Identification and Biological Activity of the Active Metabolite of Clopidogrel

TLDR

Clopidogrel, like ticlopidine, is inactive in vitro and requires intravenous or oral administration to exert antiaggregatory and antithrombotic effects. The study aims to demonstrate that clopidogrel can be converted into an active metabolite by human liver microsomes. The active metabolite’s structure was identified using LC/MS, NMR, and chiral supercritical fluid chromatography. The unstable metabolite, distinct from ticlopidine’s, irreversibly inhibits ADP‑receptor binding (IC₅₀ 0.53 µM), blocks ADP‑induced platelet aggregation (IC₅₀ 1.8 µM), and suppresses adenylyl cyclase down‑regulation, likely via a disulfide bridge with a platelet ADP‑receptor cysteine.

Abstract

Like ticlopidine, the ADP receptor antagonist clopidogrel is inactive in vitro and must be administered i.v. or orally to exhibit antiaggregatory and antithrombotic activities. We have previously shown that hepatic metabolism is necessary for activity. This study demonstrates that an active metabolite can be generated from human liver microsomes incubated with clopidogrel. Using several analytical methodologies (LC/MS, NMR, chiral supercritical fluid chromatography), we have identified its structure. In vitro, this highly unstable compound, different from that formed from ticlopidine, exhibited all the biological activities of clopidogrel observed ex vivo: Irreversible inhibition of the binding of 33P-2MeS-ADP to washed human platelets (IC50) = 0.53 microM), selective inhibition of ADP-induced platelet aggregation (IC)50 = 1.8 microM) and ADP-induced adenylyl cyclase down-regulation. The irreversible modification of the ADP-receptor site which is responsible for the biological activity could be explained by the formation of a disulfide bridge between the reactive thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor.