Drug Metabolism Reviews ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20 MERITS AND LIMITATIONS OF RECOMBINANT MODELS FOR THE STUDY OF HUMAN P450- MEDIATED DRUG METABOLISM AND TOXICITY: AN INTRALABORATORY COMPARISON T. FRIEDBERG, M. P. PRITCHARD, M. BANDERA, S. P. HANLON, D. YAO, L. A. McLAUGHLIN, S. DING, B. BURCHELL & C. R. WOLF To cite this article: T. FRIEDBERG, M. P. PRITCHARD, M. BANDERA, S. P. HANLON, D. YAO, L. A. McLAUGHLIN, S. DING, B. BURCHELL & C. R. WOLF (1999) MERITS AND LIMITATIONS OF RECOMBINANT MODELS FOR THE STUDY OF HUMAN P450-MEDIATED DRUG METABOLISM AND TOXICITY: AN INTRALABORATORY COMPARISON, Drug Metabolism Reviews, 31:2, 523-544, DOI: 10.1081/DMR-100101934 To link to this article: http://dx.doi.org/10.1081/DMR-100101934 Published online: 05 Dec 1999. Submit your article to this journal Article views: 52 View related articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [US EPA Library] Date: 16 May 2017, At: 12:47 DRUG METABOLISM, 31(2), 523±544 (1999) MERITS AND LIMITATIONS OF RECOMBINANT MODELS FOR THE STUDY OF HUMAN P450-MEDIATED DRUG METABOLISM AND TOXICITY: AN INTRALABORATORY COMPARISON T. FRIEDBERG,*,1 M. P. PRITCHARD,1 M. BANDERA,1 S. P. HANLON,1 D. YAO,1 L. A. McLAUGHLIN,1 S. DING,1 B. BURCHELL,2 and C. R. WOLF1 1Biomedical Research Centre and 2Department of Molecular and Cellular Pathology University of Dundee Ninewells Hospital and Medical School Dundee, Scotland, UK I. BACKGROUND . ................................................ 524 A. Fundamentals of Heterologous Expression in Bacteria . ..... 526 B. Fundamentals of Heterologous Expression in Yeast .......... 527 C. Fundamentals of Heterologous Expression in Mammalian Cells . ....................................................... 528 II. INTRALABORATORY COMPARISON OF MODELS FOR DRUG RESEARCH ............................................... 529 A. Optimization of Expression Strategies . ....................... 529 B. Comparison of P450 Levels and Enzymatic Activities in the Various Models ............................................... 533 * To whom correspondence should be sent. Fax: ϩ441382669993; E-mail: [email protected] 523 Copyright 1999 by Marcel Dekker, Inc. www.dekker.com 524 FRIEDBERG ET AL. C. Use of Cellular Systems Expressing Recombinant P450s as Bioreactors ................................................... 537 D. Use of Cellular Systems Expressing Recombinant P450s for Investigative Toxicology ...................................... 539 III. CONCLUSIONS . ............................................... 541 Acknowledgments . ............................................... 542 References ......................................................... 542 ABSTRACT A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism. Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development. Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions fa- cilitates risk assessment in environmental toxicology. Recently, a variety of re- combinant expression systems has been employed to study the role of human P450s in these reactions. These include insect, bacterial, yeast, and mammalian models. As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently dif®cult. To resolve this problem, we have established and characterized the latter three systems and pre- sent the key results here. In general, the catalytic properties of P450 isozymes in the various models were rather similar. However, taking technical consider- ations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism re- search, including the generation of milligram quantities of metabolites for struc- tural determinations. For toxicological studies, however, expression of P450s in mammalian cells was most appropriate. This is exempli®ed here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs. I. BACKGROUND The notion that warfare is the creator of many things is a lesson which can be drawn from evolution. During the evolutionary process, some organisms de- RECOMBINANT MODELS IN DRUG RESEARCH 525 veloped a wide array of toxins to escape the fate of being eaten; others developed strategies to deal with these toxic substances, mainly by metabolizing them [1,2]. The structural diversity of the compounds requiring detoxi®cation led to the evo- lution of a complex enzyme system which deals with these environmental chemi- cals (xenobiotics). The majority of xenobiotics are lipophilic and cannot be readily excreted. Metabolic pathways have, therefore, evolved which convert lipophilic substances, usually in several steps, into more hydrophilic metabolites. This mainly occurs in the liver and can be divided into two steps. The phase I reactions entail the introduction of a functional group into the lipophilic com- pound. In most cases, this is achieved by the introduction of an oxygen atom into a compound as the ®rst step toward detoxi®cation and elimination [3]. This reaction is mainly catalyzed by cytochrome P450s. In phase II, the resulting highly electrophilic metabolite is conjugated to a hydrophilic nucleophile such as glutathione [4], glucuronic acid [5], sulfate [6], or water [7], making it suf®- ciently water soluble to be excreted in the bile or for transport in the bloodstream and excretion in the urine. Although this cascade of metabolic events usually leads to detoxi®cation, the highly reactive intermediates often formed during this process are, in many cases, more toxic than the parent compound. Even though the P450 system and other drug-metabolizing enzymes evolved to protect against environmental agents, drugs are seen as foreign compounds and are also metabo- lized by these enzymes. The activity of this system therefore in¯uences the phar- macological as well as the toxicological actions of pharmaceuticals. To ensure the maximum therapeutic value and safety of pharmaceuticals in man, the pharmacotoxicological properties of new pharmaceuticals are ®rst tested in animals or in vitro systems which mainly rely on animal tissues. Besides being often considered ethically and economically problematic, animal-based systems have limitations partially due to large species differences in the enzymes involved in drug metabolism [8]. It is, therefore, essential to develop systems which are based on material derived from human tissues. These should signi®cantly shorten the period between the discovery of a drug and its introduction onto the market (Fig. 1). Models for human drug metabolism can be roughly classi®ed into those which try to mimic the entire cascade of drug-metabolizing events and those which try to imitate only a limited set of reactions. The former models (complex systems) include human hepatocyte and hepatoma cell cultures. The latter (simple systems) include heterologous in vivo and in vitro expression as well as enzymes puri®ed from tissues. A disadvantage of the complex models is that drug metabolism is, in all cases, altered as compared to liver, either due to culture conditions or due to phenotypic transformation in the case of hepatoma cells [9,10]. In addition, these models cannot be used to investigate the role of a speci®c isozyme in drug metabolism, because other drug-metabolizing enzymes, which may contribute to the metabolism of a drug, are present in hepatocytes. 526 FRIEDBERG ET AL. FIG. 1. Flowchart showing the applications of expression systems produc- ing recombinant drug-metabolizing enzymes in drug development and the bio- technology industry. Simple models are useful for characterizing speci®c steps in the metabolism of a drug and also the enzymes involved. Furthermore, they provide a powerful means to predict drug±drug interactions which may be observed in vivo. These systems also yield recombinant enzymes for antibody production, which allows quantitation of drug-metabolizing enzymes in human tissues and antibody inhibi- tion studies in complex systems to be made. Combined with the knowledge of the role of a particular enzyme in drug metabolism, consequences of polymor- phisms in drug metabolism can be predicted. This knowledge is essential for the individual adjustment of clinical drug treatment regimens. Heterologous expres- sion systems which provide large quantities of xenobiotic metabolizing enzymes are often a prerequisite for the structural study of these proteins. A. Fundamentals of Heterologous Expression in Bacteria Escherichia coli has been most frequently used for the bacterial expression of human drug-metabolizing enzymes. E. coli is an attractive system because high levels of expression as well as growth to very high cell densities can be achieved [11]. In addition, E. coli is easily manipulated and a wide variety of strain variants and vectors with powerful promoters are available. A limitation of the bacterial systems is that, in almost all cases, mammalian cDNAs have to be modi®ed before they can be expressed [11]. Aside from trimming the 5′ and RECOMBINANT MODELS IN DRUG RESEARCH 527 3′ untranslated regions of the P450 cDNAs, the region around the initiation codon of protein