HIROSHI YAMAZAKI, KIYOSHI INOUE, PETER M. SHAW, WILLIAM J. CHECOVICH, F. PETER GUENGERICH and TSUTOMU SHIMADA

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 2 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283: , 1997 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 1 HIROSHI YAMAZAKI, KIYOSHI INOUE, PETER M. SHAW, WILLIAM J. CHECOVICH, F. PETER GUENGERICH and TSUTOMU SHIMADA Osaka Prefectural Institute of Public Health, 3 69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan (H.Y., K.I., T.S.); PanVera Corporation, 545 Sciences Drive, Madison, Wisconsin (P.M.S., W.J.C.); and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee (F.P.G.) Accepted for publication July 25, 1997 ABSTRACT Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Correlation coefficients between omeprazole 5-hydroxylation activities (when determined at a substrate concentration of 10 M) and S-mephenytoin 4 -hydroxylation and testosterone 6 -hydroxylation activities were found to be 0.64 and 0.67, respectively, in liver microsomes of 84 human samples examined. Omeprazole sulfoxidation activities in these human samples were correlated with testosterone 6 -hydroxylation activities (r 0.86). Omeprazole 5-hydroxylation by liver microsomes of a human sample that contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-cyp3a4 antibodies, although a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive toward fluvoxamine and anti-cyp2c9 antibodies. Sulfaphenazole (at 100 M) did not affect the omeprazole 5-hydroxylation and sulfoxidation catalyzed by human liver microsomes. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low K m and high V max values for the former enzyme and high K m and low V max values for the CYP3A4. These results suggest that contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation depend upon the ratio of these two P450 levels in human liver microsomes. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and CYP3A4 enzymes. Finally, when recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined by analysis of high-performance liquid chromatography. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5- oxidation of omeprazole (at a substrate concentration of 10 M) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Received for publication April 21, This work was supported in part by Grants from the Ministry of Education, Science, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Developmental and Creative Studies from Osaka Prefectural Government, and by United States Public Health Service Grants R35 CA44353 and P30 ES P450 or CYP comprises a superfamily of the enzymes that catalyze oxidation of xenobiotic chemicals such as drugs, toxic chemicals and carcinogens and endobiotic chemicals such as steroids, fatty acids, prostaglandins and vitamins (Nelson et al., 1996; Gonzalez, 1989; Guengerich, 1991) and individual P450 enzymes have considerable but overlapping, substrate specificities (Ryan and Levin, 1990). Liver microsomes contain the highest levels and multiple forms of P450 enzymes that detoxicate, and in some instances activate, a number of xenobiotic chemicals. CYP2C and 3A subfamily enzymes are the major P450 forms in human liver microsomes and contribute significantly to the oxidation of clinically used drugs and other xenobiotic chemicals (Guengerich and Shimada, 1991; Shimada et al., 1994). Both CYP2C9 and CYP2C19 proteins have been reported to be polymorphic enzymes. The molecular bases of the genetic polymorphisms of the CYP2C9 and CYP2C19 genes have been identified, and there are ethnic-related differences in the incidence in poor metabolizer phenotypes (in vivo) (de Morais et al., 1994; Sullivan-Klose et al., 1996; Chang et al., ABBREVIATIONS: P450 or CYP, cytochrome P450; IgG, immunoglobulin G; HPLC, high-performance liquid chromatography. 434

2 1997 Oxidation of Omeprazole by CYP2C19 and 3A a). CYP2C9 catalyzes the oxidation of a number of clinically-used drugs (e.g., tolbutamide, S-warfarin, phenytoin, piroxicam, tienilic acid and torsemide) and CYP2C19 has been shown to be involved in the oxidation of a limited number of drugs including S-mephenytoin, citalopram, proguanil and omeprazole (Goldstein et al., 1994; Goldstein and de Morais, 1994; Jean et al., 1996). Omeprazole is a substituted benzimidazole derivative and a potent long-acting inhibitor of gastric acid secretion by irreversible binding to the proton pump (H,K ) ATPase in the gastric parietal cell. This drug has been shown to be oxidized by several forms of P450 in human liver microsomes in vitro and in vivo (Marinac et al., 1996; Chang et al., 1995a; Ieiri et al., 1996; Ibeanu et al., 1996; Ishizaki et al., 1994). CYP2C19 is suggested to be a major form involved in the 5-hydroxylation, while CYP3A4 catalyzes sulfoxidation of omeprazole in humans (Chiba et al., 1993; Chang et al., 1995b; Karam et al., 1996). However, several lines of evidence have suggested that CYP3A4 is also active in catalyzing formation of 5-hydroxyomeprazole as well as of omeprazole sulfone (Andersson et al., 1993; Vandenbranden et al., 1996; Karam et al., 1996; Rost and Roots, 1996). Because the levels of CYP3A4 have been shown to be more than 20-fold higher than those of CYP2C19 in human liver microsomes (Inoue et al., 1997), it is suggested that CYP3A4 may be an important enzyme in the oxidation of omeprazole in human liver microsomes, as well as CYP2C19. In this study, we examined the roles of CYP2C19 and CYP3A4 in the oxidation of omeprazole by human liver microsomes and several types of recombinant human P450 enzymes. Different human samples who were genotyped for CYP2C19 gene were used for the analysis. The results presented in this study collectively indicate that both CYP2C19 and CYP3A4 are involved in the 5-hydroxylation of omeprazole (at a substrate concentration of 10 M) by liver microsomes, depending on the contents of these P450 forms in the different human samples. Materials and Methods Chemicals. S-Mephenytoin was purchased from Ultrafine Chemicals Co. (Manchester, UK). Omeprazole and its metabolites were kindly donated by Dr. T. Ishizaki of International Medical Center of Japan. Other drug substrates, their oxidation products, and reagents used in this study were obtained from sources as described previously or of highest qualities commercially available (Shimada et al., 1989; Shimada et al., 1994; Mimura et al., 1993; Yamazaki et al., 1994a). Enzyme preparation. Human liver samples were obtained from organ donors or patients undergoing liver resection as described previously (Shimada et al., 1994; Mimura et al., 1993). Liver microsomes were prepared as described and suspended in 10 mm Tris-Cl buffer (ph 7.4) containing 1.0 mm EDTA and 20% glycerol (v/v) (Guengerich, 1994). Recombinant CYP2C19 and CYP3A4 in microsomes of human lymphoblast cells and in yeast microsomes were purchased from Gentest Co. (Woburn, MA) and from Sumitomo Chemical Co. (Osaka, Japan), respectively. Insect microsomes containing CYP2C19 and 3A4 were prepared as described elsewhere (P. M. Shaw, N. A. Hosea, D. V. Thompson, J. M. Lenius and F. P. Guengerich, submitted for publication). Briefly, Trichoplusia ni A cells were infected with a baculovirus containing cdna inserts for human CYP2C19 and CYP3A4 and rabbit NADPH-P450 reductase as previously described (Lee et al., 1995), with the resulting ratio of reductase: P The infected cells were harvested by centrifugation and microsomes prepared using a procedure described elsewhere (Penman et al., 1993). Rabbit anti-p450 antibodies raised against human CYP2C9 and CYP3A4 were prepared and the IgG fractions were obtained as described (Shimada et al., 1986; Guengerich et al., 1986). Enzyme assays. Omeprazole 5-hydroxylation and sulfoxidation activities were determined as described elsewhere, with slight modification (Birkett et al., 1996; Karam et al., 1996). The standard incubation mixture consisted of microsomal protein (0.5 mg/ml) in a final volume of 0.25 ml of 50 mm potassium phosphate buffer (ph 7.4) containing an NADPH-generating system and omeprazole (10 or 400 M). Incubations were carried out at 37 C for 15 min and terminated by adding 6 vol. of CH 2 Cl 2 and 0.3 M NaCl. Product formation was determined by HPLC with a reverse-phase Nucleosil C 8 (5 m) column ( mm, Chemco Scientific, Osaka, Japan) in a mobile phase consisting of a mixture of CH 3 OH/CH 3 CN/H 2 O (40:8:52, v/v) with a flow rate of 1.2 ml/min. Detection was by UV absorbance at 302 nm. S-Mephenytoin 4 -hydroxylation and testosterone 6 -hydroxylation were determined using HPLC as described (Yamazaki et al., 1994b; Shimada et al., 1985; Shimada et al., 1994). P450 contents were estimated spectrally by the original method (Omura and Sato, 1964). The contents of human P450 proteins in liver microsomes were estimated by coupled sodium dodecyl sulfatepolyacrylamide gel electrophoresis/immunochemical development ( Western-blotting ) (Guengerich et al., 1982). The intensities of the immunoblots were measured with an Epson GT-8000 Scanner equipped with NIH Image/Gel Analysis Program adapted for Macintosh computers. Protein concentrations were estimated by the method of Lowry et al. (1951). Statistical analysis. Kinetic parameters for the omeprazole oxidation by human P450 enzymes were estimated using a computer program (KaleidaGraph program from Synergy Software, Reading, PA) designed for nonlinear regression analysis. The correlations between activities of S-mephenytoin 4 -hydroxylation, testosterone 6 -hydroxylation and omeprazole 5-hydroxylation and omeprazole sulfoxidation in different human liver microsomal preparations were analyzed using a linear regression analysis program (InStat program from GraphPad Software, San Diego, CA). Statistical analysis was analyzed by Student s t test. Results Oxidation of omeprazole by liver microsomes of different human samples. Omeprazole (10 M concentration) was incubated in vitro with liver microsomes of 84 human samples and the levels of 5-hydroxyomeprazole and omeprazole sulfone thus formed were compared with activities of S-mephenytoin 4 -hydroxylation and testosterone 6 -hydroxylation, two prototypic reactions catalyzed by CYP2C19 and CYP3A4, respectively (fig. 1) (Goldstein and de Morais, 1994; Guengerich and Shimada, 1991; Guengerich et al., 1986). Correlation coefficients between activities of omeprazole 5-hydroxylation and S-mephenytoin 4 -hydroxylation and of omeprazole 5-hydroxylation and testosterone 6 -hydroxylation in these human samples were found to be 0.64 and 0.67, respectively, suggesting that CYP2C19 and 3A4 enzymes may play important roles for the omeprazole 5- hydroxylation reaction. There was good correlation between omeprazole sulfoxidation and testosterone 6 -hydroxylation activities in these human samples (r 0.86). To determine which P450 forms are more active in catalyzing oxidation of omeprazole in human liver microsomes, three human samples who showed relatively high levels of omeprazole oxidation activities were selected for further

3 436 Yamazaki et al. Vol. 283 Fig. 1. Correlation between S-mephenytoin 4 -hydroxylation and omeprazole 5-hydroxylation (A), testosterone 6 -hydroxylation and omeprazole 5-hydroxylation (B) and testosterone 6 -hydroxylation and omeprazole sulfoxidation (C) in liver microsomes of 84 human samples. analysis (table 1). A human sample, HL-C15, which contained high levels of CYP2C19 had higher activities for S- mephenytoin 4 -hydroxylation, and CYP3A4-rich samples HL-C6 and HL-C19 showed high activities for testosterone 6 -hydroxylation in liver microsomes (table 1). These three samples also had relatively high omeprazole 5-hydroxylation and sulfoxidation activities, although the ratio of activities at substrate concentrations of 10 and 400 M differed somewhat, depending on the samples used. For example, the ratio of activities of omeprazole 5-hydroxylation between 10 and 400 M omeprazole was high in liver microsomes of HL-C15, indicating that different P450 enzymes contribute to the oxidation of omeprazole in human liver microsomes, depending on the substrate concentrations determined. Effects of fluvoxamine, ketoconazole and sulfaphenazole on oxidations of S-mephenytoin, testosterone, and omeprazole by human liver microsomes. The effects of fluvoxamine, ketoconazole, and sulfaphenazole, known inhibitors of CYP2C19, CYP3A4 and CYP2C9 (fig. 2) (Guengerich and Shimada, 1991; Xu et al., 1996; Yamazaki et al., 1996b; Brian et al., 1989), respectively, were examined TABLE 1 P450 contents and substrate oxidation activities of liver microsomes of three human samples towards oxidation of S-mephenytoin (fig. 2A), testosterone (fig. 2D), and omeprazole in liver microsomal samples HL- C15 and HL-C6. Fluvoxamine was found to be inhibitory towards omeprazole 5-hydroxylation catalyzed by liver microsomes of human sample HL-C15 (fig. 2B), although it very weakly inhibited the omeprazole 5-hydroxylation by those of human sample HL-C6 (fig. 2C). 5-Hydroxylation of omeprazole by human sample HL-C6 was significantly inhibited by ketoconazole (fig. 2C). Omeprazole sulfoxidation activities were inhibited very significantly by ketoconazole in both human samples (figs. 2E and F). None of the activities were inhibited by sulfaphenazole at concentration of 100 M. Effects of anti-cyp2c9 and anti-cyp3a4 on omeprazole 5-hydroxylation and sulfoxidation by human liver microsomes. Effects of anti-cyp2c9 and anti-cyp3a4 on oxidation of omeprazole by human liver microsomes were studied in samples HL-C15 and HL-C6 (fig. 3). Anti-CYP2C9 antibodies have already been characterized and shown to inhibit CYP2C19-dependent S-mephenytoin 4 -hydroxylation activities as well as CYP2C9-dependent tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activ- P450 HL-C6 HL-C15 HL-C19 (pmol P450/mg protein) Total P CYP2C CYP3A Substrate oxidation (pmol product formed/min/mg protein) S-Mephenytoin 4 -hydroxylation (400 M) a Testosterone 6 -hydroxylation (200 M) a Omeprazole 5-hydroxylation (10 M) a (400 M) a (Ratio) b (0.19) (0.34) (0.20) Omeprazole sulfoxidation (10 M) a (400 M) a (Ratio) b (0.14) (0.13) (0.14) a Substrate concentrations. b Ratio of activities of omeprazole oxidation when determined at substrate concentrations of 10 and 400 M.

4 1997 Oxidation of Omeprazole by CYP2C19 and 3A4 437 ities in human liver microsomes (Shimada et al., 1986; Brian et al., 1989). Anti-CYP2C9 was found to significantly inhibit omeprazole 5-hydroxylation catalyzed by liver microsomes of a human sample HL-C15 (fig. 3A), but not of HL-C6 (fig. 3B). This antibody failed to inhibit omeprazole sulfoxidation activities in both samples. However, anti-cyp3a4 inhibited omeprazole sulfoxidation activities of both human samples (figs. 3C and D) and completely inhibited omeprazole 5-hydroxylation catalyzed by a human sample HL-C6 (fig. 3D), although the inhibition of omeprazole 5-hydroxylation by anti-cyp3a4 in sample HL-C15 was only 50% (fig. 3C). Omeprazole hydroxylation activities by recombinant human CYP2C19 and CYP3A4 expressed in different chimeras. To define the roles of CYP2C19 and CYP3A4 more clearly, we examined the omeprazole hydroxylation activities by recombinant human P450 enzymes (fig. 4). The recombinant P450 enzymes used were obtained from three different sources, e.g., microsomes of human lymphoblastoid cells (Gonzalez et al., 1991; Crespi et al., 1990), yeast Fig. 2. Effects of fluvoxamine, ketoconazole and sulfaphenazole on activities of S-mephenytoin 4 -hydroxylation (A), testosterone 6 -hydroxylation (D), omeprazole 5-hydroxylation (B and C) and omeprazole sulfoxidation (E and F) in liver microsomes of human samples HL-C15 (A, B and E) and HL-C6 (C, D and F). Concentrations of two inhibitors used are shown in the left side of the figures. Substrate concentrations used were 400 M for S-mephenytoin, 200 M for testosterone, and 10 M for omeprazole. microsomes (Imaoka et al., 1996), and baculovirus microsomes (P. M. Shaw, N. A. Hosea, D. V. Thompson, J. M. Lenius and F. P. Guengerich, submitted for publication). HPLC analysis showed that recombinant CYP2C19 produced 5-hydroxylated product of omeprazole (peak a in the chromatograms), although turnover numbers varied with source of enzymes used (fig. 4 A-C). Insect microsomes showed the highest turnover numbers for the formation of the 5-hydroxy product and also the unidentified metabolite (peak c; possibly 5-O-desmethylomeprazole) (Birkett et al., 1996). Recombinant CYP3A4 also produced formation of 5-hydroxyomeprazole as well as of omeprazole sulfone (fig. 4D to F), although the turnover numbers for 5-hydroxylation activities were lower than those catalyzed by CYP2C19. Of the three CYP3A4 enzyme systems examined, the baculovirus sample showed the highest turnover numbers for omeprazole 5-hydroxylation and sulfoxidation and formation of the unidentified metabolite (peak b; possibly 3-hydroxyomeprazole) (Birkett et al., 1996). Fig. 3. Effects of anti-cyp2c9 (A and B) and anti-cyp3a4 (C and D) on omeprazole 5-hydroxylation (E) and sulfoxidation (F) catalyzed by liver microsomes of HL-C15 (A and C) and HL-C6 (B and D). Preimmune IgG did not inhibit the microsomal catalytic activities determined (data not shown). Oxidation of omeprazole was determined at a substrate concentration of 10 M.

5 438 Yamazaki et al. Vol. 283 were determined to be 6.6 M and 8.3 nmol/min/nmol P450 for CYP2C19 and 60 M and 2.4 nmol/min/nmol P450 for CYP3A4, respectively. Omeprazole sulfoxidation was catalyzed by recombinant CYP3A4 with K m and V max values of 140 M and 13 nmol/min/nmol P450. Prediction of omeprazole hydroxylation by human liver microsomes based on kinetic parameters of recombinant P450 enzymes. To define the potential contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation by three human samples (HL-C6, C15 and C19), we calculated the predicted activities of human liver microsomal omeprazole 5-hydroxylation activities based on the kinetic parameters of CYP2C19 and CYP3A4 and estimated contents of these P450 proteins by immunoblotting. The equation used for the prediction of expected activities was from the previous method (Iwatsubo et al., 1997b): Fig. 4. Omeprazole hydroxylation activities by recombinant human CYP2C19 (4 nm P450; A, B and C) and CYP3A4 (40 nm P450; D, E and F) expressed in human lymphoblast cells (A and D), in yeast (B and E) and in baculovirus (C and F). Peak a, 5-hydroxyomeprazole; peak b, unknown (suggested to be 3-hydroxyomeprazole); peak c, unknown (suggested to be 5-O-desmethylomeprazole); peak d, omeprazole sulfone. Substrate concentration used was 10 M. Because baculosomal P450 enzymes examined in this study had high catalytic activities for these drug oxidation reactions, we determined kinetic parameters for omeprazole 5-hydroxylation activities by recombinant CYP2C19 and CYP3A4 in insect microsomes (fig. 5). K m and V max values v A [V max1 S/(K m1 S)] B [V max2 S/(K m2 S)] where v predicted activities of omeprazole 5-hydroxylation (pmol product formed/min/mg protein); A CYP2C19 content (nmol/mg protein); B CYP3A4 content (nmol/mg protein); S substrate concentration; K m1 and V max1, kinetic parameters from recombinant CYP2C19; and K m2 and V max2, kinetic parameters from recombinant CYP3A4. With the estimated contents of CYP2C19 and CYP3A4 shown in table 1 and kinetic parameters from recombinant CYP2C19 and CYP3A4 (fig. 5), we found that the omeprazole 5-hydroxylation activities obtained from human liver microsomes fitted with those obtained from predictions using kinetic parameters of recombinant P450 enzymes and the levels of CYP2C19 and CYP3A4 determined immunochemically (fig. 6). We also compared the omeprazole 5-hydroxylation activities (at a substrate concentration of 10 M) in experimental and prediction results in 28 human samples and found that there were some relations between these activities (fig. 7). Omeprazole 5-hydroxylation activities by recombinant CYP2C19 and CYP3A4 when these two P450 enzymes were mixed at ratio similar to those in human Fig. 5. Kinetic analysis of omeprazole 5-hydroxylation by recombinant human CYP2C19 (E) and CYP3A4 (F) expressed in baculosomes. K m and V max values were determined to be 6.6 M and 8.3 nmol/min/nmol P450 for CYP2C19 and 60 M and 2.4 nmol/min/nmol P450 for CYP3A4, respectively. Fig. 6. Omeprazole hydroxylation by liver microsomes of human samples HL-C6 (E), HL-C15 (F) and HL-C19 ( ). Different concentrations of omeprazole were used for the determinations of omeprazole 5-hydroxylation activities. Symbols, measured activities; lines, predicted values.

6 1997 Oxidation of Omeprazole by CYP2C19 and 3A4 439 Fig. 7. Correlation between measured and predicted values of omeprazole hydroxylation activities in liver microsomes of 28 human samples. The substrate concentration used was 10 M. liver microsomes. Because both CYP2C19 and CYP3A4 are suggested to be involved in the 5-hydroxylation of omeprazole in human liver microsomes, we determined the omeprazole oxidation by recombinant P450 enzymes mixed at ratio similar to those estimated in liver microsomal samples HL-C6, HL-C15 and HL-C19 (fig. 8). The HPLC profiles of omeprazole oxidation formed by recombinant CYP2C19 and CYP3A4 mixtures were found to be resemble those in human liver microsomes. We also determined the omeprazole oxidation by recombinant CYP3A4 and CYP2C19 enzymes when these two forms were mixed at ratio of 1, 3, 30, 100 and 300 (fig. 9). In this experiment, two substrate concentrations of 10 and 400 M omeprazole were used. At a 10 M omeprazole concentration, formation of 5-hydroxylated product (and unidentified peak c) was decreased and that of omeprazole sulfone (and unidentified peak b) was increased with increasing the ratio of levels of CYP3A4 and CYP2C19 in the incubation mixture (figs. 9A-E). However, at a 400 M omeprazole, such changes in chromatographic profile of omeprazole oxidation by the mixture of CYP3A4 and CYP2C19 were not so drastic, probably due to the preferential role of CYP3A4 at this substrate concentration (figs. 9 F-J). Discussion Our results supported the view that both CYP3A4 and CYP2C19 are involved in the 5-hydroxylation of omeprazole (when determined at a substrate concentration of 10 M) in human liver microsomes, on the basis of the following lines of evidence. First, there were some correlations between omeprazole 5-hydroxylation activities and S-mephenytoin 4 -hydroxylation (r 0.64) and testosterone 6 -hydroxylation (r 0.67) in liver microsomes of 84 human samples. Second, both fluvoxamine and ketoconazole, known inhibitors of CYP2C19 and CYP3A4, respectively (Xu et al., 1996; Yamazaki et al., 1996b; Guengerich and Shimada, 1991), inhibited the formation of 5-hydroxyomeprazole, depending on which P450 enzymes are more abundant in liver microsomes of different human samples examined. Third, a similar tendency was also noted when specific antibodies raised against CYP2C9, Fig. 8. Oxidation of omeprazole (at a concentration of 10 M) by liver microsomes of human samples of HL-C6 (A), HL-C15 (B), and HL-C19 (C) and recombinant human P450 mixtures containing 0.5 pmol of CYP2C19 and 49.5 pmol of CYP3A4 (D), 9.7 pmol of CYP2C19 and 40.3 pmol of CYP3A4 (E), and 1.4 pmol of CYP2C19 and 48.6 pmol of CYP3A4 (F). Peak a, 5-hydroxyomeprazole; peak b, unknown (suggested to be 3-hydroxyomeprazole); peak c, unknown (suggested to be 5-O-desmethylomeprazole); peak d, omeprazole sulfone. which inhibit both CYP2C9 and 2C19 activities (Shimada et al., 1986; Brian et al., 1989), and CYP3A4 antibodies were used for the effects on omeprazole 5-hydroxylation activities by human liver microsomes. The role of CYP2C9 in the oxidation of omeprazole by human liver microsomes was ruled out, because sulfaphenazole, a selective inhibitor of CYP2C9, did not inhibit the activities by liver microsomes. Finally, both recombinant human CYP2C19 and CYP3A4 had omeprazole 5-hydroxylation activity (at low substrate concentrations); the former enzyme was an enzyme with low K m and high V max and the CYP3A4 gave high K m and low V max values. Average levels of CYP2C19 and CYP3A4 in liver microsomes have been determined to be about 1 and 30%, respectively, of total P450 in different human samples examined (Inoue et al., 1997; Shimada et al., 1994). Although the K m value of recombinant CYP2C19 for omeprazole 5-hydroxylation in baculosomes was about one-tenth that of CYP3A4, the contribution of these P450 enzymes in the catalytic activities may be affected by the levels of these two P450 forms in human liver microsomes. In fact, when the recombinant

7 440 Yamazaki et al. Vol. 283 Fig. 9. HPLC profiles of omeprazole oxidation by recombinant (insect microsomal) CYP3A4 and CYP2C19, when these two forms were mixed at ratio of 1 (A and F), 3 (B and G), 30 (C and H), 100 (D and I) and 300 (E and J) in the incubation mixture. Sum of the levels of CYP3A4 and CYP2C19 in the incubation mixture was set up to be 50 pmol P450 in each of these cases. Substrate concentrations determined were 10 M omeprazole (A-E) and 400 M omeprazole (F-J). Other details are referred to the legend to figure 8. Omeprazole metabolites in chromatograms A to E were detected at range between 0 to 0.004, while those in chromatograms F to J were between 0 to CYP2C19 and CYP3A4 were mixed at ratio similar to those found in liver microsomes of different humans, HPLC profiles of oxidation of omeprazole were found to be resemble to those in intact microsomal incubations. These results suggest that CYP3A4 as well as CYP2C19 is involved in the oxidation of omeprazole, depending on the human samples examined. It has been reported that contributions of several P450 forms toward oxidations of xenobiotics in vitro are affected by the substrate concentrations determined (Yamazaki et al., 1994a, 1996a; Kato and Yamazoe, 1994; Iwatsubo et al., 1997a). In this study, we observed that at a 10 M substrate concentration, 5-hydroxyomeprazole formation was found to be decreased and omeprazole sulfone formation was increased with the increasing the ratio of levels of recombinant CYP3A4 and CYP2C19 in the incubation mixture (fig. 9). However, at a 400 M substrate concentration, both omeprazole 5-hydroxylation and sulfoxidation activities were determined largely by the presence of CYP3A4, but not CYP2C19. These results suggest that at higher substrate concentrations, both 5-hydroxylation and sulfoxidation of omeprazole are catalyzed principally by CYP3A4 in human liver microsomes. Extrapolation from in vitro data to intrinsic metabolism of clinically used drugs in humans has been a subject of interest in many laboratories, because of the potential usefulness in predicting pharmacological actions and safety of these drugs in vivo (Iwatsubo et al., 1997a, 1997b; Crespi, 1995). Kinetic parameters obtained from recombinant human P450 enzymes toward oxidation of drugs are generally used for the calculation, because kinetic parameters of the drug oxidation activities by structurally defined P450s can be determined (Iwatsubo et al., 1997a; Crespi, 1995). However, it must be assumed that all of the recombinant P450 enzymes obtained from different chimeric organisms give the same kinetic parameters, i.e., K m and V max values (Crespi, 1995), to use this approach in a valid manner. In fact, our results show that recombinant CYP2C19 and CYP3A4 expressed in three different chimeras such as human lymphoblastoid cells, yeast and baculosomes have different turnover numbers for the oxidation of omeprazole. Using kinetic parameters from baculovirus-expressed CYP2C19 and CYP3A4 and levels of human liver P450 proteins determined immunochemically, however, we obtained good correlations between predicted and experimental values of omeprazole 5-hydroxylation by liver microsomes; the calculation was based on a suggested procedure reported recently (Iwatsubo et al., 1997a, 1997b). These results again support the view that both CYP2C19 and CYP3A4 are involved in omeprazole 5-hydroxylation by human liver microsomes, and that predictions can be made if kinetic parameters for the oxidation of drugs by recombinant P450 enzymes and levels of individual P450 forms in human liver microsomes have already been determined. However, estimation of the modeling to the in vivo situation is yet another step, and other assumptions must be considered. In this study, we used the P450 inhibitors ketoconazole and fluvoxamine for studies of inhibition of CYP3A4- and CYP2C19-dependent oxidations of omeprazole in human liver microsomes. It is well known that ketoconazole is a potent and selective inhibitor of CYP3A enzymes in humans and experimental animals both in vitro and in vivo (Xu et al., 1996; Yamazaki et al., 1996b; Guengerich and Shimada, 1991). However, detailed studies are lacking regarding which chemicals are selective and potent inhibitors of CYP2C19 in humans, although sulfaphenazole has been determined to be a potent and relatively selective inhibitor of another CYP2C member, CYP2C9 (Brian et al., 1989). Recent studies have shown that fluvoxamine, a potent inhibitor of CYP1A2 (Br sen et al., 1993; Gjerving et al., 1995; Jeppesen et al., 1996b), is also an inhibitor of CYP2C19 in vivo in humans (Jeppesen et al., 1996a; Xu et al., 1996). In this work, fluvoxamine was found to inhibit CYP2C19-dependent S-mephenytoin 4 -hydroxylation activities by human liver microsomes. Inhibition of omeprazole 5-hydroxylation activities by fluvoxamine and anti-cyp2c9 in liver microsomes of a human sample HL-C15, a sample having a high level of CYP2C19 in the liver, supported the view that omeprazole 5-hydroxylation is catalyzed mainly by CYP2C19 in this subject. However, omeprazole 5-hydroxylation by liver microsomes of a human sample HL-C6 (which had a high level of CYP3A4) was inhibited weakly by fluvoxamine and anti-cyp2c19, but very strongly by ketoconazole and anti-cyp3a4, suggesting that CYP3A4 is a major form involved in the 5-hydroxylation of omeprazole in this human sample. Our studies have been done in liver microsomes, which contain both CYP2C19 and CYP3A4. However, the in vivo situation may also be influenced by the extent of omeprazole oxidation that occurs in the small intestine. CYP3A4 is known to be highly abundant in the small intestine (Kolars et al., 1994; Watkins et al., 1987) but the concentration of CYP2C19 in small intestine has not been measured, to our knowledge. In conclusion, our results supported the view that CYP3A4 as well as CYP2C19 is involved in the 5-hydroxylation of omeprazole in human liver microsomes. The contribution of these two P450 forms in the omeprazole 5-hydroxylation reaction may be determined by the levels and ratio of CYP2C19 and CYP3A4 in liver microsomes of different hu-

8 1997 Oxidation of Omeprazole by CYP2C19 and 3A4 441 man samples. Using kinetic parameters from recombinant CYP2C19 and CYP3A4 and levels of liver microsomal CYP2C19 and CYP3A4 determined immunochemically, predicted values for the 5-hydroxylation of omeprazole were roughly related to the activities of intact liver microsomes of different human samples. Acknowledgments The authors thank Dr. T. Ishizaki of International Medical Center of Japan for supplying omeprazole and its metabolites and Dr. Y. Sugiyama of University of Tokyo for preprint of a paper submitted. References ANDERSSON, T., MINERS, J. O., VERONESE, M. E., TASSANEEYAKUL, W., MEYER, U. A. AND BIRKETT, D. J.: Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism. Br. J. Clin. Pharmacol. 36: , BIRKETT, D. J., ANDERSSON, T. AND MINERS, J. O.: Assays of omeprazole metabolism as a substrate probe for human CYP isoforms. Methods Enzymol. 272: , BR sen, K., Skjelbo, E., Rasmussen, B. B., Poulsen, H. E. and Loft, S.: Fluvoxamine is a potent inhibitor of cytochrome P4501A2. Biochem. Pharmacol. 45: , BRIAN, W. R., SRIVASTAVA, P. K., UMBENHAUER, D. R., LLOYD, R. S. AND GUENGERICH, F. P.: Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae. Biochemistry 28: , CHANG, M., DAHL, M. -L., TYBRING, G., GOTHARSON, E. AND BERTILSSON, L.: Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: Comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype. Pharmacogenetics 5: , 1995a. CHANG, M., TYBRING, G., DAHL, M. -L., GOTHARSON, E., SAGAR, M., SEENSALU, R. AND BERTILSSON, L.: Interphenotype differences in disposition and effect on gastrin levels of omeprazole -suitability of omeprazole as a probe for CYP2C19. Br. J. Clin. Pharmacol. 39: , 1995b. CHIBA, K., KOBAYASHI, K., MANABE, K., TANI, M., KAMATAKI, T. AND ISHIZAKI, T.: Oxidative metabolism of omeprazole in human liver microsomes: cosegregation with S-mephenytoin 4 -hydroxylation. J. Pharmacol. Exp. Ther. 266: 52 59, CRESPI, C. L., LANGENBACH, R. AND PENMAN, B. W.: The development of a panel of human cell lines expressing specific human cytochrome-p450 cdnas. In Mutation and the Environment, Part B: Metabolism, Testing Methods, and Chromosomes, ed. by M. L. Mendelsohn and R. J. Albertini, pp , Wiley Liss, New York, CRESPI, C. L.: Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research. Adv. Drug Res. 26: , DE MORAIS, S. M. F., WILKINSON, G. R., BLAISDELL, J., MEYER, U. A., NAKAMURA, K. AND GOLDSTEIN, J. A.: Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese. Mol. Pharmacol. 46: , GJERVING, K., POULSEN, H. E., DOEHMER, J.AND LOFT, S.: Kinetics and inhibition by fluvoxamine of phenacetin O-deethylation in V79 cells expressing human CYP1A2. Pharmacol Toxicol 76: , GOLDSTEIN, J. A. AND DE MORAIS, S. M. F.: Biochemistry and molecular biology of the human CYP2C subfamily. Pharmacogenetics 4: , GOLDSTEIN, J. A., FALETTO, M. B., ROMKES-SPARKS, M., SULLIVAN, T., KITAREEWAN, S., RAUCY, J. L., LASKER, J. M. AND GHANAYEM, B. I.: Evidence that CYP2C19 is the major (S)-mephenytoin 4 -hydroxylase in humans. Biochemistry 33: , GONZALEZ, F. J.: The molecular biology of cytochrome P450s. Pharmacol. Rev. 40: , GONZALEZ, F. J., CRESPI, C. L. AND GELBOIN, H. V.: cdna-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment. Mutat. Res. 247: , GUENGERICH, F. P., WANG, P. AND DAVIDSON, N. K.: 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, F. P., MARTIN, M. V., BEAUNE, P. H., KREMERS, P., WOLFF, T. AND WAXMAN, D. J.: Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem. 261: , GUENGERICH, F. P.: Reactions and significance of cytochrome P-450 enzymes. J. Biol. Chem. 266: , GUENGERICH, F. P. AND SHIMADA, T.: Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol. 4: , GUENGERICH, F. P.: Analysis and characterization of enzymes. In Principles and Methods of Toxicology, ed. 3, ed. by A. W. Hayes, pp , Raven Press, New York, IBEANU, G. C., GHANAYEM, B. I., LINKO, P., LI, L., PEDERSEN, L.G.AND GOLDSTEIN, J. A.: Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole hydroxylase activity. J. Biol. Chem. 271: , IEIRI, I., KUBOTA, T., URAE, A., KIMURA, M., WADA, Y., MAMIYA, K., YOSHIOKA, S., IRIE, S., AMAMOTO, T., NAKAMURA, K., NAKANO, S. AND HIGUCHI, S.: Pharmacokinetics of omeprazole (a substrate of CYP2C19) and comparison with two mutant alleles, CYP2C19 m1 in exon 5 and CYP2C19m2 in exon 4, in Japanese subjects. Clin. Pharmacol. Ther. 59: , IMAOKA, S., YAMADA, T., HIROI, T., HAYASHI, K., SAKAI, T., YABUSAKI, Y. AND FUNAE, Y.: 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, F. P. AND SHIMADA, T.: 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: , ISHIZAKI, T., SOHN, D. -R., KOBAYASHI, K., CHIBA, K., LEE, K. H., SHIN, S. -G., ANDERSSON, T., REGARDH, C. -G., LOU, Y. -C., ZHANG, Y., DAHL, M.-L. AND BERTILSSON, L.: Interethnic differences in omeprazole metabolism in the two S-mephenytoin hydroxylation phenotypes studied in Caucasians and Orientals. Ther. Drug Monit. 16: , IWATSUBO, T., HIROTA, N., OOIE, T., SUZUKI, H., SHIMADA, N., CHIBA, K., ISHIZAKI, T., GREEN, C. E., TYSON, C. A. AND SUGIYAMA, Y.: Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol. Ther. 73: 1 25, 1997a. IWATSUBO, T., SUZUKI, H., SHIMADA, N., CHIBA, K., ISHIZAKI, T., GREEN, C. E., TYSON, C. A., YOKOI, T., KAMATAKI, T. AND SUGIYAMA, Y.: Prediction of in vivo hepatic metabolic clearance of YM796 from in vitro data using human liver microsomes and recombinant P-450 isozymes. J. Pharmacol. Exp. Ther. 282: , 1997b. JEAN, P., LOPEZ-GARCIA, P., DANSETTE, P., MANSUY, D. AND GOLDSTEIN, J. L.: Oxidation of tienilic acid by human yeast-expressed cytochromes P-450 2C8, 2C9, 2C18 and 2C19. Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9. Eur. J. Biochem. 241: , JEPPESEN, U., GRAM, L. F., VISTISEN, K., LOFT, S., POULSEN,H.E.AND BR sen, K.: Dose-dependent inhibition of CYP1A2, CYP2C19, and CYP2D6 by citalopram, fluoxetine, fluvoxamine and paroxetine. Eur. J. Clin. Pharmacol. 51: 73 78, 1996a. JEPPESEN, U., LOFT, S., POULSEN, H.E.AND BR sen, K.: A fluvoxamine-caffeine interaction study. Pharmacogenetics 6: , 1996b. KARAM, W. G., GOLDSTEIN, J. A., LASKER, J. M. AND GHANAYEM, B. I.: Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes. Drug Metab. Dispos. 24: , KATO, R. AND YAMAZOE, Y.: The importance of substrate concentration in determining cytochromes P450 therapeutically relevant in vivo. Pharmacogenetics 4: , KOLARS, J. C., LOWN, K. S., SCHMIEDLIN-REN, P., GHOSH, M., FANG, C., WRIGHTON, S. A., MERION, R. M. AND WATKINS, P. B.: CYP3A gene expression in human gut epithelium. Pharmacogenetics 4: , LEE, C. A., KADWELL, S. H., KOST, T. A. AND SERBJIT-SINGH, C. J.: CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. Arch. Biochem. Biophys. 319: , LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: , MARINAC, J. S., BALIAN, J. D., FOXWORTH, J. W., WILLSIE, S. K., DAUS, J. C., OWEN, R. AND FLOCKHART, D. A.: Determination of CYP2C19 phenotype in black Americans with omeprazole: Correlation with genotype. Clin. Pharmacol. Ther. 60: , MIMURA, M., BABA, T., YAMAZAKI, H., OHMORI, S., INUI, Y., GONZALEZ, F. J., GUENGERICH, F. P. AND SHIMADA, T.: Characterization of cytochrome P450 2B6 in human liver microsomes. Drug Metab. Dispos. 21: , NELSON, D. R., KOYMANS, L., KAMATAKI, T., STEGEMAN, J. J., FEYEREISEN, R., WAXMAN, D. J., WATERMAN, M. R., GOTOH, O., COON, M. J., ESTABROOK, R.W., GUNSALUS, I. C. AND NEBERT, D. W.: P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6: 1 42, OMURA, T. AND SATO, R.: The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239: , PENMAN, B. W., REECE, L., SMITH, T., YANG, C. S., GELBOIN, H. V., GONZALEZ,F.J. AND CRESPI, C. L.: Characterization of a human cell line expressing high levels of cdna-derived CYP2D6. Pharmacogenetics 3: 28 39, ROST, K. L. AND ROOTS, I.: Nonlinear kinetics after high-dose omeprazole caused by saturation of genetically variable CYP2C19. Hepatology 23: , RYAN, D. E. AND LEVIN, W.: Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacol. Ther. 45: , SHIMADA, T., SHEA, J. P. AND GUENGERICH, F. P.: A convenient assay for mephe-

9 442 Yamazaki et al. Vol. 283 nytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450. Anal. Biochem. 147: , SHIMADA, T., MISONO, K. S. AND GUENGERICH, F. P.: 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., IWASAKI, M., MARTIN, M. V. AND GUENGERICH, F. P.: Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA1535/pSK1002. Cancer Res. 49: , SHIMADA, T., YAMAZAKI, H., MIMURA, M., INUI, Y. AND GUENGERICH, F. P.: 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: , SULLIVAN-KLOSE, T. H., GHANAYEM, B. I., BELL, D. A., ZHANG, Z. Y., KAMINSKY, L. S., SHENFIELD, G. M., MINERS, J. O., BIRKETT,D.J.AND GOLDSTEIN, J. A.: The role of the CYP2C9-Leu 359 allelic variant in the tolbutamide polymorphism. Pharmacogenetics 6: , VANDENBRANDEN, M., RING, B. J., BINKLEY, S. N. AND WRIGHTON, S. A.: Interaction of human liver cytochromes P450 in vitro with LY307640, a gastric proton pump inhibitor. Pharmacogenetics 6: 81 91, WATKINS, P. B., WRIGHTON, S. A., SCHUETZ, E. G., MOLOWA, D. T. AND GUZELIAN, P. S.: Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man. J. Clin. Invest. 80: , XU, Z. -H., XIE, H.-G.AND ZHOU, H. -H.: In vivo inhibition of CYP2C19 but not CYP2D6 by fluvoxamine. Br. J. Clin. Pharmacol. 42: , YAMAZAKI, H., GUO, Z., PERSMARK, M., MIMURA, M., INOUE, K., GUENGERICH, F. P. AND SHIMADA, T.: Bufuralol hydroxylation by cytochrome P450 2D6 and 1A2 enzymes in human liver microsomes. Mol. Pharmacol. 46: , 1994a. YAMAZAKI, H., MIMURA, M., SUGAHARA, C. AND SHIMADA, T.: Catalytic roles of rat and human cytochrome P450 2A enzymes in testosterone 7 - and coumarin 7-hydroxylations. Biochem. Pharmacol. 48: , 1994b. YAMAZAKI, H., INOUE, K., MIMURA, M., ODA, Y., GUENGERICH, F. P. AND SHIMADA, T.: 7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes. Biochem. Pharmacol. 51: , 1996a. YAMAZAKI, H., URANO, T., HIROKI, S. AND SHIMADA, T.: Effects of erythromycin and roxithromycin on oxidation of testosterone and nifedipine catalyzed by CYP3A4 in human liver microsomes. J. Toxicol. Sci. 21: , 1996b. Send reprint requests to: Dr. T. Shimada, Osaka Prefectural Institute of Public Health, 3 69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan.