence of adequate levels of glutathione or when the kinetics are favorable for protein binding, the formation of chemicalprotein conjugates can lead to systemic toxicity.
The prototypical reactive intermediate is the quinone-imine formed from metabolism of acetaminophen. In a classic series of papers, Brodie and coworkers revealed the metabolic fate of acetaminophen and its potential tissue-binding metabolite [36 –39]. While this compound is known to be hepatotoxic and readily binds to protein in vitro, it nonetheless remains a best-selling analgesic. Generally, at recommended doses, acetaminophen is efficiently removed by conjugation or, after oxidation, by glucuronidation and/or reaction with glutathione. At elevated doses or in conjunction with CYP2E1 induction, high levels of quinone-imine can lead to tissue damage [40].
Bioanalytical
The progress in drug metabolism is paralleled by, indeed dependent on, the advances in bioanalytical techniques. For most of the first century of drug metabolism research, identification of metabolites involved isolation, purification, and chemical manipulation leading to characterization. At the end of World War II, new technology developed during the war came into use in metabolism research. A study on the distribution of radioactivity in the mouse after administration of 14 C-dibenzanthracene set new standards for metabolic research [41]. The use of high-speed centrifuges to separate cellular components was another legacy of the Manhattan Project. The development of liquid-liquid partition chromatography by Martin and Synge [42] heralded the addition of new separation tools, including paper, thin layer, and gas chromatography. Spectrometry in biological media became routine with the Cary 14 spectrophotometer.
Mass spectrometry moved from the hands of specialists to the analytical laboratory with the launch of the LKB 9000 GC/MS. A crucial development for the eventual linking of mass spectrometry and liquid chromatography was the discovery of electrospray ionization by Fenn in 1980 [43]. LC/MS instruments from Sciex and Finnegan revolutionized the bioanalytical laboratories leading to increasingly more rapid and more efficient delineation of metabolic pathways. Newer analytical techniques permit the analysis of the chemical bound to protein and the characterization of the proteins involved. For example, Shin and coworkers recently identified binding of electrophiles to 263 proteins in human microsomal incubations [44]. Doss and Baillie [45] have suggested that drug developers use in vitro binding ability as a screen for potential reactive intermediates.
Enzymology—Mechanisms of Metabolism
Conjugation
The discovery of cofactor structures started with acetyl coA. This important cofactor, vital for intermediary metabolism, was identified using the acetylation of sulfanilamide as an assay. Lippman and coworkers painstakingly isolated and identified coenzyme A as the energy-containing component driving acetylation [46]. The principle of active cofactors led researchers to solving the structures of 3ʹ-phosphoadenosine-5ʹ-phosphosulfate (PAPS) [47], uridinediphosphoglucuronic acid (UDPGA) [48], and S -adenosylmethionine (SAM) [49]. Defining mercapturic acid formation took slightly longer because of the fact that the actual conjugating moiety was altered before elimination. The actual structure of mercapturic acids was solved when Baumann correctly identified the acetyl cysteine moiety [16]. Glutathione, originally isolated by M.J. de Rey Pailhade [50], was fully characterized by Hopkins
in 1929 [51]. But it was not until 1959 when the relationship between glutathione conjugation and the formation of mercapturic acids was elucidated by Barnes and associates [52]. In 1961, Booth, Boyland, and Sims published data on the enzymatic formation of glutathione conjugates [53]. As a variation in the theme, it became clear that conjugation with amino acids such as glycine or glutamine involved initial activation of the substrate rather than the linking agent. The unique ability of humans and Old World monkeys to conjugate with glutamine was found to be due to the specificity of acyl transferase enzymes found in the mitochondria [54].
The structures of most of the human conjugating enzymes have now been elucidated, and in many cases, the enzymes have been cloned. Crystal structures have been slow to emerge for glucuronyl transferases because of the membrane-bound nature of these enzymes. There are 13 human sulfotransferases [55], 16 glucuronyl-transferases [56], multiple N -, O -, and S -methyl transferases, 2 N -acetyl transferases [57 ], and 24 glutathione transferases [58]. The multiplicity of isozymes and overlapping specificities require extensive evaluation to understand which enzymes may be critical for a given drug.
Reduction
Some of the earliest in vitro experiments in metabolism dealt with enzymatic reduction. The importance of the azo derivatives of sulfanilamide led to initial experiments using neoprontosil as a model substrate. Bernheim reported the reduction of neoprontosil by liver homogenates [59]. Mueller and Miller studied the metabolism of the carcinogen dimethylaminoazobenzene (DAB) and found that in rat liver homogenates DAB was hydroxylated and demethylated and the azo linkage was reduced [60]. The reducing enzyme was shown to require reduced nicotinamide adenine dinucleotide phosphate (NADPH) and to reside in the particulate portion of the fragmented cells [61].
Oxidation
The incorporation of oxygen into drugs and endogenous molecules was found to occur directly from molecular oxygen using isotopically labeled oxygen [62 ,63]. This led to the definition of a new category of oxidases termed “monooxygenases” by Hayaishi and “mixed-function oxidases” by Mason. These oxidases required oxygen and a reductant, usually NADPH.
In Vitro Methodology/Enzymology
The unraveling of the secrets of the cell began with the development of the techniques of gently breaking the cell developed by Potter and Elvejham and then fractionating the fragments and components of the cell by differential centrifugation [64]. The first drug to be studied using these techniques was amphetamine. Axelrod examined the deamination of amphetamine and showed the activity to be dependent on oxygen and NADPH and to reside in the microsomal fraction of the cell [65]. Brodie’s laboratory quickly examined a number of drug substrates and found them to be metabolized by the same microsomal system [66].
Further examination of microsomes by Klingenberg and Garfinkel revealed the presence of a pigment that had some of the properties of a cytochrome and a peak absorbance after reduction in the presence of CO at 450 nm [67,68]. The pigment was shown by Omura and Sato to be a cytochrome [69]. Estabrook Cooper and Rosenthal used light activation of the CO-inhibited system to prove that cytochrome P450 was the terminal oxidase in the oxidation of many classes of drug substrates [70]. The use of microsomes became standard practice in drug metabolism studies. However, the enzymes defied purification because of the fact that they were embedded in the membrane and solubilization inevitably led to denaturation.
Lu and Coon solved the problem of releasing the enzyme from the membrane using sodium deoxycholate in the presence of dithiothreitol and glycerol, rapidly expanding our knowledge of multiple
P450s [71]. Coon’s laboratory, Wayne Levin and associates, and Fred Guengerich were among the pioneers in the separation and purification of P450s [72]. Nebert and coworkers developed a unifying nomenclature on the basis of the degree of similarity of the P450s, and the field began to blossom [73]. The culmination of the efforts to define human P450s came with the sequencing of the human genome. At that point, it was clear that there were 57 variants of human P450. The major ones involved in drug metabolism have been well characterized, while there are still some isozymes whose function is yet to be defined [72].
The physical characteristics of the enzymes are rapidly being defined. The first P450 to be crystallized and the first structure determined were from a pseudomonad, Pseudomonas putida [74]. This provided a blueprint for all the structures to come. Human P450s resisted crystallization until they were modified by shortening the amino terminus. It is this portion of the protein that binds the membrane, and by removing the amino terminal segment, it was possible to obtain a soluble, active P450 that could be crystallized and analyzed. The crystal structures of all of the major drug-metabolizing P450s now have been determined [75]. The knowledge of the crystal structures has helped in our understanding of the broad specificity of this class of enzymes, helped to determine the necessary properties of potential substrates, and led to the development of computer programs to predict whether a compound will be a substrate or not [76].
While the P450s have been the stars of drug metabolism, there are many other oxidative enzymes that can play a role, sometimes dominant, in the fate of new molecules. The flavin monooxygenases (FMOs), which are often involved in the metabolism of heterocyclic amines, sulfur, or phosphorous-containing compounds, have been characterized and five forms identified [77]. Aldehyde oxidase, a molybdenumcontaining enzyme oxidizes nitrogen heterocycles and aldehydes [78], xanthine oxidase and xanthine dehydrogenase mainly involved in the production of uric acid [79], aldehyde dehydrogenase (3 classes, at least 17 genes [80], and alcohol dehydrogenase [23 distinct human forms]) [81] are among the enzymes most prominent in drug metabolism.
Genetic Characteristics of Drug-Metabolizing Enzymes
The drug-metabolizing enzymes showed early indications of genetic polymorphism on the basis of the individual variations in therapeutic effectiveness. The discovery of the utility of isoniazid in the treatment of tuberculosis was quickly followed by the realization that a significant portion of the patient population had elevated levels of isoniazid in the plasma. This was traced to a genetically determined deficiency in the N-acetyl transferase responsible for the inactivation of isoniazid [82]. Similarly, genetic variations in serum cholinesterase led to altered susceptibility to the effects of the muscle-relaxant succinyl choline [83]. A major breakthrough in the enzymes involved with drug oxidation came with the studies of Smith and coworkers on debrisoquine metabolism [84] and the work of Eichelbaum and coworkers on sparteine metabolism [85]. These discoveries led to a broad range of population studies on debrisoquine hydroxylase, later to be identified as CYP450 2D6. The variation in blood levels of these agents could be traced to whether patients had diminished levels of CYP2D6 or, in some cases, enhanced levels of CYP2D6.
As we learned more about the role of the isozymes of P450, genetic variations became a major topic of study. Significant polymorphic variations in CYP2C9, CYP 2C19, CYP2A6, and CYP2B6 must be taken into account for substrates of these enzymes [86]. Other oxidizing enzymes such as FMOs [87 ] and dihydropyrimidine dehydrogenase [88] also show genetic variation leading to drug toxicity. Conjugating enzymes such as thiopurine methyl transferase, N -acetyl transferase, and glucuronyl transferase have variants that have been shown to be important in altered response to drugs [89].
The message for drug development is clear. It is vital to know the enzymes involved in the breakdown of the administered drug. If a specific isozyme is responsible for either the majority of the inactivation
or for the activation of an agent, then appropriate studies are required to determine the efficacy and/or toxicity of the drug over a spectrum of the population, including the genetic variants.
Induction-Control Mechanisms
One of the most striking features of drug-metabolizing enzymes is their ability to adapt to the substrate load. Early studies showed that ethanol administration to rats increased the ability of the kidney to metabolize ethanol [90], while borneol administration to dogs or menthol administration to mice led to increased β -glucuronidase activity in these species [91]. Conney et al. discovered enzyme induction by aromatic hydrocarbons [92], while Remmer and Merker reported phenobarbital induction of smooth endoplasmic reticulum in rabbits, rats, and dogs [93]. Studies on the induction phenomenon eventually led to discovery of the Ah receptor [94]. The mechanism of transcriptional regulation has been elucidated and forms the basis for our understanding of this superfamily of regulators [95]. Other receptors integral to the initiation of induction include peroxisome proliferator activated receptor (PPAR) [96], constitutive androstane receptor (CAR), and pregnane X receptor (PXR). Interactions between CAR and PXR have recently been reviewed [97]. The crystal structure of the human PXR ligand-binding domain in the presence and absence of ligands has been reported [98,99].
Inhibitors
Compounds that had broad specificity as inhibitors of P450 played a major role in the understanding of this class of enzymes. The discovery of SKF525a in the laboratories of SKF and its expanded use by Brodie and coworkers defined the microsomal oxidases before the discovery of P450 [100,101]. That one inhibitor could decrease the metabolism of so many diverse compounds argued for an enzyme with broad specificity or multiple enzymes with a common site of inhibition. Other inhibitors such as metyrapone, ketoconazole, and AIA were similarly employed. Attention was drawn to the role of inhibition in drug interactions when cimetidine, a popular proton pump inhibitor, was found to be a weak inhibitor of P450-catalyzed reactions [102]. The observation that grapefruit juice had inhibitory properties stimulated the studies of endogenous and environmental inhibitors resulting in adverse drug reactions [103]. Once the multiplicity of P450s was clear, specific inhibitors of individual isozymes were used to define activity [35]. These inhibitors included antibodies with unique specificities [104]. The field of specific inhibitors for targeted therapy is rapidly developing, as the roles of all 57 P450s are unraveled [105,106].
Transporters
The discovery of P-glycoprotein (P-gp or mdr1) in 1976 created an enhanced appreciation of the role of transporters in drug disposition [107). In addition to playing an important role in drug penetration through the intestine, pgp plays a significant role in controlling the penetration of many drugs into the brain [108]. Umbenhauer and coworkers showed that a genetic deficiency in pgp correlated with the penetration of avermectin into the brain in mice [109,110]. Later studies showed a wide range of compounds controlled in a similar fashion. There are many other transporters that have yet to be characterized with regard to drug disposition. A total of 770 transporter proteins were predicted from analysis of the human genome. The ABC family, which contains pgp, consists of 47 members. The latest information and structural details on transporters can be found in the transporter protein analysis database [111]. The first crystal structure of an S. aureus ABC transporter was reported in 2007 [112]. In the drug development process, knowledge as to whether the candidate compounds are substrates for transporters is crucial to predicting bioavailability.
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—Dans quel guêpier suis-je donc, s’écria-t-il? Tout s’embrouille autour de moi; je n’y reconnais plus rien; jamais pareil fait ne s’est produit; jamais aussi ce stupide règlement dépourvu de sanction ne m’a paru aussi ridicule qu’aujourd’hui. Je ne veux dénoncer personne, mais je ne veux pas qu’on me dénonce; cela n’en finirait plus. Continuez à jouer aux cartes, jouez toute la journée, si le cœur vous en dit, mais laissez-nous la paix.
—Avant de vous en aller, voulez-vous me dire pourquoi la Compagnie a imposé ce règlement? Pouvez-vous lui trouver une excuse, j’entends une excuse rationnelle, plausible, et qui ne soit pas l’élucubration d’un cerveau d’idiot?
—Certainement, je le puis; la raison en est bien simple. C’est pour ne pas heurter les sentiments des autres voyageurs, de ceux qui ont des principes religieux; ceux-ci ne supporteraient pas que le jour du Sabbat fût profané en jouant aux cartes en wagon.
—C’est juste ce que je pensais. Ils ne regardent pas, eux, à voyager le dimanche, mais ils ne veulent pas que les autres...
—Par Dieu! vous voyez juste! je n’y avais jamais pensé avant; au fond quand on y réfléchit, ce règlement paraît stupide.
Sur ces entrefaites le surveillant du train arriva et fit mine de vouloir faire cesser le jeu; mais le conducteur du wagon l’arrêta et le prit à part pour lui expliquer la situation. Tout en resta là.
Pendant onze jours, je séjournai à Chicago, malade dans mon lit; je ne vis donc rien de la foire et je dus retourner dans l’est, dès qu’il me fut possible de voyager. Le major prit la précaution de retenir un wagon-salon pour me donner plus de place et rendre mon voyage plus confortable; mais quand nous arrivâmes à la gare, par suite d’une erreur, notre wagon n’était pas attaché au train. Le conducteur nous avait bien réservé une section du compartiment, mais, nous assura-t-il, il lui avait été impossible de faire mieux. Le major déclara que rien ne nous pressait et que nous attendrions jusqu’à ce qu’on ait accroché un wagon. Le conducteur lui répondit avec une certaine ironie:
—Possible que vous ne soyez pas pressé, comme vous le dites, mais nous n’avons pas de temps à perdre; veuillez monter, messieurs, et ne nous faites pas attendre.
Mais le major refusa de monter en wagon et il m’engagea fort à l’imiter. Il déclara qu’il voulait son wagon et qu’il l’aurait; le conducteur impatienté
s’écria:
—Nous ne pouvons mieux faire, nous ne sommes pas tenus à l’impossible. Vous occuperez ces places réservées ou vous ne partirez pas. On a commis une erreur qui ne peut être réparée au dernier moment. Le fait se produit quelquefois et personne n’a jamais fait autant de difficultés que vous.
—Ah! précisément; si tous les voyageurs savaient faire valoir leurs droits, vous n’essaieriez pas aujourd’hui de trépigner les miens avec une pareille désinvolture. Je ne tiens pas spécialement à vous causer des désagréments, mais il est de mon devoir de protéger mon prochain contre cette sorte d’empiètement. J’aurai mon wagon-salon ou bien j’attendrai à Chicago et je poursuivrai la Compagnie pour violation de son contrat.
—Poursuivre la Compagnie pour une telle bagatelle?
—Certainement.
—Vous le feriez réellement?
—Oui.
Le conducteur regarda le major avec étonnement et ajouta:
—Décidément vous avez raison; j’y vois clair maintenant, je n’y avais jamais songé auparavant. Tenez, je vais envoyer chercher le chef de gare.
Ce dernier arriva et parut plutôt ennuyé de la réclamation du major (mais pas du tout de l’erreur commise).
Il accueillit la plainte du major avec brusquerie et sur le même ton que le conducteur du train au début; mais il ne sut fléchir le major qui réclama plus énergiquement que jamais son wagon-salon. Cependant le chef de gare s’amadoua, chercha à plaisanter, et esquissa même un semblant d’excuses. Cette bonne disposition facilitait un compromis, le major voulut bien faire une concession. Il déclara qu’il renoncerait au wagon-salon retenu par lui à l’avance, à condition qu’on lui en fournît un autre. Après des recherches ardues on finit par trouver un voyageur de bonne composition qui consentit à échanger son wagon-salon contre notre section de compartiment. Dans la soirée, le surveillant du train vint nous trouver et, après une causerie très courtoise, nous devînmes bons amis. Il souhaitait, nous déclara-t-il, que le public fît plus souvent des protestations; cela produirait un très bon effet d’après lui, les Compagnies de chemin de fer ne se décideraient à soigner
les voyageurs qu’autant que ces derniers défendraient eux-mêmes leurs propres intérêts.
J’espérais que notre voyage s’effectuerait maintenant sans autres «incidents réformateurs», mais il n’en fut rien.
Au wagon-restaurant, le matin, le major demanda du poulet grillé; le garçon lui répondit:
—Ce plat ne figure pas sur le menu, monsieur, nous ne servons que ce qui est sur le menu.
—Pourtant je vois là-bas un voyageur qui mange du poulet grillé.
—C’est possible, mais ce monsieur est un inspecteur de la Compagnie.
—Raison de plus pour que j’aie du poulet grillé; je n’aime pas ces récriminations, dépêchez-vous et apportez-moi du poulet grillé.
Le garçon appela le maître d’hôtel qui expliqua très poliment que la chose était impossible; des règlements très sévères s’y opposaient.
—Soit, mais alors vous devez appliquer impartialement ces règlements ou les violer avec la même impartialité. Vous allez enlever à ce monsieur son poulet ou m’en apporter un.
Le maître d’hôtel resta aussi ébahi qu’indécis. Il esquissait une argumentation incohérente lorsque le conducteur survint et demanda de quoi il s’agissait. Le maître d’hôtel expliqua qu’un voyageur s’obstinait à avoir du poulet, tandis qu’il n’y en avait pas sur la carte et que le règlement s’y opposait. Le conducteur répondit:
—Cramponnez-vous au règlement, vous n’avez pas autre chose à faire.
—Mais un instant, s’agit-il de ce voyageur? Dans ce cas, continua-t-il en riant, croyez-moi, ne vous occupez plus du règlement; donnez-lui ce qu’il demande et ne le laissez pas énumérer tous ses droits. Oui, donnez-lui tout ce qu’il demande et si vous ne l’avez pas, arrêtez le train pour vous le procurer.
Le major mangea son poulet, mais il avoua qu’il l’avait fait uniquement par devoir, pour établir un principe, car il n’aimait pas le poulet.
J’ai manqué la foire, il est vrai, mais j’ai recueilli dans mon sac un certain nombre de tours diplomatiques qui, plus tard, pourront m’être très utiles; le lecteur les trouvera sans doute comme moi aussi pratiques que subtils.
UN VEINARD!
Ceci se passait à un banquet donné à Londres en l’honneur d’un des plus illustres noms de l’armée anglaise de ce siècle. Pour des raisons que le lecteur connaîtra plus tard, je préfère tenir secrets le nom et les titres de ce héros, et je l’appellerai le lieutenant général Lord Arthur Scorosby V. C. K. C. B. etc.... Quel prestige exerce un nom illustre! Là, devant moi, était assis en chair et en os l’homme dont j’entendis parler plus d’un millier de fois, depuis le jour où son nom, s’élevant d’un champ de bataille de Crimée, monta jusqu’au zénith de la gloire. Je ne pouvais me rassasier de contempler ce demi-dieu; j’étais en extase devant lui, je le buvais des yeux; son calme, sa réserve, son attitude digne, la profonde honnêteté qui s’exhalait de toute sa personne faisaient mon admiration; ce héros n’avait pas conscience de sa valeur; il semblait ne pas se douter que des centaines d’yeux admirateurs étaient fixés sur lui et que de toutes les poitrines des assistants montait vers lui un culte profond d’adoration.
Le Clergyman assis à ma gauche était une de mes vieilles connaissances. Clergyman aujourd’hui, il avait passé la première moitié de sa vie dans les camps et sur les champs de bataille, comme instructeur à l’école militaire de Woolwich.
A ce moment un éclair singulier illumina ses yeux, se penchant vers moi il murmura confidentiellement à mon oreille, en désignant d’un geste discret le héros du banquet:
—Entre nous, sa gloire est un pur accident; il la doit à un coup de veine incroyable.
Cette déclaration me causa une grande surprise; s’il s’était agi de Napoléon, de Socrate ou de Salomon, mon étonnement n’eût pas été plus grand. Quelques jours plus tard, le Révérend me fournit l’explication suivante de son étrange remarque:
—Il y a environ 40 ans j’étais instructeur à l’école militaire de Woolwich; le hasard voulut que je me trouvasse là lorsque le jeune Scorosby passa son examen préliminaire; sa nullité m’inspira une profonde pitié: tandis que les autres élèves de sa section répondaient tous
brillamment, lui se montra d’une ignorance crasse. Il me fit évidemment l’effet d’un brave garçon, doux et sans astuce, mais c’était navrant de le voir planté debout comme un piquet et décocher des réponses d’une stupidité et d’une ignorance prodigieuses. J’eus vraiment compassion de lui et je me dis: «La prochaine fois qu’il passera un nouvel examen il sera certainement renvoyé; aussi serait-il plus charitable d’adoucir sa chute autant que possible.»
Je le pris à part et m’aperçus qu’il savait quelques mots de l’histoire de César, mais c’était là tout son bagage; je me mis donc à l’œuvre et lui rabâchai un certain stock de questions sur César, qui devaient infailliblement être posées aux élèves. Vous me croirez si vous voulez: le jour de l’examen il se montra transcendant dans ses réponses, si transcendant qu’il recueillit force compliments pour ce «gavage» purement superficiel; tandis que les autres, mille fois plus instruits que lui, répondirent mal, et furent fruit-sec. Avec une veine fantastique qui ne se reproduira peut-être pas deux fois dans un siècle, il n’eut pas à répondre à d’autres questions. C’était stupéfiant. Pendant le temps que dura son examen, je restai à côté de lui avec la sollicitude qu’éprouve une mère pour son enfant estropié; il se tira toujours d’affaire comme par enchantement.
A n’en pas douter, les mathématiques allaient le couler et décider de son sort; toujours par bonté d’âme pour adoucir sa chute, je le pris de nouveau à part et je lui serinai un certain nombre de questions que l’examinateur ne manquerait pas de poser; puis je l’abandonnai à son triste sort. Eh bien! vous me croirez si vous voulez: à ma grande stupéfaction il mérita le premier prix et reçut une véritable ovation de compliments.
Pendant une semaine il ne me fut plus possible de dormir: ma conscience me torturait nuit et jour; par pure charité j’avais essayé de rendre moins dure la déconfiture de cet infortuné jeune homme sans me douter du résultat qui allait se produire. Je me sentais coupable et misérable: comment, par mon fait, cette pauvre cervelle bornée allait se trouver en tête d’une promotion et supporter de graves responsabilités! A n’en pas douter, à la première occasion, un effondrement ne manquerait pas de se produire.
La guerre de Crimée venait d’être déclarée.
«Quel malheur, pensai-je, voici maintenant la guerre; ce pauvre âne va avoir l’occasion d’étaler au grand jour sa nullité.» Je m’attendais à un désastre: ce désastre se produisit: j’appris avec terreur que le jeune
Scorosby venait d’être nommé capitaine d’un régiment de marche. Qui eût pu supposer qu’un tel poids de responsabilité dût peser sur des épaules aussi faibles et aussi jeunes? J’aurais encore compris sa nomination au grade de porte-étendard, mais à celui de capitaine, songez quelle folie! Je crus que mes cheveux allaient en devenir blancs. Moi qui aime tant la tranquillité et l’inaction, je me tins le triste raisonnement suivant: «Je suis responsable de ce malheur vis-à-vis de ma patrie; j’accompagnerai donc cet incapable, je resterai à ses côtés pour sauver ma patrie dans la mesure du possible.» Je rassemblai le pauvre petit capital péniblement économisé pendant mes années de dur labeur, je me mis en route avec un gros soupir et j’achetai un grade de porte-étendard dans son régiment. Ainsi nous partîmes tous deux pour la guerre.
Là, mon cher, quel spectacle effroyable! Il ne fit que des bévues, inepties sur inepties; mais, voyez-vous, personne ne connaissait à fond cet individu, personne n’avait mis au point ses capacités; aussi prit-on ses bévues navrantes pour des traits de génie. Le spectacle de ses sottises me fit crier de rage et délirer dans ma fureur; j’étais exaspéré de voir que chaque nouvelle insanité de sa part augmentait sa réputation; je me disais: «Le jour où les yeux de ses admirateurs s’ouvriront, sa chute sera aussi grande que celle du soleil tombant du firmament.» Montant de grade en grade, il passa par-dessus les cadavres de ses supérieurs; au plus chaud de la bataille, notre colonel tomba frappé, et mon cœur se mit à battre affreusement, car Scorosby allait prendre sa place. «Pour le coup, pensai-je, avant dix minutes nous serons tous perdus.»
Le combat fut acharné; sur tous les points du champ de bataille les alliés lâchaient pied. Notre régiment occupait une position de la plus haute importance et la moindre bévue pouvait tout perdre. A ce moment critique, notre fatal insensé fit quitter au régiment la position qu’il occupait, et le lança à la charge contre la colline opposée où on ne voyait pas trace d’ennemis.
«C’est la fin de tout, pensai-je cette fois. Le régiment s’ébranla; nous avions franchi le faîte de la colline avant que ce mouvement insensé ait pu être découvert et arrêté. Nous trouvâmes de l’autre côté une armée russe de réserve au grand complet, dont personne ne soupçonnait l’existence. Qu’arriva-t-il? Nous avions 95 chances sur 100 d’être massacrés. Mais non, les Russes en conclurent que jamais un seul régiment ne se serait hasardé dans une passe aussi dangereuse; ce devrait être l’armée anglaise tout
entière! Se croyant bloqués et découverts, les Russes firent demi-tour, repassèrent la colline dans un affreux désordre. Nous les serrions de près dans notre poursuite; arrivés sur le champ de bataille, ils se heurtèrent au gros de l’armée ennemie; ce fut un chaos et une confusion épouvantables, et la défaite des alliés se transforma en une éclatante victoire. Le maréchal Canrobert contemplait ce spectacle avec ravissement, émerveillé, trépignant de joie. Il fit appeler Scorosby, l’embrassa et le décora sur le champ de bataille en présence de toutes les troupes.
Quelle avait donc été la fameuse bévue de Scorosby? Il avait tout bonnement pris sa droite pour sa gauche, et rien de plus.
Il avait reçu l’ordre de se porter en arrière pour soutenir notre droite; au lieu de cela, il chargea en avant et escalada la colline par la gauche. Il acquit ce jour-là la réputation d’un grand génie militaire; la gloire de son nom répandue dans tout le monde brillera dans les annales de l’histoire. Aux yeux de tous, c’est un homme bon, doux, aimable et modeste, mais, en réalité, il est au-dessous de tout comme incapacité. Une veine phénoménale l’a servi jour par jour, année par année. Pendant un demi-siècle il a passé pour un soldat des plus brillants; sa carrière militaire est émaillée d’un nombre incalculable de bévues, cela ne l’a pas empêché de devenir chevalier, baron, voire même lord; voyez plutôt sa poitrine, elle est constellée de décorations. Eh! bien, monsieur, chacune de ces décorations représente une gaffe colossale; prises dans leur ensemble, elles constituent nettement la preuve qu’avant tout, pour réussir en ce monde, il faut être né «veinard»!
ACHEVÉ D’IMPRIMER
DE FRANCE
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