Yumiko Konno

Functional characterization of human xanthine oxidase allelic variants.

Objective Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In this study, we speculated that individual variations in XO activity are caused by genetic variations in the XO gene. Methods To investigate the genetic variations in XO in 96 Japanese participants, denaturing high-performance liquid chromatography was used. To assess the effects of these variations on enzymatic activity, wild-type XO and 21 types of variant XO – including those in the database and those just discovered – were transiently expressed in COS-7 cells. Results Three nonsynonymous single nucleotide polymorphisms, including 514G>A (Gly172Arg), 3326A>C (Asp1109Thr), and 3662A>G (His1221Arg) were identified in Japanese participants. Functional characterization of 21 XO variants showed a deficiency in enzyme activity in two variants (Arg149Cys and Thr910Lys); low activity (intrinsic clearance, CLint: 22–69% compared with the wild-type) in six variants (Pro555Ser, Arg607Gln, Thr623Ile, Asn909Lys, Pro1150Arg, and Cys1318Tyr); and high activity (CLint: approximately two-fold higher than that in the wild-type) in two variants (Ile703Val and His1221Arg). Conclusion These results suggest that several single nucleotide polymorphisms in the XO gene are involved in individual variations in XO activity. In addition, such findings will be useful to identify xanthinuria patients.

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