Short Communication Characterization of Human Cytochrome P450 Enzymes Involved in the Metabolism of Cilostazol

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (Km 5.26 M, intrinsic clearance (CLint) 0.34 l/pmol P450/min), CYP1B1 (Km 11.2 M, CLint 0.03 l/pmol P450/min), and CYP3A5 (Km 2.89 M, CLint 0.05 l/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (Km 1.60 M, CLint 0.57 l/pmol P450/min), CYP2C19 (Km 5.95 M, CLint 0.16 l/pmol P450/ min), CYP3A4 (Km 5.35 M, CLint 0.10 l/pmol P450/min), and CYP2C8 (Km 33.8 M, CLint 0.009 l/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively. Cilostazol is a cyclic nucleotide phosphodiesterase type 3 inhibitor with antiplatelet, antithrombotic, and vasodilating properties. It also exhibits antiproliferative effects on smooth muscle cells (Tsuchikane et al., 1997) and has beneficial effects on high-density lipoprotein cholesterol and triglyceride levels in vivo (Elam et al., 1998). The major pharmacologically active metabolites of cilostazol identified in the plasma of humans are OPC-13015 and OPC-13213 (Bramer et al., 1999). OPC-13015 is 3 times more potent than cilostazol with regard to inhibition of platelet aggregation, whereas OPC-13213 is 3 times less potent than cilostazol (Okuda et al., 1993). On the other hand, in vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route (Fig. 1) (Akiyama et al., 1985). The pharmacological potency of both OPC-13326 and OPC-13217 remains unclear. An early in vitro study with human liver microsomes and selective P450 inhibitors indicated that cilostazol is extensively metabolized to OPC-13326 via the hepatic enzyme CYP3A and to OPC-13217 via CYP2C19 (Abbas et al., 2000). It has also been reported that the other P450s, CYP1A2 and CYP2D6, may be partially involved in the metabolism of cilostazol (Abbas et al., 2000). Furthermore, significant drug interactions are observed when cilostazol is coadministered with other agents that inhibit CYP3A (e.g., erythromycin) (Suri et al., 1999) or CYP2C19 (e.g., omeprazole) (Suri and Bramer, 1999). To our knowledge, the specific P450 isozymes (e.g., CYP3A4 and/or CYP3A5) involved in the metabolism of cilostazol have not yet been identified by using individual P450 isozymes, and the enzyme kinetic parameters such as Km, Vmax, and intrinsic clearance (CLint) have not been determined. The objectives of this study were to identify and kinetically characterize the human P450 isozymes responsible for the two major metabolites of cilostazol, namely, OPC13326 and OPC-13217, by using recombinant human P450 isozymes. Materials and Methods Reagents and Chemicals. Cilostazol, OPC-13015, OPC-13213, OPC13217, OPC-13325, and OPC-3930 were provided by Otsuka Pharmaceutical Company (Tokushima, Japan). NADPH was purchased from Oriental Yeast Co. (Tokyo, Japan). Microsomes prepared from baculovirus-infected insect cells expressing CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were obtained from BD Gentest (Woburn, MA). Microsomes prepared from baculovirus-infected insect cells expressing CYP1A1, CYP1B1, CYP2A6, CYP2C8, CYP2J2, and CYP4A11 were obtained from Invitrogen (Carlsbad, CA). All recombinant P450s had been coexpressed with NADPH P450 oxidoreductase. Recombinant CYP2A6, CYP2C8, and CYP2J2 were also coexpressed with cytochrome b5. The other chemicals and reagents used in this study were of research grade. This work was supported by the High-Tech Research Center Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.016758. ABBREVIATIONS: P450, cytochrome P450; CLint, intrinsic clearance; SNP, single-nucleotide polymorphism; cilostazol, OPC-13013, 6-[4(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone); OPC-13326, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4hydroxy-2(1H)-quinolinone); OPC-13217, 3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone); OPC-3930, 6-(3-(1-cyclohexyl-1H-tetrazol-5-yl)propoxy-2(1H)-quinolinone). 0090-9556/07/3510-1730–1732$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 10 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 16758/3259002 DMD 35:1730–1732, 2007 Printed in U.S.A. 1730 at A PE T Jornals on M ay 3, 2017 dm d.aspurnals.org D ow nladed from Enzyme Assay. In vitro screening of cilostazol with the 14 human P450 baculosomes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11 was performed using a constant amount of cytochrome P450 (5 pmol/tube) and 0.2 to 50 M cilostazol. All incubations were carried out for 20 min, and the incubated mixture consisted of baculosomes, 3.3 mM magnesium chloride, 1.3 mM NADPH, and cilostazol in 250 l of 100 mM potassium phosphate buffer (pH 7.4). After preincubation at 37°C for 1 min, the reactions were initiated by the addition of substrate; it was then allowed to proceed at 37°C and was terminated with ice-cold acetonitrile. OPC-3930 was added as an internal standard (Tata et al., 2001). The incubation mixtures were centrifuged at 14,000 rpm for 5 min. After centrifugation, the supernatant was transferred into vials. The samples were stored at 4°C until analysis and 50 l of the sample was injected into the high-performance liquid chromatography system described below for determination of cilostazol and its metabolite concentrations. Analytical Procedures. The system used for analysis consisted of an Alliance 2695 separation module (Waters, Milford, MA), 2996 photodiode array detector (254 nm; Waters), and a Sunfire C18 column (particle size of 5 m, 4.6 mm 150 mm; Waters). The column temperature was maintained at 40°C. The mobile phase, consisting of water with 25% acetonitrile (A) and 60% acetonitrile (B), was operated at a constant flow rate (1.0 ml/min). The elution gradient was a linear increase to 68% B in 20 min. The retention times of OPC-13213, OPC-13217, OPC-13326, internal standard (OPC-3930), OPC13015, and cilostazol in this analytical condition were 5.6 min, 6.2 min, 12.3 min, 12.8 min, 15.3 min, and 17.4 min, respectively. Calculations. The apparent Vmax and Km parameters were calculated using a nonlinear regression program, SigmaPlot (HULINKS, Tokyo, Japan), fitted to the Michaelis-Menten and Eadie-Hofstee plots. The means of the data were obtained by at least 3 determinations. Results and Discussion The metabolic activities of the 14 human recombinant P450 isozymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11 toward cilostazol were evaluated. As shown in Fig. 2, CYP3A4 was found to be the most efficient for the production of OPC-13326, whereas CYP3A5 and CYP2C19 were the most efficient for the production of OPC-13217. CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6, CYP2E1, and CYP3A5 showed detectable activity for the production of OPC-13326, and CYP2B6, CYP2C8, CYP2D6, CYP2J2, and CYP3A4 showed detectable activity for the production of OPC-13217. Among those tested, the five most efficient human P450 isozymes, CYP1B1, CYP3A4, CYP3A5, CYP2C8, and CYP2C19, for cilostazol metabolism were selected to further kinetically characterize their metabolic activity. The kinetics of both OPC-13326 and OPC-13217 obtained from cilostazol by all the tested recombinant P450 isozymes were consistent with the Michaelis-Menten model. Furthermore the Eadie-Hofstee plots for the formation of metabolites showed a monophasic profile in all recombinant P450 isozymes tested (data not shown). As shown in Table 1, CYP3A4 was identified as the most efficient isozyme for generating OPC-13326, with the highest Vmax values for the metabolite. The Km, Vmax, and CLint values for the production of OPC-13326 from cilostazol by the CYP3A4 isozyme were 5.26 M, 1.93 pmol/pmol P450/min, and 0.34 l/pmol P450/ min, respectively. CYP3A5 had the highest affinity (i.e., the lowest Km values) (Km 1.60 M), and CYP2C19 had the highest Vmax (Vmax 0.94 pmol/pmol P450/min) for cilostazol in the production of FIG. 1. Metabolic pathways of cilostazol involving human hepatic P450 isozymes. FIG. 2. Biotransformation of cilostazol via OPC-13326 (A) and OPC-13217 (B) by the cDNA-expressed human P450 isozymes. Cilostazol (50 M) was metabolized by individual P450 isozymes (20 pmol/ml) with NADPH at 37°C for 20 min. The specific activities are expressed as picomoles of product generated per picomole of P450 isozyme per minute. The data shown are the mean S.E.M. (n 3). 1731 CILOSTAZOL METABOLISM BY HUMAN P450s at A PE T Jornals on M ay 3, 2017 dm d.aspurnals.org D ow nladed from OPC-13217. This accounts for their greater CLint values relative to the other P450 isozymes. The metabolism of cilostazol was examined by using microsomes expressing individual human P450 enzymes to kinetically characterize the P450 enzymes involved in the metabolic pathways of cilostazol. The results presented above indicate a major contribution of CYP3A4 to the formation of OPC-13326 from cilostazol and of CYP3A5 and CYP2C19 to the formation of OPC-13217. Although it was thought that cilostazol would be metabolized to OPC-13217 mainly via CYP2C19 (Abbas et al., 2000), CYP3A5 should be the key enzyme involved in the formation of OPC-13217 from cilostazol. Interestingly, both CYP3A5 (Kuehl et al., 2001; Xie et al., 2004; Roy et al., 2005) and CYP2C19 (de Morais et al., 1994a,b) are genetically polymorphic enzymes. For CYP3A5 variant alleles, the CYP3A5*3 allele is characterized by an A to G single nucleotide polymorphism in intron 3 of the CYP3A5 gene that creates a cryptic consensus splice site, and exon 3B causes a splicing defect (Kuehl et al., 2001). The allelic frequency of the inactive CYP3A5*3 has been reported to be 74% in Japanese, 93% in Caucasians, and 32% in African Americans (Hiratsuka et al., 2002; Roy et al., 2005). Numerous functional polymorphisms in the CYP2C19 gene have also been identified; some alleles (e.g., CYP2C19*2 and CYP2C19*3) are associated with poor metabolism of CYP2C19 substrate drugs (Ieiri et al., 1996). The individuals with poor CYP2C19 metabolism comprise 20% of the Japanese and 3% of Caucasians (Kimura et al., 1998). If CYP3A5 and CYP2C19 have a significant role in the metabolism of cilostazol in vivo as well as in vitro, the individuals with poor CYP3A5 and/or CYP2C19 metabolism might have higher plasma cilostazol concentrations than those who can extensively metabolize via CYP3A5 and/or CYP2C19. However, because both OPC-13015 and OPC-13213 are active, it would be difficult to assess the clinical outcome in subjects who polymorphically express CYP3A5 or CYP2C19 without in vivo data. To understand the mechanistic basis of our findings further, it would be of great value to clinically examine the relationship between the genotypes of both CYP3A5 and CYP2C19 and the plasma concentration of cilostazol and its metabolites. Acknowledgments. We acknowledge Chiharu Yokoyama, Junji Ikeda, Naoki Fujio, and Yasuo Ogawa (Otsuka Pharmaceutical Co.) for their support. Department of Clinical Pharmacotherapeutics (M.H., T.S., M.I.) and Department of Clinical Pharmaceutics (M.H., Y.H., T.S., Y.K., M.M.), Tohoku Pharmaceutical University, Sendai, Japan; and Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan (K.I.) MASAHIRO HIRATSUKA YUDAI HINAI TAKAMITSU SASAKI YUMIKO KONNO KENICHI IMAGAWA MASAAKI ISHIKAWA MICHINAO MIZUGAKI