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Donglu Zhang, PhD, is a senior scientist in the Biotransformation Depart-ment at Bristol-Myers Squibb. His research interests include metabolite characterization, drug metabolism enzymology, LC/MS methodologies, and microbial biotransformation.

MINGSHE ZHU, PhD, is a drug metabolism scientist with more than ten years of experience in drug discovery and development. His research interests include metabolic activation, LC/MS technology, and regulatory drug metabolism.

W. Griffith Humphreys, PhD, is the Director of the Biotransformation Department at Bristol-Myers Squibb. His research interests include optimizationof ADME properties, metabolic activation, and new methodologies for metabolite characterization.

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The essentials of drug metabolism vital to developing new therapeutic entities

Information on the metabolism and disposition of candidate drugs is a critical part of all aspects of the drug discovery and development process. Drug metabolism, as practiced in the pharmaceutical industry today, is a complex, multidisciplinary field that requires knowledge of sophisticated analytical technologies and expertise in mechanistic and kinetic enzymology, organic reaction mechanism, pharmacokinetic analysis, animal physiology, basic chemical toxicology, preclinical pharmacology, and molecular biology. With chapters contributed by experts in their specific areas, this reference covers:

• Basic concepts of drug metabolism

• The role of drug metabolism in the pharmaceutical industry

• Analytical techniques in drug metabolism

• Common experimental approaches and protocols

Drug Metabolism in Drug Design and Development emphasizes practical considerations such as the data needed, the experiments and analytical methods typically employed, and the interpretation and application of data. Chapters highlight facts, common protocols, detailed experimental designs, applications, and limitations of techniques.

This is a comprehensive, hands-on reference for drug metabolism researchers as well as other professionals involved in pre-clinical drug discovery and development.

Fragmento. © Reproducción autorizada. Todos los derechos reservados.

Drug Metabolism in Drug Design and Development

John Wiley & Sons

Chapter One

1.1 INTRODUCTION

It is interesting to contrast contemporary pharmaceutical biotransformation with that practiced by R.T. Williams. The fundamental objectives are virtually unchanged, to characterize the disposition of a drug in animals. In addition, then and now the routes of excretion and overall molecular transformation are still, arguably, the most important aspects of the discipline. However, in the intervening years the scope of technological advancement, scientific breadth of knowledge, and range of impact has expanded in a manner that could not have been foreseen. This chapter will give an overview of biotransformation as it is practiced in the pharmaceutical industry today.

The role of any pharmaceutical biotransformation scientist is to characterize the disposition of a drug to relate this to overall safety and efficacy. The range of information needed to characterize overall disposition is so broad that it is unlikely any single scientist will accomplish the entire characterization alone. However, it is critically important that the entire disposition process is thoroughly understood, and then intelligently integrated with other pertinent aspects of the drug's behavior. The history of contemporary pharmaceutical industry is replete with examples of how the lack of fundamental scientific knowledge (e.g., mechanism and effects of enzyme induction), appreciation of known metabolic effects (e.g., metabolic activation to toxic reactive metabolites), or incomplete integration of existing information (e.g., drug-drug interactions) led to drastically adverse outcomes. It could be argued that proper integration of information is both more difficult and important than the process of collecting the data itself. Thus, the challenge to the scientist today is to be able to comprehend decades of scientific knowledge, master an array of sophisticated technology, and integrate a diverse range of information to form a sound understanding of a drug's ultimate clinical behavior.

1.2 TECHNOLOGY

There is now an awe-inspiring array of technology available to aid the study of drug disposition. Consider that what once may have taken Williams nearly 6 months to accomplish, might only take about 20 min for a contemporary biotransformation scientist. This modern armamentarium has done much to integrate the power of biotransformation into pharmaceutical discovery and development. However, this tremendous evolution in technology presents its own set of dilemmas.

Taking full advantage of any technology requires an understanding of the technology itself. Fortunately, software and hardware engineering have greatly simplified common use of very sophisticated technologies. The LC/MS/MS instrument today is as common as the HPLC diode array UV instrument 15 years ago. This easy accessibility was greatly facilitated through robust instrument design and great software engineering.

Increasingly, the dilemma is not so much instrument access, as it is a thoughtful choice of exactly what experimental approaches and technology should be chosen to answer the question at hand. The biotransformation scientist is obliged to stay aware of technological innovations of all sorts, including instrumentation. However, the ultimate challenge should always be how to answer the most critical questions in the soundest way. True mastery of technology allows the scientific approach to follow naturally. The temptation to throw technological "sleights of hand" at a problem is often hard to resist.

Every technology has its inherent limits. Often, the specificity that enables prodigious sensitivity can also be a powerful filter of other important information. A rigorous biotransformation scientist is able to stand back and thoughtfully interrogate the strength of her own conclusions, including the technological blind spots of the approach. With thoughtful consideration, complementary technology may be applied judiciously to either flesh out a previous area of ambiguity or address the question from an entirely different perspective. In either case, scientific credibility is served well.

1.3 BREADTH OF SCIENCE

1.3.1 Chemistry

Biotransformation is fundamentally a chemical process. Likewise, the most frequently employed and valuable studies make heavy use of analytical and bioorganic chemistry. Over time, the underlying technology has become sufficiently complex that subspecialization in individual analytical techniques is common. For example, nuclear magnetic resonance spectroscopy (NMR) is invaluable for many unambiguous metabolite structural assignments. In most pharmaceutical companies, NMR specialists are employed to completely master the various facets of the technology. In many cases, these scientists will create sophisticated coupling and decoupling sequences to provide highly specific structural information. Often, their training also makes them most qualified to interpret all forms of NMR spectroscopic data. However, the "complete" biotransformation scientist will, at a minimum, know how to employ NMR spectroscopy to advance their structural understanding of a metabolite. Increasingly, the use of heteronuclear decoupling experiments is considered almost routine in the art.

Furthermore, biotransformation scientists are often fully capable of interpreting the spectra to deduce structure and are also able to recognize when such spectra still leave absolute structural assignments tentative. When one then considers the broader range of additional spectroscopic and chromatographic techniques employed in biotransformation studies, one soon recognizes the degree of technical sophistication required to be an effective biotransformation scientist.

Often, the definitive elucidation of a molecule's metabolic pathway is considered the ultimate goal of biotransformation studies. Proper application of analytical techniques, for the most part, will often be sufficient to achieve this goal. However, as often as it is "good enough" to simply define what has happened to a molecule, there are probably twice as many instances where it is also important to understand how these changes happened. The best biotransformation scientists are usually good "electron pushers." That is, their knowledge of bioorganic chemistry allows them to understand the mechanism of the molecular rearrangements taking place in each biotransformation process. They are able to both rationalize most biotransformations in a mechanistic sense and recognize when a proposed metabolite structure seems untenable. It is not uncommon to encounter a set of spectroscopic data that seems quite inconsistent with the parent molecule. In these cases, the fundamental principles of bioorganic chemistry are employed to rationalize putative structures that would be consistent with the data.

Increasingly, the roles of medicinal chemists and biotransformation scientists intersect in the discipline of bioorganic chemistry. Frequently, they share a mutual interest in decreasing metabolic liability through structural modification as well as avoiding creation of reactive metabolites through informed molecular design. Fortunately, their common understanding of bioorganic chemistry also greatly facilitates the intelligent redesign of structures to mitigate these liabilities. At its best, this requires the best of both disciplines and each scientist can develop a deeper fundamental understanding of the other's craft.

1.3.2 Enzymology and Molecular Biology

Although each of these disciplines could be discussed separately, for the contemporary biotransformation scientist these areas are intimately intertwined. Since biotransformations are enzyme mediated, complete understanding of xenobiotic disposition is only achieved when one also considers the role and impact of the individual enzymes involved.

Enzymological techniques allow the study of individual enzymatic reactions as well as the role of individual enzymes in complex systems. Each of the questions "What happens?" "What enzymes contribute?" "How does it happen?" will require separate techniques. It is not unusual to ask and answer these questions in a very short period of time. This obviously requires a certain degree of breadth, versatility, and flexibility along with a fundamentally strong understanding of the literature.

Cells and subcellular fractions from humans and many preclinical species are readily available. These reagents make it possible to make interspecies extrapolations easily. At one time, a major reason cited for early drug attrition was pharmacokinetic failure, attributable to the difficulty in extrapolating pharmacokinetic behavior from animals to humans. In this author's experience, unexpected pharmacokinetic performance in humans is now a rare event. In addition, it is now commonplace to obtain very mechanistic information revealing the probability of observing quite specific molecular events (e.g., toxicity) in humans (Mutlib et al., 2000).

While the availability of trans-species enzyme systems has had a major impact, advances in molecular biology have also enabled the query of increasingly sophisticated questions. Molecular biological methods have made it possible to clone and express enzymes to study reactions at a molecular level. This has improved our ability to study enzyme reactions at a fine molecular level, to discern the contributions of individual enzymes in complex systems, and even to employ them as "bioreactors" to generate small quantities of metabolite standards.

The basis for many metabolizing enzyme polymorphisms is becoming better understood, allowing one to anticipate potential interindividual disposition differences. Molecular biological techniques have defined the basis for polymorphisms and have described the distribution of the variants in a population. It is now quite easy to discern whether a drug may behave differently in one individual compared to another and to even exclude anticipated poor responders from trials in a controlled fashion (Murphy et al., 2000).

The means by which enzyme systems are regulated are now being appreciated and studied in a mechanistic fashion. Tools available today make it possible to screen against enzyme inducers as well as inhibitors in a relatively inexpensive, well-defined fashion.

1.4 IMPACT OF DRUG METABOLISM ON EFFICACY AND SAFETY

Even in the simplest case, a drug that is injected intravenously and excreted completely unchanged in the urine, there are likely important implications to the human risk/benefit evaluation. Is the excretion so fast that efficacy is compromised? Will the dose need to be adjusted in patients with compromised renal function? Will high drug concentrations in the urinary tract lead to important safety concerns? The biotransformation scientist who only asks "What is happening?" without "What could it mean?" is missing an opportunity to play a larger role in making important decisions. In fact, it can be argued that the biotransformation scientist is perhaps best suited to raise these concerns and is neglecting a critical aspect of their profession by not leading these discussions.

1.4.1 Efficacy

At the earliest stages of drug discovery, an important transition must be made from the screening well to the functioning cell. Even at this stage, there are often significant hurdles related to biotransformation. At every step along the way to higher levels of biological organization, biotransformation inevitably imposes further challenges to the goal of therapeutic efficacy. Understandably then, significant time and resource in drug discovery is spent optimizing a molecule's disposition properties. Perhaps it is more precise to say that much effort is put into the overall process of molecular optimization to yield a molecule with acceptable disposition properties. This distinction, though subtle, is critically important. For once a molecule is made, its properties are cast and its biological fate cannot be changed. Thus, it is critically important in drug discovery to get the optimization done right.

Few would argue that molecular optimization to achieve adequate pharmacokinetic properties is a high priority in early discovery. Practically speaking, much of this work could be accomplished with little biotransformation insight. By using in vitro and/or in vivo models, a chemistry team may certainly achieve the necessary degree of optimization. However, even when the optimization comes as a result of a well-developed sense of SAR, one recognizes that substantial amounts of intuition and good fortune were also necessary. Luck is fleeting and intuition has its limits. This is particularly true when there is little baseline data and the problem is complex. Thus, a purely empirical pharmacokinetic approach is not likely to be the most efficient path for success.

Pharmacokinetic optimization can be greatly aided with further biotransformation information. A limited disposition study can be extremely useful. Simply looking for intact drug in urine and bile, one may be able to discern significant clearance by renal or biliary excretion. Neither of these disposition routes are normally modeled by high throughput in vitro clearance assays. One may quickly learn that information from in vitro screening is not likely to have the desired benefits. Unfortunately, given current state of knowledge of transporter ligand affinity, screening in these instances is likely to remain a largely "black box" screening effort with in vivo models.

A simple study designed to identify biotransformation "hot spots" is frequently invaluable during pharmacokinetic optimization. Samples from either in vitro or in vivo studies analyzed by HPLC with parallel UV/MS detectors can often quickly identify those aspects of a chemotype most susceptible to metabolism. Now the challenge becomes an exercise of molecular modifications, informed by knowledge of the area of the molecule needing attention.

1.4.2 Safety

By definition, xenobiotic metabolism considers how an organism disposes of a foreign chemical. It is the study of what the body does to the drug. Whether intentional or unintentional, these xenobiotics often have physiological effects. Thus, a major role for biotransformation is to understand how metabolic processes terminate or limit desired physiological effects (efficacy) as well as how other processes may lead to unintended consequences (toxicity).

A drug's duration of action, its intensity of action, and interindividual variability in responsiveness are frequently related to its disposition properties. For drugs with a narrow therapeutic index, these sources of variability can and do lead to adverse effects and may significantly limit the full therapeutic usefulness of the product. Likewise, drug-drug interactions also lead to unintended effects. As an inhibitor or inducer of enzymes involved in the disposition of other co-medications the drug may cause exacerbated pharmacological effects (inhibitors) or therapeutic lapses (inducers). Again, drugs of this nature may have severely restricted use, depending on the therapeutic utility and the co-medication environment in which they would be used. Thus, without even considering how a drug is metabolized, safety can be affected.

Dr. James Gillette, the Millers, their coworkers and colleagues, and generations after them have documented how molecular biotransformation leads to toxicity (Brodie et al., 1971; Miller and Miller, 1955). Molecular activation (or biological reactive intermediates) is one of the most intensively studied aspects of both drug metabolism and toxicology. Thousands of publications have documented the breadth of reactions leading to reactive metabolites, and thousands of others have shown the breadth of impact throughout the body and among all species. Consequently, there is a well-developed basis for anticipating structural features that may predispose a molecule to form reactive metabolites. Once discovered, reactive metabolites can often be avoided or minimized by judicious molecular redesign. In fact, both biotransformation scientists and medicinal chemists are obligated to know this area. This knowledge facilitates design of molecules without known liabilities, or at least guides the incorporation of certain worrisome features in a way that can be carefully evaluated.

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