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SHORT COMMUNICATION

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

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.)

(Received May 21, 2007; accepted July 20, 2007)

	Abstract

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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 (K_m = 5.26 µM, intrinsic clearance (CL_int) = 0.34 µl/pmol P450/min), CYP1B1 (K_m = 11.2 µM, CL_int = 0.03 µl/pmol P450/min), and CYP3A5 (K_m = 2.89 µM, CL_int = 0.05 µl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K_m = 1.60 µM, CL_int = 0.57 µl/pmol P450/min), CYP2C19 (K_m = 5.95 µM, CL_int = 0.16 µl/pmol P450/min), CYP3A4 (K_m = 5.35 µM, CL_int = 0.10 µl/pmol P450/min), and CYP2C8 (K_m = 33.8 µM, CL_int = 0.009 µl/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.

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).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Metabolic pathways of cilostazol involving human hepatic P450 isozymes.

	Materials and Methods

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Reagents and Chemicals. Cilostazol, OPC-13015, OPC-13213, OPC-13217, 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.

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 µM 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 µl 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 µl of the sample was injected into the high-performance liquid chromatography system described below for determination of cilostazol and its metabolite concentrations.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Biotransformation of cilostazol via OPC-13326 (A) and OPC-13217 (B) by the cDNA-expressed human P450 isozymes. Cilostazol (50 µM) 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).

Calculations. The apparent V_max and K_m 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

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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 V_max values for the metabolite. The K_m, V_max, and CL_int values for the production of OPC-13326 from cilostazol by the CYP3A4 isozyme were 5.26 µM, 1.93 pmol/pmol P450/min, and 0.34 µl/pmol P450/min, respectively. CYP3A5 had the highest affinity (i.e., the lowest K_m values) (K_m = 1.60 µM), and CYP2C19 had the highest V_max (V_max = 0.94 pmol/pmol P450/min) for cilostazol in the production of OPC-13217. This accounts for their greater CL_int 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.

	Footnotes

 
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.

doi:10.1124/dmd.107.016758.

ABBREVIATIONS: P450, cytochrome P450; CL_int, 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-4-hydroxy-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).

Address correspondence to: Dr. Masahiro Hiratsuka, Department of Clinical Pharmacotherapeutics, Tohoku Pharmaceutical University, 4-4-1 Komataushima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan. E-mail: mhira{at}tohoku-pharm.ac.jp

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.016758v1 35/10/1730    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hiratsuka, M. Articles by Mizugaki, M. Search for Related Content PubMed PubMed Citation Articles by Hiratsuka, M. Articles by Mizugaki, M.