Identification and Biological Activity of the Active Metabolite of Clopidogrel

Abstract

SummaryLike 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

P2 receptors and platelet function

“…The thienopyridine compounds are prodrugs which have to be metabolized by the liver in order to generate active metabolites. The active metabolite of clopidogrel [107] covalently binds cysteine residues of the P2Y 12 receptor, thus precluding the binding of ADP [108][109][110]. Moreover, it has been recently reported that clopidogrel's active metabolite disrupts homopolymers of the P2Y 12 receptor expressed in lipid rafts and partitions them out of lipid rafts [111], pointing to the importance of oligomerization and membrane localization on the function of this receptor.…”

Historical perspective on ADP-induced platelet activation

“…Concern about the toxic effects of ticlopidine (neutropenia, thrombotic thrombocytopenic purpura) led to the development of a similar prodrug, clopidogrel, that did not have these adverse effects and has become widely used. Both ticlopidine and clopidogrel exert their inhibitory effect through an active metabolite that has a thiol group that reacts irreversibly with a cysteine residue of P2Y 12 [138].…”

Atorvastatin does not affect the antiplatelet potency of clopidogrel when it is administered concomitantly for 5 weeks in patients with acute coronary syndromes

“…1 Recently, it was shown that cytochrome P-450, isoform 3A4 (CYP3A4), is primarily responsible for the metabolism and activation of clopidogrel in vivo. 2 Several studies have shown that clopidogrel decreases the incidence of coronary artery stent thrombosis and is approved for reduction of myocardial infarction, stroke, and vascular death in patients with atherosclerotic vascular disease.…”