Glutaredoxin is involved in the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate.
Clopidogrel is a thienopyridine antiplatelet agent that is converted to the active metabolite, R-361015, in vivo. Clopidogrel is first oxidized to a thiolactone intermediate R-115991. R-115991 is thought to be metabolized to a GSH conjugate of R-361015 (R-361015-SG) and then is reduced to R-361015 in the presence of GSH. In this study, we investigated the enzyme-mediated formation of R-361015 from R-361015-SG in human liver microsomes and cytosols. After incubation of R-115991 in human liver microsomes, the formation of R-361015-SG, and subsequently of R-361015, was observed. The apparent formation rate of R-361015-SG was markedly decreased when human liver cytosols were added. Fitting the data to the kinetic model showed that the rate constant of R-361015-SG reduction to R-361015 in human liver microsomes was approximately 20-fold higher in the presence of human liver cytosols (6.56 min⁻¹) than in the absence of cytosols (0.326 min⁻¹). In addition, the formation rate of R-361015 from R-361015-SG was higher in human liver cytosols (2843 ± 1176 pmol · min⁻¹ · mg⁻¹) compared with in human liver microsomes (508 ± 396 pmol · min⁻¹ · mg⁻¹). The formation of R-361015 from R-361015-SG in human liver microsomes or cytosols was inhibited by anti-human glutaredoxin antibody in a concentration-dependent manner. Recombinant human glutaredoxin mediated the formation of R-361015 from R-361015-SG with the K(m) and V(max) values of 30.0 ± 1.3 μM and 381.6 ± 209.8 pmol · min⁻¹ · μg⁻¹, respectively. The intrinsic clearance value (V(max)/K(m)) was 12.9 ± 7.5 μl · min⁻¹ · μg⁻¹. In conclusion, we found that human glutaredoxin is a main contributor to the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate in human liver.
Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-Ku, Tokyo 140-8710, Japan. hagihara.katsunobu.fc@daiichisankyo.co.jp
Katsunobu
K
Hagihara
Miho
M
Kazui
Atsushi
A
Kurihara
Toshihiko
T
Ikeda
Takashi
T
Izumi
D001711:Biotransformation; D000077144:Clopidogrel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054477:Glutaredoxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008954:Models, Biological; D010084:Oxidation-Reduction; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000378:metabolism
D004791:Enzyme Inhibitors; D054477:Glutaredoxins; D010975:Platelet Aggregation Inhibitors; D011355:Prodrugs; D011994:Recombinant Proteins; D000077144:Clopidogrel; D005978:Glutathione; D013988:Ticlopidine