PT  - JOURNAL ARTICLE
AU  - Haoming Zhang
AU  - Wei C Lau
AU  - Paul F. Hollenberg
TI  - Formation Of The Thiol Conjugates And Active Metabolite of Clopidogrel By Human Liver Microsomes
AID  - 10.1124/mol.112.079061
DP  - 2012 May 14
TA  - Molecular Pharmacology
PG  - mol.112.079061
4099  - http://molpharm.aspetjournals.org/content/early/2012/05/14/mol.112.079061.short
4100  - http://molpharm.aspetjournals.org/content/early/2012/05/14/mol.112.079061.full
AB  - We have previously reported 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 (Zhang et al, Mol. Pharmacol. 80:839-847 (2011)). In this work, we have extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four different reductants: GSH, L-cysteine (L-Cys), N-acetyl-L-cysteine (NAC) and ascorbic acid (ASC). Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amount of the AM formed was increased in the order of NAC (17%) < L-Cys (53%) < ASC (61%) < GSH (100%). In addition, 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 were dependent on the concentrations of GSH with similar Km values in the range of ~0.5 mM, indicating that formation of the thiol conjugates constitutes an integral part of the bioactivation processes of clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate with a t½ of ~10 hrs. Addition of dithiothreitol to the reaction mixture reversed the conversion resulting in a decrease in the AM-thiol conjugate with a concomitant increase in the AM, whereas addition of NAC led to formation of the AM-NAC with a concomitant decrease in AM-GSH. 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 potentially affect the effective concentration of the AM in vivo.