ENHANCEMENT OF CYTOCHROME P-450 3A4 CATALYTIC ACTIVITIES BY CYTOCHROME b 5 IN BACTERIAL MEMBRANES

Transcription

1 /99/ $02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 27, No. 9 Copyright 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. ENHANCEMENT OF CYTOCHROME P-450 3A4 CATALYTIC ACTIVITIES BY CYTOCHROME b 5 IN BACTERIAL MEMBRANES HIROSHI YAMAZAKI, MIKI NAKAJIMA, MAMI NAKAMURA, SATORU ASAHI, NORIAKI SHIMADA, ELIZABETH M. J. GILLAM, F. PETER GUENGERICH, TSUTOMU SHIMADA, AND TSUYOSHI YOKOI Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan (H.Y., M. Nakajima, M. Nakamura, M. Nakajima, T.Y.); Takeda Chemical Industries, Ltd., Osaka, Japan (S.A.); Daiichi Pure Chemicals Co., Ibaraki, Japan (N.S.); Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland, Australia (E.M.J.G.); Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee (F.P.G.); and Osaka Prefectural Institute of Public Health, Osaka, Japan (T.S.) (Received March 9, 1999; accepted June 2, 1999) This paper is available online at ABSTRACT: Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6 -hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b 5 (b 5 ) and apolipoprotein b 5 further enhanced the testosterone 6 -hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when Multiple forms of cytochrome P-450 (P-450) 1 exist in mammals, and these P-450 enzymes play important roles in the oxidation of structurally diverse xenobiotic chemicals and endobiotics (Guengerich and Shimada, 1991; Guengerich, 1995; Nelson et al., 1996). P-450s are not self-sufficient enzymes, and the microsomal enzymes require a NADPH-P-450 reductase (NPR) as an electron carrier to function as monooxygenases. In human livers, levels of each of the P-450 forms are different and roles in various substrate oxidations vary. CYP3A4 is the major P-450 enzyme involved in the oxidation of a large number of compounds (Wrighton and Stevens, 1992; Gonzalez and Gelboin, 1994; Shimada et al., 1994). Recently, recombinant P-450 enzymes from different sources e.g., microsomes of human lymphoblastoid cells (Gonzalez et al., 1991; Crespi, 1995), yeast (Renaud et al., 1993; Imaoka et al., 1996), This work was supported in part by grants from the Japanese Ministries of Education, Science, Sports, and Cultures; and Health and Welfare. 1 Abbreviations used are: P-450, cytochrome P-450; NPR, NADPH-P-450 reductase; CYP3A4/NPR membranes, membranes prepared from bacteria coexpressing CYP3A4 and NPR from a bicistronic vector; apo b 5, apolipoprotein cytochrome b 5 ; LB, Luria-Bertani medium; TB, Terrific Broth. Send reprint requests to: Hiroshi Yamazaki, Ph.D., Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa , Japan. yamazak@kenroku.kanazawa-u.ac.jp 999 added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b 5 ). Apparent maximal activities of testosterone 6 -hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b 5 was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6 -hydroxylation, nifedipine oxidation, and midazolam 4- and 1 -hydroxylation activities in CYP3A4/NPR membranes plus b 5 systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/ NPR membrane systems containing b 5 are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations. and insect cells infected with baculovirus systems (Buters et al., 1994; Lee et al., 1995) and reconstitution systems containing purified P-450 enzymes from Escherichia coli membranes (Gillam et al., 1993; Shaw et al., 1997) have been used widely for drug metabolism research. We have shown that a 1:2:1 molar ratio of CYP3A4/NPR/cytochrome b 5 (b 5 ) is optimal in the reconstitution of drug oxidation (Yamazaki et al., 1996b; Shimada and Yamazaki, 1998) using purified CYP3A4. However, the marker activities or kinetic parameters of CYP3A4 reported from different research laboratories and commercial enzymes manufacturers are not always similar. In this study, we have determined optimal catalytic activities for testosterone 6 -hydroxylation, nifedipine oxidation, and midazolam 4- and 1 -hydroxylation by CYP3A4/ NPR membranes (obtained using a bacterial bicistronic CYP3A4 expression system) fortified with b 5 or NPR and compared the activities of several types of recombinant CYP3A4 preparations before and after addition of b 5 and/or NPR. Enhancement of CYP3A4-dependent activities in membranes by addition of b 5 from different sources, apolipoprotein b 5 (apo b 5 ), and kinetic parameters for testosterone 6 -hydroxylation are also reported. Materials and Methods Chemicals. Midazolam and its metabolites were kindly donated by Yamanouchi Pharmaceuticals Co., Ltd. (Tokyo, Japan). Testosterone, nifedipine, and their metabolites and reagents used in this study were obtained from sources

2 1000 YAMAZAKI ET AL. TABLE 1 Catalytic activities for testosterone 6 -hydroxylation by several preparations of CYP3A4/NPR membranes obtained from E. coli transformed with a bicistronic CYP3A4/NPR expression vector a Culture Conditions CYP3A4/ NPR Preparation LB Time Medium TB Time rpm b CYP3A4 NPR NPR/CYP3A4 Testosterone 6 - Hydroxylation h h nmol/g cells nmol/liter culture nmol/g cells nmol/liter culture Molar ratio nmol/min/nmol CYP3A4 A B C D E F a The LB cultures were incubated for 7 to 15 h at 37 C with shaking at 170 rpm and then diluted 1:100 into TB. The TB cultures (100 ml) were grown at 30 C with shaking at 120 to 180 rpm for 24 to 32 h in 500-ml triple-baffled flasks. Results are presented as means of duplicate determinations. b Agitation speed (rpm) for TB culture. described previously or were of the highest qualities commercially available (Yamazaki et al., 1996b; Shimada and Yamazaki, 1998). Enzyme Preparations. Membranes were prepared from E. coli into which CYP3A4 and NPR cdnas had been introduced as described previously (Parikh et al., 1997). Briefly, single transformed colonies of E. coli strains DH5 and JM109 were used to inoculate starter Luria-Bertani medium (LB)/ ampicillin (25 g/ml) cultures. The starter cultures were incubated for 7 to 15 h at 37 C, with shaking at 170 rpm, and then diluted 1:100 into Terrific Broth (TB)/ampicillin (100 g/ml) medium containing additives (0.5 mm -aminolevulinic acid, 1.0 mm isopropyl -D-thiogalactoside, trace salts, and 1.0 mm thiamine; Guengerich et al., 1996). The expression cultures (100 ml) were grown at 30 C with shaking at 120 to 180 rpm for 24 to 32 h in 500-ml triple-baffled flasks. Membrane fractions were prepared from the bacterial pellets by a series of fractionation and high-speed centrifugation steps (Guengerich et al., 1996) and suspended in one volume of 10 mm Tris-HCl buffer (ph 7.4) containing 0.10 mm EDTA and 20% glycerol (v/v). Recombinant monocistronic CYP3A4, CYP1A2, and NPR were purified from membranes as described elsewhere (Gillam et al., 1993; Dong et al., 1996; Parikh et al., 1997). Rabbit NPR (Guengerich et al., 1981) and rabbit (Strittmatter et al., 1978), rat, and human b 5 (Shimada et al., 1986) were purified from liver microsomes by the methods described. Recombinant human NPR and b 5 were purified using similar methods. Apo b 5 was prepared from rabbit b 5 as described previously (Yamazaki et al., 1996a). Horse heart cytochrome c was purchased from Sigma. Human liver microsomes were prepared in 10 mm Tris-HCl buffer (ph 7.4) containing 0.10 mm EDTA and 20% glycerol (v/v) as described previously (Guengerich, 1994; Inoue et al., 1997). Recombinant CYP3A4 expressed in microsomes of insect cells infected with baculovirus containing human P-450 and rabbit or human NPR cdna inserts were obtained from PanVera (Madison, WI) or Gentest (Woburn, MA). Recombinant CYP3A4 in microsomes from lymphoblastoid cells coexpressing NPR was obtained from Gentest. The P-450 contents were used as described in the data sheets provided by the manufacturers. Enzyme Assays. Testosterone hydroxylation, nifedipine oxidation, and midazolam hydroxylation activities were determined as described (Kronbach et al., 1989; Brian et al., 1990) with slight modifications (Shimada and Yamazaki, 1998). The standard incubation mixture (final volume of 0.25 ml) contained M recombinant CYP3A4, M NPR, M b 5, and 100 mm potassium phosphate buffer (ph 7.4), an NADPH-generating system consisting of 0.5 mm NADP, 5 mm glucose 6-phosphate, and 0.5 U of glucose 6-phosphate dehydrogenase/ml, and 200 M testosterone (or nifedipine) or 100 M midazolam. In some cases, human liver microsomes were used at the same CYP3A4 concentrations as the recombinant system. Incubations were carried out at 37 C for 10 min and terminated by adding 1.5 ml of CH 2 Cl 2 and 0.3 M NaCl. Reactions with nifedipine were incubated at 37 C for 5 min and terminated by adding 1.5 ml of CH 2 Cl 2, 0.2 M NaCl, and 0.1 M Na 2 CO 3. Organic phases were evaporated under a nitrogen stream, and product formation was determined by HPLC with a C 18 (5- m) analytical column ( mm). Reactions with midazolam were incubated at 37 C for 5 min and terminated by adding 0.25 ml of CH 3 OH. The elution was conducted with a mixture of 64% CH 3 OH/36% H 2 O (v/v) at a flow rate of 1.2 ml/min, and the detection was by UV absorbance at 240 nm (testosterone) and 254 nm (nifedipine). The elution of midazolam metabolites was conducted with a mixture of 27% CH 3 OH/18% CH 3 CN/55% 10 mm potassium phosphate buffer (ph 7.4) (v/v) at a flow rate of 1.5 ml/min, and detection was by UV absorbance at 220 nm. Other Assays. Concentrations of P-450 and b 5 and protein were estimated spectrally by the described methods (Lowry et al., 1951; Omura and Sato, 1964). NADPH-cytochrome c reduction activities were determined as described (Williams and Kamin, 1962; Yasukochi and Masters, 1976) using mm 1 cm 1 and an assumed specific activity of 3.0 mol reduced cytochrome c/min/nmol NPR based on purified human and rabbit NPR preparations (Parikh et al., 1997). The contents of CYP3A4 in human liver microsomes were estimated by coupled SDS-polyacrylamide gel electrophoresis/immunochemical development (Western blotting) (Guengerich et al., 1982). Kinetic analyses for substrate oxidations by P-450 enzymes were estimated from the fitted curves using a computer program (KaleidaGraph program; Synergy Software, Reading, PA) designed for nonlinear regression analysis. Results Recovery of CYP3A4 and NPR in Membranes of E. coli. CYP3A4 and NPR were coexpressed in E. coli from a bicistronic vector using six different culture conditions (Table 1). With regard to CYP3A4 expression levels, long incubation for both LB and TB cultures with vigorous shaking resulted in good yields (Table 1, lot F). On the other hand, the final yield of NPR was highest when the culture time in LB was 7 h, followed by 32 h with mild shaking, in TB medium (Table 1, lot B). Catalytic activities of CYP3A4/NPR membranes for testosterone 6 -hydroxylation were higher in samples B and A. Effects of Exogenous b 5 and NPR on Catalytic Rates of CYP3A4/NPR Membranes. Because a 1.1:1 molar ratio of NPR/ CYP3A4 in membrane preparations appeared to give highest catalytic activities for CYP3A4-dependent testosterone 6 -hydroxylation among the conditions tested (Table 1), the effects of b 5 on activity were investigated mainly using this preparation. The effect of b 5 was shown to be concentration-dependent (Fig. 1A). Both human b 5 in E. coli membranes and purified recombinant human b 5 enhanced the testosterone hydroxylation activities about 2-fold; a1to2:1molar ratio of b 5 /CYP3A4 produced the highest activities. The effects of adding recombinant human NPR (in membranes) were studied. Rates of testosterone 6 -hydroxylation by recombinant CYP3A4 (lot B) were increased 2-fold by the supplementation with

3 TESTOSTERONE, NIFEDIPINE, AND MIDAZOLAM OXIDATION BY CYP3A FIG. 1.Concentration dependence of the effects of human b 5 and NPR in membranes on testosterone 6 -hydroxylation by CYP3A4/NPR membranes. A, testosterone (200 M) was incubated with CYP3A4/NPR membranes (Table 1, preparation B, M CYP3A4) fortified with membranes expressing human NPR (final, M) in the presence of recombinant human b 5 added in E. coli membranes (f) or as purified b 5 (Œ). B, testosterone (200 M) was incubated with CYP3A4/NPR membranes containing CYP3A4 (0.010 M) and recombinant human NPR (0.011 M) that were fortified further with different amounts of NPR, added in bacterial membranes to the final ratios shown, in the absence (E) or presence of added, purified human b 5 (Œ) orb 5 in membranes (f) (0.010 M). At the zero molar ratio of NPR to CYP3A4, E. coli membranes expressing only CYP3A4 were used. recombinant human NPR to an 8-fold excess of NPR over CYP3A4 in membranes in the absence of b 5 (Fig. 1B). In the presence of b 5, apparent optimal activities were observed after the addition of a 2:1 final molar ratio of NPR to CYP3A4 (Fig. 1B). Stimulating effects of b 5 also were observed upon addition of either rabbit, rat, or human b 5 purified from liver microsomes as well as recombinant human b 5 (Table 2). Apo b 5 and recombinant CYP1A2 also enhanced the catalytic activities of CYP3A4/NPR as well as b 5, but cytochrome c did not. Recombinant human NPR, purified from E. coli membranes, catalyzed NADPH-dependent cytochrome c reduction and supported CYP3A4-dependent testosterone 6 -hydroxylation (Table 3); however, purified human NPR-supported CYP3A4 activity for testosterone hydroxylation was lower in a reconstituted system containing a 1:2:1 molar ratio of CYP3A4/NPR/b 5 than was that supported by native rabbit NPR. The effects of supplementation with purified native rabbit NPR and cholate on CYP3A4 expressed in membranes were investigated (Fig. 2). Cholate enhanced dose-despondently the exogenous rabbit NPR-supported activities of CYP3A4/NPR membranes in the presence of b 5 (Fig. 2A). When purified native rabbit NPR was used, testosterone 6 -hydroxylation was improved in the presence of both b 5 and cholate (Fig. 2B). The apparent maximal activities were similar after addition of purified rabbit NPR ( 80 min 1 ) (Fig. 2B) and recombinant human NPR (in membranes) (Fig. 1B). Comparison of Bacterial CYP3A4/NPR Membranes with Other Recombinant Proteins and Human Liver Microsomes. To compare testosterone 6 -hydroxylation activities among recombinant CYP3A4 systems, we determined the rates of other recombinant CYP3A4 systems and human liver microsomes after addition of purified b 5 and/or NPR with cholate (Table 4). Catalytic activities of microsomes of lymphoblastoid cells were increased 3-fold by the addition of a 2-fold excess of b 5 over CYP3A4. However, the rates of this system fortified with NPR were lower than those obtained with CYP3A4 expressed using the bacterial CYP3A4/NPR membranes. The activities of one of the microsomal systems from insect cells with baculovirus systems (Baculosomes; PanVera) containing a 1:4.6 molar ratio of CYP3A4 to NPR were improved by the addition of a 2-fold excess of b 5 to CYP3A4. The activities of another baculovirus system containing CYP3A4/NPR/b 5 (1:12:16) (Supersomes; Gentest) were not TABLE 2 Effects of source of b 5 and other proteins on testosterone 6 -hydroxylation activities by CYP3A4/NPR membranes NPR Membrane Added Component Testosterone 6 -Hydroxylation nmol/min/nmol P-450 Experiment 1 CYP3A4/NPR 41 CYP3A4/NPR Recombinant 80 human b 5 CYP3A4/NPR Human b 5 78 CYP3A4/NPR Rabbit b 5 73 CYP3A4/NPR Rat b 5 70 Experiment 2 CYP3A4/NPR 38 4 CYP3A4/NPR b CYP3A4/NPR apo b CYP3A4/NPR cytochrome c 41 6 CYP3A4/NPR CYP1A NPR CYP1A2 0.1 In experiment 1, cytochrome b 5 (0.020 M) purified from E. coli membranes or from liver microsomes was added to CYP3A4/NPR membranes (0.010 M P-450 and M NPR). In experiment 2, other proteins (0.010 M) were added to CYP3A4/NPR membranes. Apo b 5 and CYP1A2 were prepared from rabbit liver b 5 and E. coli membranes expressing human CYP1A2, respectively. Results are presented as means S.D. of duplicate or triplicate determinations. affected by further addition of b 5 and/or NPR. Human liver microsomes containing 71% CYP3A4 (of total P-450) also were used. Based on CYP3A4 contents, the testosterone 6 -hydroxylation activity of this sample was 85 nmol/min/nmol CYP3A4. The molar ratio of CYP3A4/NPR/b 5 in this human liver microsomal preparation was 1:0.06:0.52, and testosterone 6 -hydroxylation was only minimally affected by the addition of recombinant b 5 or NPR. Nifedipine and midazolam oxidation activities of recombinant CYP3A4 systems and human liver microsomes also were determined (Table 5). In addition to a reconstituted system, the same enzyme sources as in Table 4 were used after addition of purified b 5 and NPR, with cholate added. Activities of nifedipine oxidation and midazolam 4- and 1 -hydroxylation of CYP3A4/NPR membranes plus b 5 and in the reconstituted system were similar to those of human liver microsomes ( 30, 10, and 20 min 1, respectively), based on CYP3A4 contents. Catalytic activities of microsomes of lymphoblastoid cells were lower, and nifedipine oxidation activities of a baculovirus sys-

4 1002 YAMAZAKI ET AL. TABLE 3 Cytochrome c reduction and CYP3A4-dependent testosterone 6 -hydroxylation by purified recombinant human and native rabbit NPR NPR a Cytochrome c Reduction b Molar Ratio of CYP3A4/NPR Testosterone 6 -Hydroxylation c mol/min/nmol NPR nmol/min/nmol CYP3A4 Human 3.2 1:2 16 1:4 41 1:6 70 1:8 79 Rabbit 3.1 1:2 76 a Human and rabbit NPR were purified from membranes of E. coli-expressing human NPR and from liver microsomes of phenobarbital-treated rabbits, respectively. b Rates of cytochrome c (50 M) reduction by NPR (5 nm) were recorded at 25 C in the presence of 0.1 mm NADPH. c Hydroxylation of testosterone (200 M) was determined at 37 C for 10 min in reconstitution systems containing purified recombinant CYP3A4 (0.010 M), NPR ( M), and human b 5 (0.010 M) with lipid mixture (20 g/ml) and cholate (0.25 mm) in the presence of an NADPH-generating system. Results are presented as means of duplicate determinations. tem that coexpressed b 5 and NPR were higher than those of human liver microsomes. Kinetic Analysis of Activity Catalyzed by CYP3A4/NPR Membranes Plus b 5 and Human Liver Microsomes. Because recombinant CYP3A4 (expressed in bacterial membranes using the bacterial bicistronic system) premixed at a molar ratio of 1:2:1 of CYP3A4 to human NPR to human b 5 appeared to be a suitable model for human liver microsomal CYP3A4 with regard to catalytic activities, kinetic parameters were compared with those of human liver microsomes. Testosterone 6 -hydroxylation was dependent on CYP3A4 concentration (Fig. 3); linearity of product formation was obtained in a narrow range ( M). Kinetic analysis for testosterone 6 hydroxylation over a substrate concentration range of 10 to 500 M yielded K m and V max values of M and nmol/ min/nmol CYP3A4 for the recombinant bacterial CYP3A4 system plus b 5 (1:2:1 molar ratio of CYP3A4/NPR/b 5 ) and M and nmol/min/nmol P-450 for human liver microsomes, respectively. Discussion A number of studies have shown that CYP3A4 is a major P-450 enzyme involved in the oxidation of many clinically used drugs in TABLE 4 Effects of exogenous NPR and b 5 on testosterone 6 -hydroxylation activities in CYP3A4/NPR membranes, microsomes containing recombinant CYP3A4/NPR, and human liver microsomes Enzymes Added NPR a Added b 5 b Testosterone 6 -hydroxylation nmol/min/nmol CYP3A4 E. coli membranes c Lymphoblastoid cell 14 microsomes d Baculosomes e Supersomes f Human liver 85 microsomes g 88 (HL-110) Testosterone (200 M) was incubated with CYP3A4 (0.010 M) for 10 min, and formed product was determined by HPLC. Results are presented as means of duplicate determinations. a Purified rabbit NPR (2-fold molar excess compared with CYP3A4) was added with 0.25 mm sodium cholate. b Purified human b 5 (2-fold molar excess compared with CYP3A4) was added. c E. coli membranes containing CYP3A4/NPR (1:1.1). d Microsomes of lymphoblastoid cells containing CYP3A4/NPR (1:1) (Gentest, lot 64). e Microsomes of insect cells with baculovirus system containing CYP3A4/NPR (1:4.6) (PanVera, lot 10345). f Microsomes of insect cells with baculovirus system containing CYP3A4/NPR/b 5 (1:12:16) (Gentest, lot 18). g The CYP3A4 content (71% of total P-450) in liver microsomes was determined immunochemically, and the molar ratio of CYP3A4/NPR/b 5 was 1:0.06:0.52. human liver microsomes (Guengerich, 1995; Wilkinson, 1996; Li et al., 1997). Prediction of microsomal oxidation of drugs in human livers has been studied recently using activities or kinetic parameters obtained from recombinant systems (Iwatsubo et al., 1997; Ito et al., 1998). A relative activity factor was proposed by Crespi (1995), and calculations have been reported with other factors (Kobayashi et al., FIG. 2.Concentration dependence of the effects of cholate and purified NPR on testosterone 6 -hydroxylation by CYP3A4/NPR membranes. A, cholate (0 1.0 mm) was added to CYP3A4/NPR membranes containing CYP3A4 (0.010 M) and human NPR (0.011 M), which were fortified further with exogenous rabbit NPR (0.010 M) with or without purified human b 5 (0.010 M). These components were mixed and allowed to stand 10 min at room temperature, followed by addition of other reaction components. Testosterone 6 -hydroxylation activities (200 M testosterone) were determined. B, exogenous rabbit NPR ( M) was added to CYP3A4/NPR membranes containing CYP3A4 (0.010 M) and human NPR (0.011 M), which were fortified with purified human b 5 (0.010 M) and/or cholate (0.25 mm). Testosterone 6 -hydroxylation activities were determined as described in A.

5 TESTOSTERONE, NIFEDIPINE, AND MIDAZOLAM OXIDATION BY CYP3A TABLE 5 Nifedipine and midazolam oxidation activities in a reconstitution system, CYP3A4/NPR membranes, microsomes containing recombinant CYP3A4/NPR, and human liver microsomes a CYP3A4 System b Nifedipine Oxidation 4- Hydroxylation Midazolam 1 - Hydroxylation nmol products/min/nmol CYP3A4 Reconstitution c E. coli membranes Lymphoblastoid cell microsomes Baculosomes Supersomes Human liver microsomes a Nifedipine (200 M) and midazolam (100 M) were incubated with CYP3A4 (0.010 M) for 5 min, and product formation was determined by HPLC. Data are the means of duplicate determinations. b The enzyme source was the same as in Table 4. Purified human b 5 (1:1 molar ratio compared with CYP3A4) and rabbit NADPH-P-450 reductase (2-fold molar excess compared with CYP3A4) were added with 0.25 mm sodium cholate. c Reconstitution systems containing purified CYP3A4 (0.010 M), rabbit NPR (0.020 M), and human b 5 (0.010 M) with lipid mixture (20 g/ml) and cholate (0.25 mm). FIG. 3.Effects of concentration of CYP3A4/NPR membranes premixed with b 5 on testosterone 6 -hydroxylation. Testosterone (200 M) was incubated with different concentrations of CYP3A4/ NPR membranes ( M CYP) premixed with human NPR and b 5 (1:2:1 molar ratio of CYP3A4/NPR/b 5 ). 1997; Nakajima et al., 1998; Venkatakrishnan et al., 1998). Omeprazole oxidation by human liver microsomes was predicted by using a combination of liver microsomal contents of CYP2C19 and CYP3A4 (immunochemically determined) and kinetic parameters obtained from experiments using recombinant CYP2C19 and CYP3A4 (Yamazaki et al., 1997b), using the methods outlined by Iwatsubo et al. (1997). In these studies, catalytic activities and kinetic parameters of marker drug oxidations catalyzed by recombinant P-450 enzymes are very important for prediction; however, different activities have been reported using several expression systems for CYP3A4 (Shaw et al., 1997; Yamazaki et al., 1997b). Drug oxidation activities of purified CYP3A4 have been studied extensively, and it has been shown that some CYP3A4 activities are dependent on b 5, specific lipid mixtures, cholate, and buffer and salt compositions (Yamazaki et al., 1996a; Shimada and Yamazaki, 1998). We have shown (using the purified recombinant CYP3A4 obtained from a monocistronic bacterial expression system) that a 1:2:1 molar ratio of CYP3A4/NPR/b 5 was suitable for reconstitution of drug oxidation (Yamazaki et al., 1996b). The present results indicate that a 1:2:1 molar ratio of CYP3A4/ NPR/b 5 gives apparently optimal activities for testosterone 6 -hydroxylation, nifedipine oxidation, and midazolam 4- and 1 -hydroxylations by recombinant CYP3A4 expressed in bacterial membranes using a bicistronic system. The molar ratio of the three proteins in membranes was the same as in a reconstituted system containing purified CYP3A4. Enhancing effects of exogenous b 5 were observed with either b 5 purified from human, rabbit, or rat liver microsomes, with recombinant human b 5 added in bacterial membranes or with apo b 5, devoid of heme. Effects of b 5 on CYP3A4 activities also were observed in microsomes from lymphoblastoid cells and insect cells (lacking endogenous b 5 ). Cholate (0.25 mm) was needed to enhance rates when purified NPR was added to CYP3A4 in bacterial membranes; however, cholate appeared not to be necessary when NPR was supplemented from bacterial membranes. Testosterone 6 -hydroxylation ( 80 min 1 ), nifedipine oxidation ( 30 min 1 ), and midazolam 4- and 1 -hydroxylation ( 10 and 20 min 1 ) activities were similar among human liver microsomes and/or baculovirus systems based on CYP3A4 contents. These results indicate that the bacterial CYP3A4/NPR membranes plus b 5 should be a simple and suitable model of drug oxidation study for microsomal CYP3A4-dependent reactions in human livers. NPR is essential to P-450-dependent drug oxidation, and many catalytic activities of CYP3A4 require b 5 for optimal rates. We have shown that b 5 can stimulate some CYP3A4-catalyzed oxidations by complexing with CYP3A4 in a synthetic phospholipid mixture, cholate, and MgCl 2 and enhancing its reduction by NPR without directly transferring electrons to P-450 (Yamazaki et al., 1996a). In the present study, purified b 5, membrane b 5 as well as apo b 5 enhanced the catalytic activities of CYP3A4 in bacterial membranes. Exogenous b 5 also was effective when added to microsomal CYP3A4 systems from lymphoblastoid and insect cells (lacking coexpressed b 5 ). This suggested that insertion of rabbit, rat, or human b 5 into the phospholipid bilayers is facile, and b 5 can make a suitable complex with CYP3A4 and NPR for efficient electron transfer. Similarly, the membrane-associated NPR was able to mix with membranes containing CYP3A4 and enhance catalytic activities; however, catalytic function of exogenous-purified NPR was dependent on the presence of added b 5 and cholate. Both cholate and b 5 may be necessary for insertion of purified NPR into phospholipid membranes or for complexation of purified NPR with CYP3A4. Cholate may not be an essential component for all of the activities catalyzed by CYP3A4 in reconstituted system; however, it sometimes has been helpful for reconstitution (Yamazaki et al., 1997a). Similar enhancing effects of cholate were observed at the same concentrations (0.01% w/v, or 0.25 mm) on catalytic activities of CYP1B1 in a reconstituted system (Shimada et al., 1998). In vitro assays are used to predict in vivo drug metabolism in humans. It has been reported that assay conditions, such as different buffers, salts, ionic strengths, detergent level, and components of NADPH-generating systems may affect the CYP3A4-mediated reactions (Yamazaki et al., 1997a; Mäenpää et al., 1998; Shimada and Yamazaki, 1998). Because effects of buffer and salt concentrations are likely to be more significant in reconstituted systems containing CYP3A4 than in human liver microsomes (Yamazaki et al., 1996b), we used 100 mm potassium phosphate buffer (ph 7.4) for drug metabolism reactions with CYP3A4/NPR membranes in this study. In conclusion, the present study suggested that CYP3A4 coexpressed with NPR in bacterial membranes supplemented with additional NPR plus b 5 is a very useful model for prediction of the rates

6 1004 YAMAZAKI ET AL. for microsomal CYP3A4-dependent drug oxidations in human livers. The ratio of NPR to CYP3A4 in human liver microsomes is low and very different from that found to give apparent maximal activities using the bacterial CYP3A4/NPR membranes. We reported previously that CYP3A4-dependent testosterone 6 -hydroxylation is stimulated by CYP1A2 in reconstitution systems (Yamazaki et al., 1997a). In this study, CYP1A2 also enhanced catalytic activities of CYP3A4/ NPR membranes. These findings indicate the potential for one P-450 enzyme to influence the catalytic characteristics of another P-450 enzyme when a low amount of NPR is present in human liver microsomes. Further studies are necessary for optimization of recombinant P-450 enzyme systems for use as human liver models. References Brian WR, Sari MA, Iwasaki M, Shimada T, Kaminsky LS and Guengerich FP (1990) Catalytic activities of human liver cytochrome P-450IIIA4 expressed in Saccharomyces cerevisiae. Biochemistry 29: Buters J, Korzekwa KR, Kunze KL, Omata Y, Hardwick JP and Gonzalez FJ (1994) cdnadirected expression of human cytochrome P450 CYP3A4 using baculovirus. Drug Metab Dispos 22: Crespi CL (1995) Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research. Adv Drug Res 26: Dong M-S, Yamazaki H, Guo Z and Guengerich FP (1996) Recombinant human cytochrome P450 1A2 and N-terminal-truncated form: Construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase. Arch Biochem Biophys 327: Gillam E, Baba T, Kim B-R, Ohmori S and Guengerich FP (1993) Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch Biochem Biophys 305: Gonzalez FJ, Crespi CL and Gelboin HV (1991) cdna-expressed human cytochrome P450s: A new age of molecular toxicology and human risk assessment. Mutat Res 247: Gonzalez FJ and Gelboin HV (1994) Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins. Drug Metab Rev 26: Guengerich FP (1994) Analysis and characterization of enzymes, in Principles and Methods of Toxicology (Hayes AW ed) pp , Raven Press, New York. Guengerich FP (1995) Human cytochrome P450 enzymes, in Cytochrome P450 (Oritiz de Montellano PR ed) pp , Plenum Press, New York. Guengerich FP, Martin MV, Guo Z and Chun Y-J (1996) Purification of functional recombinant P450s from bacteria. Methods Enzymol 272: Guengerich FP and Shimada T (1991) Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol 4: Guengerich FP, Wang P and Davidson NK (1982) Estimation of isozymes of microsomal cytochrome P-450 in rats, rabbits, and humans using immunochemical staining coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry 21: Guengerich FP, Wang P and Mason PS (1981) Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductase. Biochemistry 20: Imaoka S, Yamada T, Hiroi T, Hayashi K, Sakai T, Yabusaki Y and Funae Y (1996) Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. Biochem Pharmacol 51: Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich FP and Shimada T (1997) Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S- mephenytoin 4 -hydroxylation activities in livers of Japanese and Caucasian populations. Pharmacogenetics 7: Ito K, Iwatsubo T, Kanamitsu S, Nakajima Y and Sugiyama Y (1998) Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport. Annu Rev Pharmacol Toxicol 38: Iwatsubo T, Hirota N, Ooie T, Suzuki H, Shimada N, Chiba K, Ishizaki T, Green CE, Tyson CA and Sugiyama Y (1997) Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol Ther 73: Kobayashi K, Chiba K, Yagi T, Shimada N, Taniguchi T, Horie T, Tani M, Yamamoto T, Ishizaki T and Kuroiwa Y (1997) Identification of cytochrome P450 isoforms involved in citalopram N-demethylation by human liver microsomes. J Pharmacol Exp Ther 280: Kronbach T, Mathys D, Umeno M, Gonzalez FJ and Meyer UA (1989) Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36: Lee CA, Kadwell SH, Kost TA and Serbjit-Singh CJ (1995) CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPHcytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. Arch Biochem Biophys 319: Li AP, Maurel P, Gomez-Lechon MJ, Cheng LC and Jurima-Romet M (1997) Preclinical evaluation of drug-drug interaction potential: Present status of the application of primary human hepatocytes in the evaluation of cytochrome P450 induction. Chem Biol Interact 107:5 16. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: Mäenpää J, Hall SD, Ring BJ, Strom SC and Wrighton SA (1998) Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: The effect of assay conditions and regioselective stimulation by -naphthoflavone, terfenadine and testosterone. Pharmacogenetics 8: Nakajima M, Kobayashi K, Shimada N, Tokudome S, Yamamoto T and Kuroiwa Y (1998) Involvement of CYP1A2 in mexiletine metabolism. Br J Clin Pharmacol 46: Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC and Nebert DW (1996) P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1 42. Omura T and Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239: Parikh A, Gillam EMJ and Guengerich FP (1997) Bacterial catalysis of reactions important in mammalian drug and xenobiotic metabolism: Functional co-expression of six human microsomal cytochrome P450 enzymes with NADPH-cytochrome P450 reductase in Escherichia coli. Nat Biotechnol 15: Renaud JP, Peyronneau MA, Urban P, Truan G, Cullin C, Pompon D, Beaune P and Mansuy D (1993) Recombinant yeast in drug metabolism. Toxicology 82: Shaw PM, Hosea NA, Thompson DV, Lenius JM and Guengerich FP (1997) Reconstitution premixes for assays using purified recombinant human cytochrome P450, NADPHcytochrome P450 reductase, and cytochrome b 5. Arch Biochem Biophys 348: Shimada T, Misono KS and Guengerich FP (1986) Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction. J Biol Chem 261: Shimada T, Wunsch RM, Hanna IH, Sutter TR, Guengerich FP and Gillam EMJ (1998) Recombinant human cytochrome P450 1B1 expression in Escherichia coli. Arch Biochem Biophys 357: Shimada T and Yamazaki H (1998) Cytochrome P450 reconstitution systems, in Methods in Molecular Biology (Phillips IR and Shephard EA eds) pp 85 93, Humana Press, Totowa, NJ. Shimada T, Yamazaki H, Mimura M, Inui Y and Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270: Strittmatter P, Fleming P, Connors M and Corcoran D (1978) Purification of cytochrome b 5. Methods Enzymol 52: Venkatakrishnan K, von Moltke LL and Greenblatt DJ (1998) Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: Application of the relative activity factor approach. J Pharm Sci 87: Wilkinson GR (1996) Cytochrome P4503A (CYP3A) metabolism: Prediction of in vivo activity in humans. J Pharmacokin Biopharm 24: Williams CH Jr and Kamin H (1962) Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J Biol Chem 237: Wrighton SA and Stevens JC (1992) The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxicol 22:1 21. Yamazaki H, Gillam EMJ, Dong M-S, Johnson WW, Guengerich FP and Shimada T (1997a) Reconstitution of recombinant cytochrome P450 2C10 (2C9) and comparison with cytochrome P450 3A4 and other forms: Effects of cytochrome P450 P450 and cytochrome P450-b 5 interactions. Arch Biochem Biophys 342: Yamazaki H, Inoue K, Shaw PM, Checovich WJ, Guengerich FP and Shimada T (1997b) Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: Effects of contents of these two forms in individual human samples. J Pharmacol Exp Ther 283: Yamazaki H, Johnson WW, Ueng Y-F, Shimada T and Guengerich FP (1996a) Lack of electron transfer from cytochrome b 5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo cytochrome b 5. J Biol Chem 271: Yamazaki H, Nakano M, Imai Y, Ueng Y-F, Guengerich FP and Shimada T (1996b) Roles of cytochrome b 5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes. Arch Biochem Biophys 325: Yasukochi Y and Masters BSS (1976) Some properties of a detergent-solubilized NADPHcytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J Biol Chem 251: