Merits and Limitations of Recombinant Models for the Study of Human P450- Mediated Drug Metabolism and Toxicity: an Intralaboratory Comparison

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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.

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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

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 recombinant 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 present 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 research, including the generation of milligram quantities of metabolites for structural 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 evolution 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 compound. 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 metabolized by these enzymes. The activity of this system therefore in¯uences the pharmacological 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 producing recombinant drug-metabolizing enzymes in drug development and the biotechnology 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 expression 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 biosynthesis should be modi®ed to remove rigid secondary structures, which can occur in mammalian mRNAs. The prerequisite to change the 5′ coding region of a mammalian cDNA for optimal expression in E. coli is exempli®ed by the cDNA modi®cations which were necessary to achieve high levels of functional mammalian cytochrome P450 expression. Initially, it was found that the presence of a hydrophobic cytochrome P450 anchor sequence was deleterious for bacterial expression [12,13]. In addition, it was shown that changing codon 2 to an alanine codon (GCT) as well as removing dG- and dC-rich regions particu- larly in codons 4 and 5 improved the functional expression [14]. A more detailed analysis of the P450 cDNA modi®cations required for functional expression was performed by Gillam et al. [15], where codons encoding the ®rst 35 amino acids of the CYP3A4 sequence were systematically modi®ed by point mutations and deletions. It was found that the native CYP3A4 was synthesized in E. coli at very low levels. Decreasing the potential of the 5′ coding region to form secondary structures as well as deletion of amino acids 3±24 resulted in improved CYP3A4 expression. A combination of both modi®cation strategies was found to be optimal. Similar rules have been applied to the expression of CYP2C9 [16]. It is important to note, however, that the expression of P450s in E. coli is ex- tremely sensitive to the culture conditions and the E. coli strain used. Therefore, expression rules based on such studies as this should be interpreted with caution.

To obtain a functional monooxygenase complex in E. coli, it is necessary to coexpress cytochrome P450 reductase, which is lacking in this organism. Several strategies have been developed to this end. We have expressed several P450s and P450 reductase under the control of two separate promoters [17,18]. Others have achieved coexpression of P450s and the reductase from a single transcription unit [19,20]. A third approach to obtain a functional mammalian monooxygenase system in bacteria is the expression of P450±P450 reductase fusion proteins [21,22].

B. Fundamentals of Heterologous Expression in Yeast

Bakers yeast, Saccharomyces cerevisiae, has been almost exclusively used for the heterologous expression of drug-metabolizing enzymes in yeast. S. cerevisiae has proved suitable for production of a wide variety of eukaryotic proteins for both basic research and industrial and pharmaceutical application [23]. This uni- cellular organism has some of the advantages of the bacterial expression systems and the additional advantage of being a eukaryotic cell with many similarities to mammalian cells in protein synthesis and processing and membrane compart- ment. Two types of yeast vector are available [24]: episomally replicating vectors and vectors with the potential for genomic integration. Most episomal vectors 528 FRIEDBERG ET AL. contain sequences from the 2-µm endogenous yeast plasmid that confer a high copy number and stable maintenance. Integrating plasmids lack a yeast origin of replication but contain regions of homology with the yeast genome ¯anking the cDNA cloning site. The stable integration of an ancillary protein, such as cytochrome P450 reductase, required for functional cytochrome P450 expression [25] increases the versatility of yeast for the subsequent expression of further proteins. S. cerevisiae-based expression biotechnology bene®ts from the availability of numerous selection markers for gene transfer, making it possible to cotransfect several cDNAs. There are also several powerful regulatable yeast promoters which can yield high levels of heterologous protein expression. However, some features of mammalian cDNAs are not optimal for the expression of proteins in S. cerevisiae. It has been shown, for example, that ef®cient expression of several proteins, including cytochrome P450s, requires the deletion of most of the 5′ untranslated region [25]. For example, a CYP1A1 cDNA under the control of the GAL10±CYC1 promoter containing either 15 base pairs (bp) or 5 bp of 5′ untranslated region, yielded 1 µg and 6 µg of functional CYP1A1 per mg of microsomal protein, respectively [26]. Even though it does not appear to be necessary to change the coding region of mammalian cDNAs for their expression in S. cerevisiae, certain mammalian proteins are dif®cult to express unless the amino acid sequence is modi®ed. An example is the rat cytochrome P450 reductase, which could not be stably expressed unaltered in S. cerevisiae. However, upon fusion of the N-terminal sequence of this protein with the N-terminus of the P450 reductase from S. cerevisiae, high levels of the rat P450 reductase were achieved [27]. Coexpression of mammalian P450 reductase and P450s is required to achieve a catalytically highly active monooxygenase system, as the host P450 reductase couples poorly with mammalian P450s [28].

C. Fundamentals of Heterologous Expression in Mammalian Cells

Proteins can be expressed in mammalian cells in a transient or stable manner. This categorization has direct implications for the utility of the resulting models. Transient expression of P450s has been predominantly based on either the use of vaccinia virus [29] or plasmids containing an SV40 origin of replication which leads to plasmid replication in COS 1 kidney ®broblasts. These cells express SV40 T-antigen, which is necessary for the episomal ampli®cation of the plasmid [30,31]. In both systems, multiple extra chromosomal copies of the cDNA per cell are present and, consequently, the chances of achieving high expression of the transgene are good. Expression is transient because the cells are either killed by virally induced lysis (vaccinia system) or because the plasmid is not stably maintained (COS cell system). Transient expression systems have serious limitations for toxicological studies because the cells are already damaged by viral RECOMBINANT MODELS IN DRUG RESEARCH 529 infection or because exposure to the test compounds is longer than the length of expression of the recombinant protein. Mammalian models for the stable expression of proteins have been based on Epstein±Barr virus (EBV), retroviral-mediated gene transfer and on direct gene transfer, and chromosomal integration of the transgene. One to several hundred copies of EBV based vectors can be maintained episomally, increasing the possibility of achieving high levels of recombinant protein [30]. The EBV-based vectors contain selection markers and, therefore, can be stably maintained. The use of this vector is restricted to mammalian cell lines containing the Epstein±Barr nuclear antigen (EBNA) such as the lymphoblastoid cell line AHH-1 [32], which, however, does not contain signi®cant P450 reductase activity. Valuable P450 expression models involving chromosomal integration of the cDNA have also been developed [33±35]. This involves cointegration of a selection marker which allows cells containing the transfected DNA to be selected. Almost all cell lines can be used, the only prerequisite being that a method of gene transfer has been developed. Lymphoblastoid cell lines and V79 Chinese hamster lung ®broblasts expressing P450s have been established as important models in investigative toxicology because they contain easily detectable mutation endpoints [36,37]. Using more recent expression technologies, it is now possible to amplify the genomic copy number of the cDNA, for example, by cotransfection with the dihydrofolate reductase (dhfr) gene. This allows selection for cells harboring ampli®ed copies of the transgene by culturing the transfectants in the presence of increasing concentrations of the dhfr inhibitor methotrexate [38]. Several hundred integrated copies of the cDNA can be obtained by this method, greatly increasing expression levels. Faced with the multitude of recombinant models which have been developed by different laboratories for pharmacotoxicological studies, it is dif®cult to judge their merits and limitations. To address this important issue, we have expressed several P450 isozymes in different organisms and have evaluated the use of the resulting models for pharmacokinetic and toxicological studies.

II. INTRALABORATORY COMPARISON OF MODELS FOR DRUG RESEARCH A. Optimization of Expression Strategies

Most P450 cDNAs were isolated by reverse transcription±polymerase chain reaction (RT-PCR) based on published sequences. The identity of all cDNAs used throughout this work was veri®ed by DNA sequencing. In order to obtain a functional monooxygenase system in bacteria, we have coexpressed the P450s 530 FRIEDBERG ET AL. RECOMBINANT MODELS IN DRUG RESEARCH 531 together with P450 reductase. Expression of unmodi®ed P450 reductase in E. coli was low but signi®cantly improved after fusing the enzyme to a bacterial pelB leader sequence, which, in most cases, directs proteins to the periplasm [18]. Initially, we have coexpressed P450s and P450 reductase from two separate plasmids in E. coli (Fig. 2a). In this approach (two plasmid strategy), an E. coli strain was established which expressed a human P450 reductase fused to a bacterial pelB leader sequence under the control of the IPTG inducible (tac)2 promoter in a vector (pJR7) derived from pACYC184, which confers chloramphenicol re- sistance [17]. This strain was subsequently transformed with the vector pCW containing the various P450 cDNAs under the control of the (tac)2 promoter and the gene-encoding β-lactamase as illustrated in Fig. 2 for the vector NF14 carrying the CYP3A4 cDNA. In a second approach (one plasmid strategy), we have expressed the modi®ed P450 reductase and the P450s both under the control of two separate (tac)2 promoters from pCW as illustrated in Fig. 2 for the vector pB216 carrying the CYP3A4 and the P450 reductase cDNAs [18]. The latter approach has the disadvantage that the cloning procedure is more tedious. How- ever, because the vector pCW has a higher copy number than pACYC 184, the expression level of P450 reductase from the one-plasmid system is typically threefold higher (Table 1). The yield of P450 reductase in these strains can be estimated from its level in bacterial membranes to be approximately 120 nmol P450 reductase/L culture.

Because expression of mammalian P450s in E. coli has been shown to require modi®cations of P450 cDNAs in a segment encoding the P450 membrane anchor [14], we have also tried to circumvent this potentially problematic approach by

FIG. 2. Vectors used for the expression of P450s and P450 reductase in E. coli (a), in S. cerevisiae (b), and in CHO cells (c). The vectors NF14 and pB216 were derived from the vector pCW [14,18] and the vector pJR7 was derived from pACYC184 [17]. In these vectors, the expression of the recombinant protein is controlled by the IPTG inducible (tac)2 promoter. pMA91±P450 is derived from the episomally replicating vector pMA91 [24] and from the integrative vector pFL35 [24]. pDHFR P450 is derived from pcDNA by replacing the G-418 selection marker with the dihydrofolate reductase cDNA [41]. P450s and P450 reductase were modi®ed for bacterial expression using different strategies as exempli- ®ed here for CYP3A4. In one strategy (17α-), P450s with modi®cations within their membrane anchor were expressed [14,18]. Other strategies employed the fusion of P450s and P450 reductase to bacterial (pelB-, ompA-) leader sequences [39]. The ompA- sequence was further modi®ed [ompA(ϩ2)-] to allow more ef®- cient processing by the bacterial signal peptidase. 532 FRIEDBERG ET AL. fusing the P450 sequences in frame to the bacterial ompA leader sequence, which, in the case of ompA±CYP2D6 but not in the case of ompA±CYP3A4, was found to be cotranslationally removed [39]. This prompted us to opimize the region around the cleavage site to allow ef®cient processing by the bacterial signal peptidase [40]. This resulted in the ompA(ϩ2)±P450 fusion constructs (Fig. 2a). Inter- estingly, removal of the leader sequence was essential to achieve high monooxygenase activity. Initial characterization of the membrane topology of the bacterially expressed P450s revealed that the 17α-P450s and the P450s fused to the bacterial leader sequences are mainly located at the cytosolic side of the inner membrane. Generally, we have found that the yield of spectrally active P450 in bacterial membranes was higher with the P450s being expressed from constructs encoding P450s optimally fused to bacterial leader sequences and that, in most cases, the turnover number of these constructs was higher than that of the 17α- P450s. The expression levels, the yield, and the catalytic properties of the human P450s which have been expressed in E. coli are given for the most completely characterized constructs in Table 1. In most cases, the P450 expression levels per milligram of bacterial protein were higher than the level of total P450 in human liver microsomes (ϳ200 pmol/mg microsomal protein), with the exception of CYP2E1. However, we have found that during the isolation of bacterial membranes, CYP2E1 denatured, a process which could be inhibited by the addition of CYP2E1 ligands such as ethanol. The P450 expression levels reported here were, for most P450 isozymes, similar to those published recently for the coexpression of P450s and P450 reductase in E. coli from a single transcriptional unit [19]. However, unlike us, this group ®nds that E. coli producing a low yield of a given P450 isozyme also produced low levels of P450 reductase. For example, where the yield of P450 was low [e.g., CYP1A1 (27 nmol/L)], the level of reductase was also low (16 nmol/L). This yield is about eightfold lower than the level of reductase obtained with our one-plasmid expression strategy. However, we found that for unknown reasons, the levels of P450 reductase were low in strains expressing either the 17α-CYP1A2 or ompA±CYP1A2, even though the level of this P450 was similar to that of CYP3A4 or CYP2D6. We have established a functional monooxygenase system in S. cerevisiae by stable integration of the P450 reductase linked to the 3′ phosphoglycerol±kinase (PGK) promoter into the yeast genome. The resulting strain had a cytochrome c reductase activity of more than 200 nmol/min/mg microsomal protein, which was 20-fold higher than the activity in the parental strain. The recombinant strain was subsequently transformed with an episomally replicating vector containing the various P450 cDNAs under the control of the powerful constitutive PGK promoter (Fig. 2b). In the absence of human reductase, the human P450s were catalytically inactive; however, upon expression of this ancillary factor, CYP3A4 and CYP2D6 displayed an activity toward prototypical substrates such as testosterone and bufuralol, respectively. Interestingly, the expression level of the vari- RECOMBINANT MODELS IN DRUG RESEARCH 533 ous P450 isozymes varied widely, with the level of CYP2C9 being only 91 pmol/ mg microsomal protein, whereas the level of CYP3A4 in microsomes was found to be 500 pmol/mg. As yet, we have not characterized the strain which coexpressed CYP2C9 and P450 reductase for P450-mediated enzyme activity. How- ever, we found that this P450 displayed a diclofenac hydroxylase activity when coexpressed together with a yeast±rat reductase fusion protein [27] in S. cerevisiae (data not shown). Because the integrative vector and the vector (pMA91) containing the P450 cDNAs carried auxotrophic markers (Leu and Trp), it was necessary to select the recombinant strains on media de®cient in these amino acids. However, we discovered that growing these strains subsequently in rich medium increased the yield of P450 by a factor of 3. In order to achieve high levels of human P450s in CHO cells, we used a strategy which allowed the genomic ampli®cation of the P450 cDNA (Fig. 2c). In this approach, the cDNA is inserted into a mammalian expression vector containing the dihydrofolate reductase (DHFR) cDNA and the resulting construct is transfected into DHFRϪ CHO cells. These were subsequently grown on increasing concentrations of the DHFR inhibitor methotrexate. Resistant clones were analyzed by immunoblotting and by enzyme assays for P450 expression. Clones with the highest expression level were then transfected with a mammalian expression vector carrying the P450 reductase and the G-418 selection marker. G-418 and MTX-resistant clones were subsequently analyzed for P450 and P450 reductase expression [41]. Cells which displayed the highest expression level were further analyzed. We found that the level of the various P450 isozymes and of P450 reductase remained stable for several weeks, even in the absence of selective pressure, with the exception of the cell line which expressed CYP2C9. In this cell line, the P450 level was slightly decreased after 2 weeks of continuous culture in selective medium and halved after 4 weeks of culture, thus necessitating either the preparation of a large batch for subsequent experiments or recloning of the cell line to improve P450 levels.

B. Comparison of P450 Levels and Enzymatic Activities in the Various Models

Table 1 displays the P450 isozymes which have been expressed by us in the different models, their expression levels, and their enzymatic characterization. P450 isozymes which have been expressed in at least two systems (bacteria, yeast, and mammalian cells) are highlighted in bold. The levels of recombinant P450s in the different cellular systems were surprisingly similar, taking into account that in mammalian cells, this parameter was determined in total cellular protein which contains approximately 10±20% of protein from the endoplasmic reticu- lum in which most P450 isozymes reside. However, we found that the level of 534 FRIEDBERG ET AL. ) d m M K µ )( 1 Ϫ number b Yield Catalytic activity

TABLE 1 a 70 190 n.d. n.d. n.d. 91 0.9 n.d. n.d. n.d. 210 370 1,200 5.7 11.1 286 498 2,400 8.7 n.d. 204 218300100500 210 63 2.6 4.4 0.3 12,000 n.d. 209 40 104 8.6 69 1.5 11.1 99 P450 expression Turnover c c 2-2E1 2-2C8 1,700 440 n.d. n.d. n.d. 2-1A1 235 170 725 3.6 n.d. 2D6 2C9 - 3A4 -1A2 ±1B1 195 287 180 0.92 n.d. ± ±2A6 190 150 550 2.9 n.d. ± ϩ ϩ ϩ 2C9 2D6 3A4 α α ␣ ␣ 17 17 17 17 ompA ompA ompA ompA Catalytic Activities and Levels of Cytochrome P450s Expressed in Different Models S. cerevisiae Expression level systemE. coli Expressed protein (pmol/mg) ompA (nmol/L) (pmol/min/mg) (min RECOMBINANT MODELS IN DRUG RESEARCH 535 bold. -hydroxy- ′ coexpressing reduced/min/ c reductase activities found S. cerevisiae c 80 7 n.d. reduced/min/mg for the two-plasmid c . -hydroxylase; CYP2D6, bufuralol 1 ′ reduced/min/mg membrane protein. CHO cells c E. coli d reductase activity of 100±200 nmol/min/mg total cellular c

68502018 n.a. n.a. n.a. n.a. 362 974 600 900 6 19 30 50 n.d. 29.8 n.d. 65 11 n.a. (230,000, Ref. 55). c -hydroxylase (5000, Ref. 44); CYP2E1, chlorzoxazone 6-hydroxylase (1500± β not applicable. ϭ -dealkylase (95, Ref. 55); CYP2C9, diclofenac 4 O 2E1 2C9 2D6 3A4 1A2 not determined; n.a. strains coexpressing P450s and P450 reductase (hOR) were typically 1200±1600 nmol cytochrome ϭ : Heterologous expression levels and enzyme activities for P450s in the bacterial and yeast systems were determined The P450 content wasThe determined following spectrophotometrically enzyme and activities have is been expressed determined as for pmol/mg the different membrane P450 protein. isoenzymes (the speci®c activities reported n.d. P450s and the reductase were expressed from the same plasmid in Note a b c d The various P450 isozymes were coexpressed with human P450 reductase. The cytochrome E. coli 4000, Ref. 56); P450 reductase, cytochrome lase (70, ref. 46); CYP3A4, testosterone 6 in literature forCYP1A2, hepatic 7-ethoxyresoru®n microsomes are given in parentheses and are expressed as pmol/min/mg microsomal protein): in in bacterial membranes and microsomes,cells respectively. (catalytic For activity) the or mammaliancells models, in (bacterial these total and values cellular yeast were protein systems). determined P450 (P450 in isozymes cultured level). which The have yield been was expressed calculated in from at the least P450 two content models in are whole shown in CHO ®broblasts mg membrane protein for the one-plasmid system and 400±600 nmol cytochrome P450s and hOR, the levels of reductase were 200±250 nmol cytochrome system with the exception of strains expressing CYP1A2 for which this value was 70±120. In coexpressing P450s and hORprotein typically with displayed the a exception cytochrome of the cell line expressing CYP2C9 for which this value was 25. 536 FRIEDBERG ET AL. some P450s in E. coli can be increased to about 1000 pmol/mg protein when growing the cells in the controlled environment of a biofermenter. This level represents approximately 5% of the bacterial membrane protein and translates into a yield of 1000 nmol P450/L culture. Similarly, it has been shown by others that the yield of P450s in S. cerevisiae can be improved to about 500 nmol/L culture when using vectors that are stably maintained in rich medium, which allows the cells to grow to very high cell densities [28]. CHO cells can be adapted to growth in suspension; however, it is unlikely that P450 yields similar to those obtained with microorganisms can be obtained using this approach. Therefore, E. coli and S. cerevisiae are ideally suited for the production of suf®cient quantities of enzymes for structural studies. The testosterone 6β-hydroxylase and the bufuralol 1′-hydroxylase activity found in membranes isolated from bacteria which expressed CYP3A4 or CYP2D6, respectively, were 2- and 30-fold, respectively, higher than in human liver microsomes (see Table 1). For pharmacokinetic investigations, it is essential that the catalytic properties of the recombinant P450 isozymes are similar across the various recombinant models. The data presented in Table 1 show that the Km values of the CYP3A4- mediated 6β-hydroxylation of testosterone and the CYP2D6-mediated 1′- hydroxylation of bufuralol were highly similar in all three models developed in this work. These values are also in good agreement with the Km reported for CYP2D6 expressed either in the baculovirus system or in human lymphoblastoid cells

which were 4.7 µM and 18 µM, respectively [42,43]. Similarly, the Km of

CYP3A4 expressed in the three models presented here agrees with the Km reported for CYP3A4 expressed in a baculovirus system which was 56 µM [44]. However, in many cases, catalytic properties of recombinant P450s cannot be directly compared with those of P450s in human liver microsomes, because, here, several P450s can contribute to the metabolism of a given substrate. For example, it is known that the bufuralol 1′- hydroxylation is catalyzed by CYP2D6 but also with a lower af®nity by CYP1A2 [42]. Similarly, the testosterone 6β-hydroxylation is not only catalyzed by CYP3A4 but also by CYP3A5 [44]. Generally, it can be said that except for CYP1A2, the substrate turnover numbers of the P450 isozymes expressed in E. coli differed by a factor of less than 4 from those of P450s expressed in mammalian cells. The relatively low turnover number of bacterially expressed CYP1A2 for the O-dealkylation of 7-ethoxyresoru®n is most likely due to the low level of P450 reductase in this particular strain of E. coli, which was at least fourfold lower than in the other E. coli lines. Lower levels of reductase may have also been partially responsible for the low substrate turnover number of CYP3A4 expressed in S. cerevisiae. For this comparison, it should be noted that the level of P450 reductase in mammalian cells was determined in total cellular protein, whereas, in yeast, this enzyme activity was determined in microsomal protein. Another reason for the low enzyme activity of CYP3A4 in yeast membranes could be the absence of cytochrome b5, which RECOMBINANT MODELS IN DRUG RESEARCH 537 has been shown to stimulate the activity of CYP3A4 toward several substrates. It should be noted that another group has coexpressed CYP3A4 together with cytochrome b5 and P450 reductase [45]; however, the turnover number given in that report is similar to our value for the testosterone 6β-hydroxylase activity of CYP3A4.

Interestingly, cytochrome b5 is absent in E. coli, in which CYP3A4 displayed a high catalytic activity. One may speculate that an E. coli protein substituted for cytochrome b5 in stimulating the activity of CYP3A4 [18]. The substrate turnover numbers of the recombinant CYP3A4 and of the recombinant CYP2D6 expressed in either E. coli or in CHO cells appears to be rather similar to the substrate turnover number estimated for these enzymes in human liver microsomes, which are 55 minϪ1 [44] and 10 minϪ1 [46], respectively. However, one caveat in this comparison is that more than one P450 may metabolize a given substrate and that P450 isozymes are dif®cult to quantitate exactly in human liver microsomes.

Because the catalytic properties (Km, turnover number) of the P450 isozymes are rather independent of the cellular system used for their expression, recombinant models could be very valuable in predicting P450-mediated metabolism in humans. However, it remains to be seen whether the substrate speci®cities of the P450 isozymes are preserved in the expression systems. In order to address this question, we, in collaboration with several pharmaceutical companies, are currently assaying various recombinant P450s using a panel of isozyme speci®c substrates.

C. Use of Cellular Systems Expressing Recombinant P450s as Bioreactors

Drug development requires the detailed characterization of metabolic pathways. This type of analysis necessitates the structural characterization of metabolites which are also needed as standard compounds. For these purposes, milligram quantities of metabolites need to become available, a task which until now had to be performed by elaborate chemical synthesis. It is obvious that recombinant P450s are ideally suited for the production of suf®cient quantities of P450 metabolites, provided that the cellular system expresses these enzymes at high levels in a catalytically highly active form. In addition, it is important that the catalysis proceeds for a suf®cient time and that the fermentation system is suitable for scale-up. The latter target is dif®cult to ful®ll with mammalian cellular systems. Our results show that the activity (expressed as pmol/min/mg membrane protein) of either CYP2D6 or of CYP3A4 in membranes isolated from recombinant bacteria were by a factor of 6- and 110-fold, respectively, higher than that of membranes isolated from yeast. In addition, the yield of P450 in E. coli was between 538 FRIEDBERG ET AL.

50- and 100-fold higher than in S. cerevisiae. Thus, at least in our hands, E. coli appears to be more suitable as a bioreactor system than S. cerevisiae, even though it appears likely that under growth conditions which give higher yields of P450 [28], the yeast system could also be employed as biofermenter. An important factor which determines the ef®ciency of cellular systems for the large-scale generation of metabolites is the permeability of the cellular membrane for P450 substrates. Figure 3 shows the time course for the formation of 6β-hydroxytestosterone in intact E. coli DH5α strains expressing CYP3A4 and in membranes isolated from these cells. The initial reaction proceeded in membranes and in intact cells at similar rates. Metabolism in intact cells did not require the addition of NADPH and slowed down considerably only after 3 h. At this time, approximately 500 nmol 6β-hydroxytestosterone were formed per nanomole of P450. From these data and the yield of CYP3A4 given in Table 1, it can be calculated that approxi-

FIG. 3. 6β-Hydroxytestosterone formation by membranes (ᮀ) or intact cells (᭹)ofE. coli expressing CYP3A4 and human P450 reductase. Incubations were carried out in 1 ϫ TSE (membranes) or 1 ϫ M9 salts (intact cells) at 37°C with shaking (100 rpm). Membranes were supplemented with NADPH generating system and intact cells with 10 mM glucose. Reactions were initiated by the addition of testosterone (500 µM). RECOMBINANT MODELS IN DRUG RESEARCH 539 mately 100 µmol corresponding to 30 mg of 6β-hydroxytestosterone were produced per liter of culture. Similarly, linearity of P450-mediated reactions for up to 2 h were also seen using chlorzoxazone and nifedipine as substrates. Presently, we are investigating the reasons for the decrease in substrate turnover after 2 h of cultureÐone possibility being the depletion of NADPH. However, from our data and the data reported by another group [19], it can be concluded that E. coli, which contain a functional P450 monooxygenase system, may soon replace chemical synthesis of metabolites on a semipreparative scale. In the future, these systems need to be optimized for the large-scale production of metabolites which are useful as intermediate products in the synthesis of pharmaceuticals.

D. Use of Cellular Systems Expressing Recombinant P450s for Investigative Toxicology

P450s control important toxication and detoxication pathways of drugs and environmental pollutants. Because the target structures for toxic effects in mammalian cells may be either not present or different in bacteria and other microorganisms, mammalian cells are the ideal model for investigating toxic mechanisms with relevance to humans. This is nicely illustrated by a study designed to investigate the role of P450s in the toxicity of vinca alkaloids. These chemotherapeutic agents are known to bind to tubulin, thereby inhibiting the assembly of microtubules. It was known that vinca alkaloids are metabolized by CYP3A enzymes in human liver microsomes and it was speculated that because the level of CYP3A4 showed a 10-fold interindividual variability, this P450 could be responsible for the interpatient variability observed for the clearance of this drug in man [47]. However, until now, it was not known if P450-mediated metabolism could alter the toxicity of these compounds. In order to address this question, we have determined the cytotoxicity of vincristine in a panel of CHO cells expressing different human P450 isozymes. The mammalian model was chosen for this study, because bacteria do not have microtubules and the composition of this cellular structure in yeast is different from mammalian cells. This could also be the reason why vinca alkaloids are not cytotoxic to S. cerevisiae. The data presented in Fig. 4 show that cells which coexpressed CYP3A4 and P450 reductase were signi®- cantly better protected from the cytotoxic effects of vincristine than the parental cell line and the cell line which expressed P450 reductase alone. A small protec- tive effect was noted in the cell line which expressed CYP3A4 in the absence of human P450 reductase, even though this cell line displayed a testosterone 6β-hydroxylase activity which was 10-fold lower than that of the cell line which coexpressed CYP3A4 together with P450 reductase. Expression of CYP1A2 or CYP2D6 or of only P450 reductase did not decrease vincristine-induced cytotoxicity. It is interesting to note that increasing the concentration of vincristine from 540 FRIEDBERG ET AL.

FIG. 4. Cytotoxicity assay for vincristine. The parental DUKXB11 cells and the various recombinant cell lines were exposed to increasing concentrations of vincristine for 3 days. Subsequently, the number of viable cells was determined using the ATP bioluminescent assay (Sigma, St. Louis, MO, U.S.A.). For each cell line, the number of cells obtained in the absence of vinblastine was set as 100%. The parental DUKB11 cells (ᮀ) and the cells expressing the following proteins were assayed: hOR (᭝); CYP3A4 (᭺); CYP2D6 and hOR (᭹); CYP1A2 and hOR (■); CYP3A4 and hOR (᭡). The data represent the means of one experiment with three determinations per concentration. The standard deviation was less than 5% of the means. In a second experiment, similar results were seen. RECOMBINANT MODELS IN DRUG RESEARCH 541

10 nM to 1 µM did not increase its cytotoxicity for the cell line which coexpressed CYP3A4 together with P450 reductase. However, it is possible that the diffusion of vinca alkaloids into the cells was rate limiting as compared to their metabolism by CYP3A4. The mean uptake rate for vinblastine into hepatocytes has been reported to be 0.568 pmol/min/106 cells [48], which is more than one order of magnitude less than the CYP3A4-mediated metabolism of this compound which we have determined in the cell line which expressed CYP3A4 together with P450 reductase. From our data, it becomes evident that CYP3A4 is involved in the detoxication of vinca alkaloids and that this pathway is most likely responsible for the idiosyncratic toxicity seen during treatment with this class of chemotherapeutic compounds [49±51]. P450s are also involved in the activation of chemotherapeutic drugs to cytotoxic metabolites. The best known example is cyclophosphamide, which is activated by CYP3A4 and CYP2B6 [52,53]. Another class of chemotherapeutic drugs which are activated by P450s are doxorubicin analogues such as morpholino-doxorubicin [54]. Using the recombinant cell lines, we were able to demon- strate that cells which expressed CYP3A4 were more than 1000-fold more sensitive toward this agent than parental CHO cells or CHO cells which expressed human P450 reductase. Presently, we are studying if in a coculture of cells which express CYP3A4 together with cells which are devoid of this P450, the latter cells will display cytotoxic bystander effects in the presence of morpholino-doxorubicin.

III. CONCLUSIONS

We have found that the catalytic properties of most P450 isozymes, such as

Km, are reasonably well preserved across the recombinant expression models. However, exceptions exist, such as the relatively low substrate turnover number of CYP3A4 in yeast, which cannot be solely explained by the levels of human

P450 reductase or of cytochrome b5 found in our recombinant strains and those reported by another group [25]. Provided that P450 substrate speci®city is also retained in the recombinant models, an issue which we are currently addressing in collaboration with the pharmaceutical industry, the recombinant models provide an ideal tool to predict drug metabolism pathways in humans. The merits and limitations of the various models are largely dictated by their envisaged use. Structural studies require large quantities of pure proteins, a task which is best performed by microbial expression models. Human P450 monooxygenase systems expressed in E. coli have a potential as bioreactors for the synthesis of valuable metabolites. However, continuous production of these metabolites will need further improvements concerning the linearity of the P450-mediated reactions with time. Because toxic responses are controlled by several pathways 542 FRIEDBERG ET AL. which are best mimicked in mammalian cells, investigations into the toxicological roleof drug-metabolizingenzymes are best performedwithmammaliancells. Thus, recombinant models for human drug metabolism complement other techniques such as animal models and hepatocyte culture to improve drug development. Note Added in Proof: Recently, we have optimized the ratio of P450 reductase to cytochrome P450 in the E. coli strains which express CYP1A1, CYP1A2, and CYP1B1. Membranes isolated from these strains displayed a turnover number for ethoxyresoru®n of 20, 8, and 3 minϪ1, respectively.

ACKNOWLEDGMENTS

This work was sponsored by the Biological Sciences Research Council, the UK Department of Trade and Industry, and the LINK consortium of pharmaceutical companies: Astra, Glaxo-Wellcome, Janssen Pharmaceutica, Lilly, Novo- Nordisk, Park-Davis, P®zer, Roche Products, Sano®-Winthrop, Servier, Smith- Kline Beecham, Wyeth-Ayerst, and Zeneca. Their generous support and their helpful discussions are gratefully acknowledged.

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