Summary
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 µM), selective inhibition of ADP-induced platelet aggregation (IC50 = 1.8 µM) 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.
Abbreviations: ADP: adenosine 5’diphosphate; 2-MeS-ADP: 2-methylthioadenosine-5’-diphosphate; Bmax:
maximum binding capacity; IC50: concentration which inhibits 50% of the activity; Kd: dissociation constant; LC/MS: Liquid chromatography coupled to mass spectrometry;
NMR: Nuclear magnetic resonance
Key words
Clopidogrel - ADP - aggregation - platelets - metabolite
