Formation, reactivity, and antiplatelet activity of mixed disulfide conjugates of clopidogrel.
In this work, we investigated the formation, reactivity, and antiplatelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the 10 thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol, and 3-nitropyridine-2-thiol (NPT). The mass spectrometry (MS) and MS(2) spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M(-1)s(-1). The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pretreatment with 1 mM GSH, confirming that the AM is responsible for the antiplatelet activity of clopidogrel. Collectively, our results provide strong support for a cytochrome P450 (P450)-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate, followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored to overcome the interindividual variability in clopidogrel therapy.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-5632, USA. haom@umich.edu
Haoming
H
Zhang
D Adam
DA
Lauver
Benedict R
BR
Lucchesi
Paul F
PF
Hollenberg
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D000077144:Clopidogrel; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011817:Rabbits; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D010975:Platelet Aggregation Inhibitors; D000077144:Clopidogrel; D013988:Ticlopidine