Abstract
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 Km and Vmax values of 30.0 ± 1.3 μM and 381.6 ± 209.8 pmol · min−1 · μg−1, respectively. The intrinsic clearance value (Vmax/Km) 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.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS:
- P450
- cytochrome P450
- MPBr
- methoxyphenacyl bromide
- LC-MS/MS
- liquid chromatography/tandem mass spectrometry
- KPB
- potassium phosphate buffer
- PON-1
- paraoxonase-1.
- Received March 28, 2012.
- Accepted June 25, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|