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Advances in in vitro drug metabolism screening

Peter J. Eddershaw and Maurice Dickins
Developments in automation, analytical technologies and molecular biology are being exploited by drug metabolism scientists in order to provide enhanced in vitro systems for the study of the metabolic disposition of potential drug candidates. Routine investigation of factors such as metabolic stability and induction and inhibition of drug metabolizing enzymes is now preferred in the early stages of drug discovery. This, in turn, should provide a greater understanding of the underlying principles governing these processes and allow a greater role for drug metabolism in the design of new drug molecules. genomics giving rise to ever increasing numbers of therapeutic targets, and the routine use of combinatorial chemistry and high throughput screening to address these targets, drug metabolism groups within the pharmaceutical industry now face a new round of challenges1. The ability to synthesize and identify larger numbers of potent and selective molecules means that pharmacokinetic properties are increasingly the major criteria in the selection of lead compounds for further investigation. Unfortunately, the small amounts of material available and the time constraints that apply to most projects conspire against many of the traditional approaches used to generate such data. Furthermore, there is a growing desire to address issues such as inhibition and induction of drug metabolizing enzymes during the discovery stage.This is in an attempt to decrease the attrition rate of compounds in the later, clinical phase of development.Therefore there is an urgent need for improved in vitro systems that can provide timely metabolic information on much larger compound sets than is currently possible and prevent drug metabolism studies from becoming the bottleneck of the drug discovery process. It is not the intention to provide an exhaustive review of available in vitro systems for the study of drug metabolism, but rather to highlight some of the key advances being made in this field, from the authors personal perspective of developing and using in vitro systems within the drug discovery environment. To screen or not to screen? There has been some debate within the drug metabolism community regarding the desirability, or otherwise, of high throughput drug metabolism screening. There has been considerable support for the view that such approaches de-intellectualize the process of candidate optimization
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Peter J. Eddershaw and Maurice Dickins* Bioanalysis and Drug Metabolism Glaxo Wellcome Research and Development Park Road Ware UK SG12 0DP *tel: 44 1920 882046 fax: 44 1920 884374 e-mail: MD45773@ glaxowellcome.co.uk

w In recent years, there has been increasing recognition within the pharmaceutical industry that a successful medicine is not only potent and safe, but one which must also possess a pharmacokinetic profile that has been optimized for the given therapeutic target. The need for this balance, coupled with the increasing economic pressure to remove sub-standard drug candidates at an early stage, is reflected in the marked expansion of the role of what were previously considered Development disciplines into the earlier phases of the discovery process. Increased role of drug metabolism in drug discovery Challenges to existing practices In contrast to other associated disciplines such as toxicology and pharmaceutics, drug metabolism has been involved in early lead-optimization for some time. This involvement has been greatly enhanced by the development of in vitro systems that complement existing in vivo techniques, and provide information on factors such as metabolic stability and production of active metabolites within a timeframe that is consistent with the iterative cycle of the typical research project. However, with the well-documented advances in

1461-5347/99/$ see front matter 1999 Elsevier Science. All rights reserved. PII: S1461-5347(98)00108-4

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and should therefore be resisted. This might seem a somewhat surprising response in view of the points outlined above, and may reflect confusion over the definition of high throughput as applied to drug-metabolism studies, tinged with the natural and, some would say, healthy scepticism of most research scientists towards so-called revolutionary advances. On the issue of what constitutes high throughput, it is unlikely that the measurement of properties such as metabolic stability and enzyme inhibition will provide sufficient value during the lead identification process to warrant the development and implementation of primary screens, capable of handling tens of thousands of compounds per week. At this stage, the main aim is to identify compounds with some semblance of activity (hits), which can then be subjected to further chemistry to convert them into possible drugs (leads). It is in this latter phase that consideration of metabolic disposition becomes important, and thus the likely target capacity for drug metabolism screens is of the order of hundreds of compounds per week rather than thousands. This represents an increase of approximately 10100-fold over existing practices, which can be achieved relatively painlessly by harnessing standard automation and analytical technologies. Indeed, the increasing use of such methods by various groups within the pharmaceutical industry, including some who also purport to reject the high throughput approach to drug metabolism, would appear to highlight the confusion regarding what actually constitutes high throughput in this area! More importantly, the charge of de-intellectualization not only does disservice to the scientists involved in producing ingenious and often elegant solutions to screen design, but fails to appreciate the enormous opportunities for increasing our understanding of the physicochemical and enzymological factors that govern drug metabolism provided by such systems. The ability to study large, diverse compound sets using well defined and controlled methods provides quality-assured data that can be used to develop computational models that describe various aspects of drug metabolism. In this way, the drug metabolism scientist can have a much greater intellectual influence on the drug design process than has hitherto been possible. Use of in vitro systems In vitro systems have become an integral part of drug metabolism throughout the drug discovery process. The advantages of speed, reduction in the use of live animals, and the ability to investigate specific aspects of the metabolic disposition of a compound, often in a human-derived preparation, have been well documented24. This approach is particularly attractive when dealing with large numbers of compounds and where only limited amounts of these compounds are available,
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because both factors make routine in vivo testing difficult. In addition, the use of human tissue and recombinant human enzymes can prove useful for the later stages of drug development. In this situation, prior knowledge of factors such as isoform selectivity and enzyme induction or inhibition can be used to focus clinical studies in volunteers and obviate the need to perform vast numbers of unethical and expensive clinical interaction studies. The search for improved systems that can meet the described requirements for increased throughput has focussed mainly on the automation and miniaturization of existing methodologies. Novel approaches to the study of drug metabolism are also beginning to emerge, but it is worth stressing that any improvements in throughput are worthless unless they are supported by rigorous and continued validation of the overall screen performance. Moreover, the decision to employ a screen within a drug discovery project must come from a rational appraisal of the project requirements, rather than simply because that screen is capable of providing the desired throughput. This latter principle is explored in more detail by Rodrigues5 as part of the automation, validation, integration and database management (AVID) strategy for preclinical drug metabolism. Determining metabolic stability Metabolic stability is probably the most important factor affecting the progression of potential lead compounds. This is because it can significantly reduce the efficacy of otherwise potent molecules. As a result, in vitro methods for the study of stability are probably the most well established of drug metabolism systems. For this reason, recent advances have tended to focus on improvements in analysis and automation of basic incubation systems. In vitro systems provide an efficient means of ranking a series of molecules according to their inherent metabolic stability, in the absence of complicating factors such as blood flow, protein binding and absorption.This simplicity can conversely become a disadvantage when trying to predict in vivo disposition from in vitro studies, although findings both inhouse and from elsewhere6 suggest that reasonable estimates of in vivo half-life in humans can be obtained from simple in vitro metabolism data, albeit on limited compound sets. There are two main components to any in vitro screen for metabolic stability; namely, a system for metabolizing the compounds and a means of analysing the metabolic reaction, and it is now possible to automate both of these systems. However, for efficient routine operation and full integration into the drug discovery project environment, it is essential that systems are in place for handling the large amounts of information being supplied both to the screen (for example, compound identities, structures and concentrations) and from the screen to a suitable database.

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Choice of metabolizing system The relative merits of the various cellular and sub-cellular systems available for the performance of conventional in vitro studies have been well documented3, although the use of such systems as part of a metabolism screen gives rise to certain additional factors that require consideration when selecting the most appropriate system for use. Nevertheless, to reinforce the earlier point, it is vital that the selection of the appropriate drug metabolizing system is rationalized in terms of the specific questions being asked of the screen, rather than because a given system provides the easiest means of operation. Routine screen operation requires a ready supply of the chosen metabolizing system, ideally an off the shelf system that can be stored frozen for a significant period of time and used on demand. At present this tends to favour the use of sub-cellular systems, such as liver microsomes or homogenate, because these can be prepared in large batches and stored frozen for many months with little loss of activity. However, developments in cryopreservation techniques, such as those described by Loretz et al.7 and Li (presentation at 12th International Symposium on Microsomes and Drug Oxidations, Montpellier, France, 1998) have facilitated the routine use of hepatocytes or liver slices. The use of microsomes limits the range of drug metabolizing activities to essentially the cytochrome P450s (CYPs), flavin-containing monooxygenases (FMOs) and the glucuronosyltransferases (UGTs). However, as these enzymes are responsible for the metabolism of a large number of drugs and other xenobiotics, they are often sufficient for routine screening of compound sets. Cellular systems offer the full complement of hepatic drug metabolizing enzymes and retain a higher degree of organization than sub-cellular preparations. For instance, Phase I and Phase II systems may operate in concert to improve the overall metabolism of a given compound and to provide a means of generating both intermediate and ultimate metabolites for kinetic studies. In addition, the presence of a cell membrane in cellular systems means that the access of compounds to drug metabolizing enzymes is more akin to the in vivo situation, and may therefore provide better predictive models for human drug disposition. Unfortunately, the current paucity of data generated in either hepatocytes or liver slices has prevented any firm conclusions on this point8. Nevertheless, the development of in vitro systems that retain the practical advantages, whilst more closely reflecting the dynamics of the in vivo situation, is attracting considerable interest within the drug metabolism field at present. For example, although in situ perfusion methods using animals have been used for some time in drug metabolism, the associated practical constraints have precluded their use in routine screening. However, new approaches such as the NaviFlow system for high throughput pharmacokinetic studies (HTPkS,

Navicyte, NV, USA) claim to offer a more practical alternative to simulating in vivo dynamics for in vitro screening purposes. Much of the recent progress in the use of cell-based systems for in vitro drug metabolism has come from the development of bioartificial liver devices, which are used to support patients with acute liver failure prior to transplantation or regeneration9. In these devices, primary hepatocytes or immortalized cell lines are maintained in three-dimensional culture by means of a matrix of semi-permeable hollow fibres that provide continuous media perfusion and oxygenation10. This arrangement greatly improves the overall functionality of the cells compared with conventional, static cell culture and shows good retention of drug metabolizing activity over several days11. Moreover, the increased efficiency of the hollow fibre system makes this approach amenable to miniaturization and thus suitable for use in a screening operation. Species selection In choosing the source of a metabolizing system for in vitro screening, there is a need to balance the increased supply and cost implications of routine operation against the desire to generate information that is most relevant to the human situation. Thus, whilst rat or dog may provide a ready source of tissue, particularly for liver slice or hepatocyte preparations, it would be preferable to use human-derived material in order to develop an improved understanding of metabolic disposition in the target species. In recent years, human liver microsomes (HLM) or hepatocytes have become available from several commercial suppliers and thus the use of human preparations is a feasible option for metabolism screening. Alternatively, the use of higher animal species, such as minipig12 or primate13, which are generally considered to be physiologically closer to human, can provide a more cost effective means of generating useful information. Recombinant human enzymes such as CYPs and UGTs can also be used to good effect in metabolism screening, either as individual enzymes, to provide structureactivity relationship (SAR) information on specific pathways of metabolism, or in combination, to form a composite human that contains isoform activities typically found in human liver microsomes (Fig. 1). This latter approach provides a means of regulating the batch-tobatch performance of the microsomal system to a greater extent than is possible with conventional pooled HLM preparations. Automation and analysis Automation of in vitro drug metabolism studies performed in 96-well microtitre plate format can be achieved relatively easily through the use of a typical robotic sample preparation system.This provides an efficient means of dispensing the various components of the in vitro system, performing incubations, and
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75 Turnover (%)

Figure 1. Comparison of the in vitro turnover of a series of 20 test compounds in pooled human liver microsomes (white) and microsomes containing a mixture of the major (recombinant) human CYPs (black). The extent of metabolism of the compounds was similar in both systems.

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sampling the reaction according to a selected timecourse. The samples can then be prepared for subsequent analysis through the use of a range of standard methods, including simple protein precipitation, on-line solid phase extraction or ultrafiltration. It has been the experience of the authors that the miniaturization of in vitro studies is relatively straightforward, although the degree of agitation of the microtitre plates during incubation must be sufficient to ensure that the samples remain homogeneous throughout the experiment. Streamlining of the incubation process, particularly for large numbers of compounds, means that the analysis of the resultant samples becomes, more than ever, the bottleneck of the overall process. Furthermore, the analytical endpoint needs to be able to distinguish parent compound from as yet unknown metabolites, across the diverse physicochemical properties of large compound sets, and with minimal method development; in essence, a generic detection system. Although no ideal system is yet available, mass spectrometry (MS) undoubtedly offers the closest current technique in terms of applicability, selectivity and speed. The authors have developed a screening system that uses a rapid, single quadrupole MS system to measure test compounds present in HLM incubations (Fig. 2). Whilst by no means generic, this system typically provides an 8090% initial success rate in detecting compounds before specific method development is required. The system operates in atmospheric pressure chemical ionization mode (APCI) and measures the molecular ion of a particular compound, based on the m/z ratio calculated from the molecular formula of that compound. The extent of metabolism is determined from the ratio of parent compound remaining in the test sample to that in the control (without cofactor). In this way, each sample takes approximately two minutes to analyse, providing a total of four minutes analysis time
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per compound (control and active samples). Thus, for a compound library of 80 compounds, it is routinely possible to generate metabolic stability data inside two days, when previously this might typically have taken two weeks. An alternative screening system has recently been described by van Breemen et al.14 This system generates and identifies metabolites from microsomal incubations through pulsed ultrafiltrationMS.The microsomal incubations are performed in an array of custom-built ultrafiltration chambers with a molecular weight cut-off

Compound supply

Robotic sample processor

Bar-coded 96-well plates

Dilutions to give 5 M final concentration

Incubate at 37C for 30 min. Reaction terminated with PCA

Molecular formula; calculate molecular weight Calculate percentage turnover Post to database Determine peak heights Autosampler

LCMS

Figure 2. Schematic of an automated in vitro metabolism screen used in the authors laboratories. The system monitors the disappearance of parent compound in human liver microsomes, relative to that in a control sample (minus co-factor) using a rapid LCMS method. PCA = perchloric acid.

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of 100,000. Each reaction is sampled, in turn, for approximately three minutes and analysed by MS/MS for the presence of parent compound and possible metabolites. Sample throughput is dependent on the number of ultrafiltration chambers used, with 60 devices providing up to 60 assays per hour. Whilst the identification of metabolites can provide useful information to discovery project teams, a more complex system such as this might be more usefully employed after the generation of initial metabolic stability information. This is because even the most cavalier combinatorial chemist would hope to produce a reasonable percentage of stable molecules, producing little or no metabolites. In addition to the general ranking of compounds according to metabolic stability and tracking metabolite formation, automated in vitro screens provide an efficient means of monitoring metabolism rates on larger numbers of compounds than would normally be attempted with conventional methods. By generating larger amounts of kinetic information such as intrinsic clearance, it is possible to develop predictive computer models for the in vivo disposition of compounds and thus improve the drug design and optimization processes. The utility of this approach has been nicely demonstrated by Bouska et al.15, who established a SAR for in vitro glucuronidation of a series of 5-lipoxygenase inhibitors in monkey liver microsomes, and were thus able to identify, in a matter of weeks, a compound that gave an eight-fold improvement in duration of action after oral dosing to humans. Inhibition Drugdrug interactions In the USA, serious adverse reactions to drugs were recently estimated to be in the order of two million every year, of which 100,000 were fatal16. A significant proportion of these adverse drug effects occurred in individuals receiving a combination of pharmaceutical agents.The potential for drugdrug interactions is now studied as a matter of course following the identification of the potentially lethal terfenadineketoconazole interaction, as a result of CYP3A4 inhibition17. Consequently, terfenadine (a prodrug) has now been removed from the US list and fexofenadine, the active metabolite formed as a result of CYP3A4-mediated oxidation, has been substituted as the drug of choice, thus bypassing both the CYP enzyme system and the drugdrug interaction.The recent example of mibefradil, which was withdrawn by its manufacturers because of numerous drug interactions18, has further emphasized the need to investigate potential drugdrug interactions at an early stage. It is important that not only the enzymology of the relevant biotransformations relevant to man be elucidated wherever possible, but also the potency of these effects19. The standard accepted in vitro method for investigating inhibition takes HLM as the enzyme source. This is useful in cases in which more than one CYP may be involved in the metabolism of

a drug at clinically relevant concentrations, such as warfarin (CYPs 1A2, 2C9, 2C19, 3A4). However, the availability of heterologously expressed CYPs has now enabled the development of a system that can investigate the effects of a potential CYP inhibitor in a high throughput screen. Crespi et al.20 have developed a 96-well microtitre plate-format screen using non-specific CYP substrates that are metabolized to readily detectable fluorescent metabolites by individual recombinant CYPs. Potential CYP inhibitors are then studied for their effects on the metabolism of the probe substrates. Selective model inhibitors were shown to have similar Ki or IC50 values to those seen with HLM systems and were selective for the appropriate CYPs21. This system has been used to conduct CYP2D6 enzyme inhibition studies on a series of 62 compounds and has been validated by a comparison with an HLM system that used dextromethorphan (O-demethylation) as substrate21. A similar approach, co-developed by Glaxo Wellcome and Amersham Pharmacia Biotech, allows the rapid screening of compounds for CYP inhibition using the Scintillation Proximity Assay (SPA) format (Hopkins et al., poster presented at the 12th International Symposium on Microsomes and Drug Oxidations, Montpellier, France, 1998). Underivatized yttrium silicate beads are used to bind recombinant CYP microsomes following incubation with a radiolabelled substrate, and the radioactivity associated with the microsomes is measured through the use of scintillation counting.The reduction in this binding due to co-incubation with a test compound provides a measure of inhibitory potential. For some inhibitory drug interactions, potentially relevant metabolites may be generated only by whole cell preparations. For example, the anticonvulsant drug sodium valproate undergoes metabolism by various enzymes in different subcellular compartments; that is, UGT (microsomal), -oxidation (mitochondrial) and CYPs (minor microsomal pathways). Using isolated human hepatocytes, Odishaw et al. were able to show inhibition of lamotrigine metabolism to its major N-glucuronide metabolite22, a clinically relevant in vivo interaction23. Recent work has suggested that the application of both human hepatocytes and HLM offers the best overall approximation of the human in vivo situation24. As with metabolic stability screening, advances in MS have meant that in vitro drug metabolism inhibition studies may be performed with little sample preparation prior to analysis. Furthermore, the application of LC/MS/MS reduces the need for complete resolution of chromatographic peaks, allowing the development of a single system that is capable of measuring a range of metabolites generated from CYP model substrates25. Full scan and selected reaction monitoring (SRM) experiments were generated, and the most abundant product ion was chosen for subsequent SRM-based assays. This method has been used in the study of drugdrug interactions in which the potential inhibitory effects of a new chemical entity are studied using diagnostic CYP substrates.
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Induction Estimation of enzyme induction is an important criterion for drug discovery and development. At present, research efforts are focussing on human systems because of the known qualitative and quantitative differences in response to enzyme inducers between animals and man26,27. For this reason, primary hepatocyte cultures have been widely used since the appropriate receptors, xenobiotic response elements and transcriptional processes are present together only in these cells. However, the use of other cell types is not precluded providing that the necessary receptors and response elements can be cloned and linked to an appropriate reporter gene, which can then be used to detect inducing agents. Primary cultures of hepatocytes The limited availability of good quality human liver tissue has meant that primary human hepatocyte cultures were difficult to use as enzyme induction screens. However, recent advances in the use of sensitive detection techniques have meant that these experiments may be performed using smaller numbers of cells in 24- or 96-well plates, enabling a greater number of experiments to be performed per hepatocyte preparation2729. An alternative approach has been suggested by Maurels group30 in which liver phenotypic characteristics (including the capacity for enzyme induction) are maintained in hepatocytes for at least 35 days. Induction is assessed by following increases in the metabolism of a model substrate. Experiments can be performed that enable the study of a number of compounds using the same cultures a treatment period of up to 96 hours is followed by a drug-free period (wash out) of 72 hours, before beginning the next cycle of drug treatment. Thus, a number of cycles of enzyme induction could be performed using a single set of hepatocyte cultures. Reporter gene constructs The mechanisms for induction of CYPs 1A and 4A are now characterized and have recently been reviewed in the context of in vitro technology31. In the case of CYP1A1, a cytosolic receptor (the aryl hydrocarbon or Ah receptor) is the protein responsible for recognition of the potential inducing agent, whereas in the case of CYP4A, the receptor is a peroxisome proliferatoractivated receptor (PPAR), which is a member of the nuclear steroid receptor superfamily. In both cases, binding of the inducerreceptor complex with a second nuclear receptor (Arnt for CYP1A1 and the retinoic acid receptor, RXR, for CYP4A) results in the formation of a heterodimer complex that can bind to an appropriate regulatory DNA sequence and initiate the transcription process. It should be noted, however, that the induction mechanisms for the major CYPs induced in humans (CYP3A) and rat (CYP2B) are much less clearly understood. An exciting development has been the recent publication of a
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mouse nuclear hormone receptor that is activated by naturally occurring steroids32. This receptor (pregnane X receptor or PXR) has been cloned and shown to be activated by dexamethasone, pregnenolone 16-carbonitrile and other compounds that induce CYP3A1 in the mouse. As with PPAR, activated PXR binds to RXR and then, as a heterodimer, to the conserved direct repeat motifs identified in the 3A gene promoter sequence as steroid response elements.The human PXR (hPXR), which is also expressed predominantly in liver and gut (the major sites of expression/induction of CYP3A4), has now been cloned and shown to bind to the rifampicindexamethasone response element in the CYP3A4 promoter as a heterodimer with RXR (Ref. 33). The hPXRRXR complex is activated by known inducers of CYP3A4 and hence may be utilized as a screen for potential inducers of this important human drug-metabolizing enzyme. Because PXR responds to a diverse set of chemicals that induce CYP3A4, it could be considered to act as a master switch that regulates CYP3A4 (Ref. 34). The cell system that has been devised for the screen is shown in Fig. 3. The elements required for the process are transiently transfected into CV-1 cells. An increase in the activity of the reporter gene in the presence of a test compound, relative to control levels, would indicate a CYP3A4 inducer. The system functions as a measure of increased transcriptional activation and thus indicates whether a chemical has the capacity to induce CYP3A4 in humans. The induction of CYP2B by phenobarbitone (PB) in cultured murine hepatocytes has been difficult to reproduce at

PXR-expression plasmid PXR RXR PXRE Reporter gene

Reporter plasmid Inducer Figure 3. Schematic of a pregnane X receptor (PXR) transient transfection reporter gene system for monitoring interaction of xenobiotics with PXR. CV1 cells (monkey kidney epithelial cells) were co-transfected with the hPXR (human pregnane X receptor) expression plasmid and the pregnane X response element (PXRE) reporter plasmid [PXRE upstream of the minimal thymidine kinase promoter and chloramphenicol acetyltransferase (CAT) gene]. The cells were treated with M concentrations of a number of compounds known to induce CYP3A expression. Induction potential was assayed by measuring activation of the reporter gene CAT.

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M concentrations. However, modification of culture conditions has enabled several groups to achieve induction of CYP2B in vitro in response to PB at the level of mRNA and/or protein3537. Reporter systems that have utilized portions of the 5 -flanking sequence of the PB gene have also been used to investigate CYP2B expression38,39. It is tempting to speculate that a receptor analogous to PXR may be involved in the ability to respond to PB. A suitable candidate is the constitutive androstane receptor (CAR), for which both the mouse and human protein have been cloned4042. CAR is an orphan nuclear receptor, expressed in liver, which binds as a heterodimer with RXR and bears a close resemblance to PXR. A reporter gene assay analogous to that described for the PXR system could thus be devised to test the effects of PB and other compounds on transcriptional activation by this receptor, and hence act as a screen for CYP2B inducers. Because PB induces CYP3A in man but is a relatively weak inducer in the PXR system, CAR may be the receptor involved in the ligand-mediated transcription by CYP3A inducers that do not act primarily via PXR. One drawback of the reporter gene assays in current development is the lack of high affinity ligands. Following the identification of such a ligand for PXR and CAR, development of a scintillation proximity assay would be feasible to measure compound-receptor affinity directly by competitive displacement of labelled high-affinity ligand. Conclusions The advances in in vitro systems outlined in this review are intended to show how the perceived threats of combinatorial chemistry and high throughput screening may be turned to the advantage of the drug metabolism scientist. The utilization of current automation and analytical and molecular technologies is helping to maintain, and indeed enhance, the key role of drug metabolism within the drug discovery process and is consistent with the current industry ethos of fail fast, fail cheap. Moreover, the routine use of in vitro systems, combined with advancing in silico analysis of the large amounts of data generated, should increase understanding of the underlying principles of drug metabolism and may enable small steps to be taken towards a new ethos of fail less. Acknowledgements The authors would like to acknowledge the conceptual and technical role of Dawn Fenton, and the assistance of Andy Harris and Ivin Silver and colleagues in the development of the in vitro metabolism screen described herein. David Bell (University of Nottingham, UK), Steve Kliewer and Amanda Woodrooffe provided assistance in compiling this article, and Alan Beresford, Mike Tarbit and Martin Bayliss provided an expert critical review of the manuscript.

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