RT Journal Article
SR Electronic
T1 Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 302
OP 309
DO 10.1124/mol.112.079061
VO 82
IS 2
A1 Haoming Zhang
A1 Wei C. Lau
A1 Paul F. Hollenberg
YR 2012
UL http://molpharm.aspetjournals.org/content/82/2/302.abstract
AB We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839–847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) < l-Cys (53%) < ascorbic acid (61%) < GSH (100%). AM-thiol conjugates were observed in the presence of NAC, l-Cys, and GSH. In the case of GSH, the formation of both the AM and the glutathionyl conjugate was dependent on the GSH concentrations, with similar Km values of ∼0.5 mM, which indicates that formation of the thiol conjugates constitutes an integral part of the bioactivation processes for clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ∼10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo.