I. Introduction

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Drug research encompasses several diverse disciplines united by a common goal, namely the development of novel therapeutic agents. The search for new drugs can be divided functionally into two stages: discovery and development. The former consists of setting up a working hypothesis of the target enzyme or receptor for a particular disease, establishing suitable models (or surrogate markers) to test biological activities, and screening the new drug molecules for in vitro and/or in vivo biological activities. In the development stage, efforts are focused on evaluation of the toxicity and efficacy of new drug candidates. Recent surveys indicate that the average new chemical entity taken to market in the United States requires 10 to 15 years of research and costs more than $300 million.

Once the target enzyme or receptor is identified, medicinal chemists use a variety of empirical and semiempirical structure-activity relationships to modify the chemical structure of a compound to maximize its in vitro activity. However, good in vitro activity cannot be extrapolated to good in vivo activity unless a drug has good bioavailability and a desirable duration of action. A growing awareness of the key roles that pharmacokinetics and drug metabolism play as determinants of in vivo drug action has led many drug companies to include examination of pharmacokinetics and drug metabolism properties as part of their screening processes in the selection of drug candidates. Consequently, industrial drug metabolism scientists have emerged from their traditional supportive role in drug development to provide valuable support in the drug discovery efforts.

To aid in a discovery program, accurate pharmacokinetic and metabolic data must be available almost as early as the results of the in vitro biological screening. Early pharmacokinetic and metabolic evaluation with rapid information feedback is crucial to obtain optimal pharmacokinetic and pharmacological properties. To be effective, the turnover rate needs to be at least three to five compounds per week for the support of each program. Due to time constraints and the availability of only small quantities of each compound in the discovery stage, studies are often limited to one or two animal species. Therefore, the selection of animal species and the experimental design of studies are important in providing a reliable prediction of drug absorption and elimination in humans. A good compound could be excluded on the basis of results from an inappropriate animal species or poor experimental design.

After a drug candidate is selected for further development, detailed information on the metabolic processes and pharmacokinetics of the new drug is required by regulatory agencies. The rationale for the regulatory requirement is best illustrated by the case of active metabolite formation. Many of the currently available psychotropic drugs form one or more metabolites that have their own biological activity (Baldessarini, 1990). Pharmacokinetically, the active metabolites may differ in distribution and clearance from that of the parent drug. Pharmacologically, the parent drug and its metabolites may act by similar mechanisms, different mechanisms, or even by antagonism. An understanding of the kinetics of active metabolite formation is important not only for predicting therapeutic outcome, but also for explaining the toxicity of specific drugs.

Conventionally, the metabolism of new drugs in humans is studied in vivo using radiotracer techniques as part of clinical absorption and disposition studies. However, this approach often occurs relatively late in the development stage. Ideally, the metabolism of new drugs should be studied in vitro before the initiation of clinical studies. Early information on in vitro metabolic processes in humans, such as the identification of the enzymes responsible for drug metabolism and sources of potential enzyme polymorphism, can be useful in the design of clinical studies, particularly those that examine drug-drug interactions. It is also desirable that the comparison of metabolism between animals and humans be performed in the early stage of the drug development process to provide information for the appropriate selection of animal species for toxicity studies before these toxicity studies begin.

The advance of in vitro enzyme systems used for drug metabolism studies (Wrighton and Stevens, 1992; Guillouzo et al., 1993; Berry et al., 1992; Remmel and Burchell, 1993; Brendel et al., 1990; Chapman et al., 1993), together with the explosion of our knowledge of various drug-metabolizing enzymes including uridine-diphosphate-glucuronosyl-transferases (Cougletrie, 1992), cytochrome P-450s (Henderson and Wolf, 1992; Gonzalez and Nebert, 1990) and carboxylesterases (Wang, 1994; Hosokawa, 1990), allows us to obtain early information on the metabolic processes of new drug candidates well before the initial clinical studies. In addition, the advent of commercial liquid chromatography-mass spectrometry instrumentation and the development of high-field nuclear magnetic resonance as well as liquid chromatography-nuclear magnetic resonance techniques have further strengthened our capability to study the metabolism of new drugs in the early drug discovery stage (Fenselau, 1992; Baillie and Davis, 1993). However, the role of drug metabolism scientists in drug discovery is more than just screening compounds in vitro and in vivo. It really entails a good understanding of the basic mechanisms of the events involved in absorption, distribution, metabolism and excretion; the interaction of chemicals with the drug-metabolizing enzymes, particularly cytochrome P-450; sources of pharmacokinetic and pharmacodynamic interindividual variability; and the consequences of metabolism on potential drug toxicities.

The purpose of this paper is to review the role of pharmacokinetics and drug metabolism in drug discovery and development from an industrial perspective. The intent is to provide a comprehensive, rather than exhaustive, overview of the pertinent literature in the field. Several excellent review articles on individual topics are available and the reader is referred to the most recent articles in the text. It is hoped that with a better understanding of the fate of the drugs, a balanced in vitro/in vivo approach and an intelligent application of sound principles in pharmacokinetics and enzymology, drug metabolism scientists can contribute significantly to the development of safe and more efficacious drugs.

	II. Role of Pharmacokinetics and Metabolism in Drug Design

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The history of the pharmaceutical industry shows that many important drugs have been discovered by a combination of fortuity and luck. This serendipity is best exemplified by the discovery of isoniazid. Isoniazid was first synthesized by Meyer and Mally (1912). Its antituberculosis properties were not found until 40 years later, when Robitzek et al. (1952) gave isoniazid to 92 "hopeless" patients with progressive caseous-pneumonic pulmonary tuberculosis that had failed to show improvement after any therapy. Furthermore, both indomethacin and ibuprofen compounds were developed as antirheumatic agents even without any knowledge of their mode of action (Shen, 1972; Adams et al., 1969, 1970). The mode of action was established several years after the drugs were on the market when Vane (1971) showed that these nonsteroidal anti-inflammatory drugs acted by inhibiting the synthesis of prostaglandins.

Another example of serendipity is the discovery of anxiolytics. Diazepam and chlordiazepoxide, the most widely used benzodiazepines, were found to have anxiolytic activity in 1958 and were marketed in 1960. Efforts to determine the mechanism of benzodiazepine action were initiated only after their introduction into the clinic. It was not until 1974 that convincing evidence from behavioral, electrophysiological, and biochemical experiments was accumulated to demonstrate that benzodiazepines act specifically at synapses in which -aminobutyric acid (GABA)^b functions as a neurotransmitter (Baldessarini, 1990; Haefely et al., 1985; Williams and Olsen, 1989).

Over the past decades, through a better understanding of disease processes, mechanism-based drug design has evolved and produced drugs that interrupt specific biochemical pathways by targeting certain enzymes or receptors. This approach does not require a knowledge of the three-dimensional environment in which drugs act. Recent advances in molecular biology and protein chemistry have provided pure protein in sufficient quantities to allow structural studies to be carried out. Visualization of these structures by sophisticated computer graphics has made structure-based drug design feasible. These rational approaches of drug design have been successful historically in the fields of HIV protease inhibitors (Vacca et al., 1994), hepatic hydroxymethylglutaryl coenzyme A reductase inhibitors (Alberts et al., 1980) and angiotensin-converting enzyme (ACE) inhibitors (Patchett et al., 1980).

Today, the design of new drugs is still received by many medicinal chemists to mean maximization of the desired drug activity within certain structural limits. Sometimes, however, compounds that show very high activity in vitro may prove later to have no in vivo activity, or to be highly toxic in in vivo models. Lack of in vivo activity may be attributed to undesirable pharmacokinetic properties, and the toxicity may result from the formation of reactive metabolites. Therefore, rational drug design should also take both pharmacokinetic and metabolic information into consideration, and the information should be incorporated with molecular biochemical and pharmacological data to provide well-rounded drug design.

1. Hard drugs. The concept of nonmetabolizable drugs, or so-called hard drugs, was proposed by Ariëns (1972) and Ariëns and Simonis (1982). The hard drug design is quite attractive. Not only does it solve the problem of toxicity due to reactive intermediates or active metabolites, but the pharmacokinetics also are simplified because the drugs are excreted primarily through either the bile or kidney. If a drug is excreted mainly by the kidney, the differences in the elimination between animal species and humans will be dependent primarily on the renal function of the corresponding species giving highly predictable pharmacokinetic profiles using the allometric approach (Lin, 1995; Mordenti, 1985). A few successful examples of such hard drugs include bisphosphonates and certain ACE inhibitors.

2. Soft drugs. In contrast to the concept of hard drugs, Bodor (1984, 1982) and Bodor et al. (1980) have proposed the approach of soft drugs. A soft drug is pharmacologically active as such, and it undergoes a predictable and controllable metabolism to nontoxic and inactive metabolites. The main concept of soft drug design is to avoid oxidative metabolism as much as possible and to use hydrolytic enzymes to achieve predictable and controllable drug metabolism. Most oxidative reactions of drugs are mediated by hepatic cytochrome P-450 enzyme systems that are often affected by age, sex, disease, and environmental factors, resulting in complex biotransformation and pharmacokinetic variability (Hunt et al., 1992; Soons et al., 1992). In addition, P-450 oxidative reactions have the potential to form reactive intermediates and active metabolites that can mediate toxicity (Guengerich and Shimada, 1993). These undesirable effects attributed to oxidative metabolism may be circumvented to some extent by incorporating metabolic structural "softness."

Many of the failures of drug candidates in development programs are attributed to their undesirable pharmacokinetic properties, such as too long or too short t_1/2, poor absorption, and extensive first-pass metabolism. In a survey, Prentis et al. (1988) reported that of 319 new drug candidates investigated in humans, 77 (40%) of the 198 candidates were withdrawn due to serious pharmacokinetic problems. This high failure rate illustrates the importance of pharmacokinetics in drug discovery and development.

To ensure the success of a drug's development, it is essential that a drug candidate has good bioavailability and a desirable t_1/2. Therefore, an accurate estimate of the pharmacokinetic data and a good understanding of the factors that affect the pharmacokinetics will guide drug design. This section includes a discussion of the chemically modifiable factors that influence drug absorption and disposition.

1. Absorption. Drug absorption is influenced by many biological and physicochemical factors. The two most important physicochemical factors that affect both the extent and the rate of absorption are lipophilicity and solubility (Leahy et al., 1989). The membrane of the gastrointestinal epithelial cells is composed of tightly packed phospholipids interspersed with proteins. Thus, the transcellular passage of drugs depends on their permeability characteristics to penetrate the lipid bilayer of the epithelial cell membrane, which is in turn dependent on the lipophilicity of the drugs. As in the example of bisphosphonates, drugs with poor lipophilicity will be poorly absorbed after oral administration (Lin, 1996a). The effect of lipophilicity on oral absorption is best exemplified by the classical study of barbiturates conducted by Schanker (1960). In this study, the absorption of these compounds increased with increasing lipophilicity as a result of increased membrane permeability. Similarly, Taylor et al. (1985) have shown that the absorption rates of a series of -blockers in rat small intestine correlated well with their lipophilicity. However, it should be noted that although there is a correlation between lipophilicity and increased permeability, lipophilicity, in some cases, is not predictive of permeability because of external factors.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure of some HIV protease inhibitors. See table 1 for the substitutions (R). Reproduced with permission from Dorsey et al. (1994).

2. Prodrugs. The prodrug concept was first proposed by Albert (1958). Since then, this approach has been widely used in drug design. Although there are many reasons to use prodrugs, improvement of oral absorption is by far the most common. Antibiotic prodrugs comprise the largest group of prodrugs developed to improve oral absorption (Wermuth, 1984). Pivampicillin, talampicillin, and bacampicillin are prodrugs of ampicillin, all resulting from the esterification of the polar carboxylate group to form lipophilic, enzymatically labile esters. The absorption of these prodrugs is nearly complete (98-99%), whereas that of ampicillin is <50% (Loo et al., 1974; Clayton et al., 1974; Bodin et al., 1975).

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Desoxyacyclovir, a prodrug of acyclovir that is activated by xanthine oxidase. The ultimate target enzyme of acyclovir is the viral DNA polymerase, which is inhibited by the triphosphate metabolite of acyclovir. Reproduced with permission from Krenitsky and Elion (1982).

3. Distribution. The lipophilicity of a drug not only affects its absorption and metabolism but also its binding and distribution. Generally, the higher the lipophilicity of a drug, the stronger its binding to protein and the greater its distribution (Seydel and Schaper, 1986; Toon and Rowland, 1983). In studies with structure-related sulfonamides, Seydel et al. (1973) have shown that there was a strong positive correlation between plasma protein binding of the drugs and their lipophilicity. Watanabe and Kozaki (1978) found that the volume of distribution increased with increasing lipophilicity when administering 15 basic drugs to dogs.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Distribution of drugs with respect to their lipophilicity. Results of a survey of 257 marketed drugs based on available literature information. Reproduced with permission from Taylor & Francis (Jezequel, 1992).

5. Stereoselectivity. Although it has been long known that stereoisomers of a chiral drug often exhibit pronounced differences in their pharmacokinetic and pharmacodynamic properties both in quantitative and qualitative terms, more than 500 drugs are marketed currently as racemic mixtures without relevant pharmacokinetic and pharmacodynamic information for each individual stereoisomer. This neglect of stereochemistry in drug development was widespread until Ariëns' (1984) famous critical review of "sophisticated scientific nonsense" was published. It was Ariëns' review that finally incited drug researchers to consider the importance of stereoselective differences.

	III. Role of Metabolism in Drug Toxicity

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As part of drug development, the safety of a drug candidate has to be evaluated carefully before it can be approved. Due to ethical constraints on performing toxicity studies in humans, relevant safety assessments must be extensively studied in laboratory animals. One of the fundamental challenges drug metabolism scientists face in drug discovery and development is the extrapolation of risk assessment from animals to humans. This extrapolation is far from straightforward. As seen in the marked species differences in metabolism (Lin, 1995; Clark and Smith, 1984), drug-induced toxicity is often species-dependent, both in quantitative and qualitative terms. Some species of experimental animals have such unique mechanisms of developing toxicity that extrapolation of such toxicity assessments to the human situation would be fraudulent (Gregory, 1988; Green, 1991; Boobis, et al., 1990; Park and Kitteringham, 1990). Although there is no single method or model that can extrapolate the toxicity from animals to humans (Boxenbaum et al., 1988), the species differences in toxicity often can be explained by pharmacokinetic or pharmacodynamic effects of drugs. To make an accurate interpretation and a reasonable prediction of potential toxicity in humans, it is important to elucidate the underlying mechanisms responsible for the species differences in metabolism and pharmacokinetics.

From an evolutionary standpoint, all mammals are similar because they originate from a common ancestor, yet they have differentiated as a result of their dissimilar environmental adaptation. Biochemistry provides countless examples of similarities and differences between species, of which one of the most instructive is the structure of cytochrome P-450s. Cytochrome P-450s appear to have evolved from a single ancestral gene over a period of 1.36 billion years. To date, at least 14 P-450 gene families have been identified in mammals (Nelson et al., 1996). Although all members of this superfamily possess highly conserved regions of amino acid sequence, there are considerable variations in the primary sequences across species. Table 2 shows the homology of nucleotide and amino acid sequences between humans and animal species (Kamataki, 1995). Even small changes in amino acid sequences can give rise to profound differences in substrate specificity (Lindberg and Negishi, 1989).

Similar to cytochrome P-450s, uridine diphosphoglucose transferases (UDPGTs) and carboxylesterases also show species similarities and differences. At least 10 rat UDPGTs and 8 human UDPGTs have been defined and characterized to date by cDNA cloning (Clarke and Burchell, 1994). Comparison of the amino acid sequences of all UDPGTs indicates that they share a common C-terminal domain, but the N-terminal half of these isoforms is quite variable. Examination of each of the UDPGT isoforms has revealed that their substrate specificities are different, although they still have overlapping substrate specificities.

Carboxylesterases, enzymes that are widely distributed in the tissues of mammals, hydrolyze drugs containing ester bonds or amide linkages and play an important role in drug metabolism, particularly for ester prodrugs. Hepatic microsomal carboxylesterases exist as multiple isozymes, and there are significant species differences in the activities of the carboxylesterase (Satoh, 1987; Hosokawa et al., 1987). Hosokawa et al. (1990) have compared the amino acid sequences and substrate specificity of purified carboxylesterase from liver microsomes of mice, hamsters, rats, guinea pig, rabbits, and monkeys. Although high (80-95%) homology in amino acid sequences was shown, all carboxylesterases had a different N-terminal amino acid, and their substrate specificities were considerably different.

As a result of the species differences in the amino acid sequences of the isozymes, both the rate of drug metabolism and the metabolite pattern may differ between animal species. Similarly, the response of the enzymes to inducers, inhibitors, and hormones may vary between species due to their enzyme structural differences. This section will discuss the factors with respect to the species differences in drug metabolism.

1. Oxidation and conjugation. Indinavir (MK-639, L-735, 524), a potent HIV protease inhibitor, is subject to extensive metabolism in animals and humans. The major metabolic pathways of indinavir in humans are identified as (a) glucuronidation at the pyridine nitrogen to yield a quaternized ammonium conjugate, (b) pyridine N-oxidation, (c) para-hydroxylation of the phenylmethyl group, (d) 3'-hydroxylation of the indan, and (e) N-depyridomethylation (Chiba et al., 1996). All oxidative metabolites observed in humans also were formed in rats, dogs, and monkeys, whereas N-glucuronide was found only in monkey and human urine (Lin et al., 1996a). An additional metabolite, a cis-2'-3'-dihydroxyindan, was formed in monkeys, but not in other species. The intrinsic clearance (cL_int) (V_max/K_m) of the oxidative metabolism of indinavir was in the rank order: rat (157 mL/min/kg)  monkey (162 mL/min/kg) > dog (29 mL/min/kg) > human (17 mL/min/kg) (Lin et al., 1996a). Clearly, indinavir metabolism is qualitatively and quantitatively different among species.

2. Induction. In the mid-1950s, Conney et al. (1956) showed that the treatment of animals with 3-methylcholanthrene (3-MC) increased the animals' ability to metabolize methylated aminoazo dyes. Remmer (1958) found that tolerance to barbiturate drugs was the result of the enhancement of their own metabolism by induction of cytochrome P-450. Although the phenomenon of induction has been known for over 4 decades, only in recent years, we began to uncover the mechanism involved in induction.