Identification and Biological Activity of the Active Metabolite of Clopidogrel

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; K_d: dissociation constant; LC/MS: Liquid chromatography coupled to mass spectrometry; NMR: Nuclear magnetic resonance

Key words

Clopidogrel - ADP - aggregation - platelets - metabolite

• References

• 1 Herbert JM, Frehel D, Vallée E, Kieffer G, Gouy D, Berger Y, Necciari J, Defreyn G, Maffrand JP. Clopidogrel, a novel antiplatelet and antithrombotic agent. Cardiovasc Drug Rev 1993; 11: 180-98.
• 2 CAPRIE Steering Committee, a randomized, blinded, trial of SR25990C versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 1996; 348: 1329-39.
• 3 Féliste R, Delebassée D, Simon MF, Chap H, Defreyn G, Vallée E, Douste-Blazy L, Maffrand JP. Broad spectrum antiplatelet activity of PCR5332 and PCR4099 involves the suppression of the effects of released ADP. Thromb Res 1987; 48: 403-15.
• 4 Herbert JM, Frehel D, Bernat A, Badorc A, Savi P, Delebassée D, Kieffer G, Defreyn G, Maffrand JP. Clopidogrel hydrogenosulfate. Drug Futur 1993; 18: 107-12.
• 5 Mills DCB, Puri R, Hu C-J, Minniti C, Grana G, Freedman MD, Colman RF, Colman RW. Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase. Arterioscler Thrombos 1992; 12: 430-6.
• 6 Savi P, Laplace MC, Maffrand JP, Herbert JM. Binding of [3H]-2-methylthio ADP to rat platelets: effect of clopidogrel and ticlopidine. J Pharm Exp Ther 1994; 269: 772-7.
• 7 Savi P, Bornia J, Salel V, Delfaud M, Herbert JM. Characterization of P2X1 purinoceptors on rat platelets: effect of clopidogrel. Brit J Haematol 1997; 98: 880-6.
• 8 Savi P, Beauverger P, Labouret C, Delfaud M, Salel V, Kaghad M, Herbert JM. Role of P2Y1 purinoceptor in ADP-induced platelet activation. FEBS letter 1998; 422: 291-5.
• 9 Ohlmann P, Laugwitz KL, Nurnberg B, Spicher K, Schultz G, Cazenave JP, Gachet C. The human platelet ADP receptor activates G(I2) proteins. Biochem J 1995; 312: 775-9.
• 10 Savi P, Laplace MC, Herbert JM. Evidence for the existence of two different ADP-binding sites on rat platelets. Thromb Res 1994; 76: 157-69.
• 11 Macfarlane DE, Mills DCB, Srivastava PC. Binding of 2-azidoadenosine [b-³²P] diphosphate to the receptor on intact human blood platelets which inhibits adenylate cyclase. Biochemistry 1982; 21: 544-9.
• 12 Cristalli G, Mills DCB. Identification of a receptor for ADP on blood platelets by photoaffinity labelling. Biochem J 1993; 291: 875-81.
• 13 Savi P, Herbert JM. ADP receptors on platelets and ADP-selective antiaggregating agents. Med Res Rev 1996; 16: 159-79.
• 14 Savi P, Heilmann E, Nurden P, Laplace MC, Bihour C, Kieffer G, Nurden AT, Herbert JM. Clopidogrel: An antithrombotic drug acting on the ADPdependent activation pathway of human platelets. Clin Appl Thrombosis/ Hemostasis 1996; 02: 35-42.
• 15 Savi P, Herbert JM, Pflieger AM, Dol F, Delebassée D, Combalbert J, Defreyn G, Maffrand JP. Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. Biochem Pharmacol 1992; 44: 527-32.
• 16 Pereillo JM, Andrieu A, Savi P, Maftouh M, Uzabiaga F, Pascal M, Herbert JM, Maffrand JP, Picard C. Structure and stereochemistry of the active metabolite of clopidogrel, Submitted.
• 17 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature (Lond) 1962; 194: 927-9.
• 18 Colman RW. Methods for studying platelet shape change, aggregation, and fibrinogen binding. In: Colman RW. Modern methods in pharmacology, vol 4. Methods for studying platelets and megakaryocytes. New York: Alan R. Liss; 1987: 33-40.
• 19 Meurs H, Kauffman HF, Koeter G, Devries K. Extraction of cyclic AMP for the determination in the competitive protein binding assay. Clin Chim Acta 1980; 106: 91-7.
• 20 Combalbert J, Savi P, Rouchon MC, Berger Y, Herbert JM, Maffrand JP. Influence of hepatic cytochrome P450 subfamilies induction on the microsomal hepatic metabolism and the antiaggregating activity of the thienopyridine clopidogrel (SR25990C). Proceedings of the European Society of Biochemical Pharmacology, Thirteenth European Workshop on Drug Metabolism. 1992 Sept 21-25; Bergamo (Italy).
• 21 Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y, Herbert JM. The antiaggregating activity of clopidogrel is due to a metabolic activation by hepatic cytochrome P450-1A. Thromb Haemost 1994; 72: 313-7.
• 22 Weber AA, Reimann S, Schror K. Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro . Br J Pharmacol 1999; 126: 415-20.
• 23 Herbert JM, Savi P. Non-specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro . Thromb Haemost 1999; 82: 156-7.
• 24 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-4.
• 25 Herbert JM, Savi P, Maffrand JP. Biochemical and pharmacological properties of clopidogrel: a new ADP receptor antagonist. Eur Heart J Supp 1999; 01 Supp A: A31-A40.
• 26 Defreyn G, Gachet C, Savi P, Driot F, Cazenave JP, Maffrand JP. Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits. Thromb Haemost 1991; 65: 186-90.