Stereoselective inhibition of human platelet aggregation by R-138727, the active metabolite of CS-747 (Prasugrel, LY640315), a novel P2Y₁₂ receptor inhibitor

Summary

CS-747 (Prasugrel, LY640315) is a thienopyridine antiplatelet prodrug that is metabolized to the thiol-containing active metabolite R-138727, which binds to and irreversibly inhibits the platelet P2Y₁₂ ADP receptor. R-138727 is composed of 4 stereoisomers, (R, S)-, (R, R)-, (S, S)-, and (S, R)-isomers (the first letter for the configuration of a chiral center at the sulfur-bearing position and the second for that at the benzylic position). In the present study, we determined the stereoselectivity of P2Y₁₂ antagonist effects by assessing the antagonism of the [³H]-2-MeS-ADP that binds to human P2Y₁₂ receptors expressed in Chinese hamster ovary cells as an affinity assay, and by the inhibition of ADP-induced aggregation of washed human platelets as a functional assay. R-138727 and its 2 components, R-99224, a mixture of (R, S)- and (S, R)-isomers and R-100364, a mixture of (R, R)- and (S, S)-isomers, inhibited [³H]-2-MeS-ADP binding and platelet aggregation. The rank order of potency of these compounds were identical in both assays: R-99224>R-138727>> R-100364. Inhibition of ADP-induced platelet aggregation by R-138727 and R-99224 was concentration- and time-related. In experiments using the 4 single stereo-isomers, all isomers inhibited ADP-induced platelet aggregation, but the (R, S)-isomer was found to be the most potent, followed by the (R, R)-isomer. These in vitro studies indicate that R-138727 is an effective antagonist of P2Y₁₂ and potent inhibitor of ADP-induced platelet aggregation, and that these antiplatelet activities of R-138727 are largely dependent on its (R, S)-isomer. This suggests that the (R)- configuration of the reactive thiol group of the active metabolite of CS-747 is critical for P2Y₁₂ and platelet inhibitory activities.

Keywords

Prasugrel - P2Y₁₂ receptors - platelet aggregation - ADP - thienopyridine

• References

• 1 Gachet C. Identification, characterization, and inhibition of the platelet ADP receptors.. Int J Hematol 2001; 74: 375-81.
• 2 Hollopeter G, Jantzen HM, Vincent D. et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs.. Nature 2001; 11: 202-7.
• 3 Sugidachi A, Asai F, Yoneda K. et al. Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine- type Gi-linked P2T antagonist, CS-747.. Br J Pharmacol 2001; 132: 47-54.
• 4 Andre P, Delaney SM, LaRocca T. et al. P2Y₁₂ regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.. J Clin Invest 2003; 112: 398-406.
• 5 Foster CJ, Prosser DM, Agans JM. et al. Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.. J Clin Invest 2001; 107: 1591-8.
• 6 Dorsam RT, Kunapuli SP. Central role of the P2Y₁₂ receptor in platelet activation.. J Clin Invest 2004; 113: 340-5.
• 7 Conley PB, Delaney SM. Scientific and therapeutic insights into the role of the platelet P2Y₁₂ receptor in thrombosis.. Curr Opin Hematol 2003; 10: 333-8.
• 8 Gent M, Blakely JA, Easton JD. et al. The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke.. Lancet 1989; 1: 1215-20.
• 9 Hass WK, Easton JD, Adams HP. et al. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. Ticlopidine Aspirin Stroke Study Group.. New Engl J Med 1989; 321: 501-7.
• 10 CAPRIE Steering Committee.. A randomised, blinded, trial of clopidogrel versus aspirin in patients atrisk of ischaemic events (CAPRIE).. Lancet 1996; 348: 1329-39.
• 11 The CURE Trial Investigators.. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation.. N Engl J Med 2001; 345: 494-502.
• 12 Savi P, Pereillo JM, Uzabiaga MF. et al. Identification and biological activity of the active metabolite of clopidogrel.. Thromb Haemost 2000; 84: 891-6.
• 13 Sugidachi A, Asai F, Ogawa T. et al. The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties.. Br J Pharmacol 2000; 129: 1439-46.
• 14 Kazui M, Ishizuka T, Yamamura N. et al. Mechanism for production of pharmacologically active metabolites of CS-747, a new pro-drug ADP-receptor antagonist.. Thromb Haemost 2001; 86 (suppl.) P1916.
• 15 Baurand A, Raboisson P, Freund M. et al. Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist.. Eur J Pharmacol 2001; 412: 213-21.
• 16 Sugidachi A, Ogawa T, Asai F. et al. Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D.. Thromb Haemost 1998; 79: 614-9.
• 17 Boyer JL, Mohanram A, Camaioni E. et al. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2’-deoxyadenosine 3’,5’-bisphosphate.. Br J Pharmacol 1998; 124: 1-3.
• 18 Kunapuli SP, Ding Z, Dorsam RT. et al. ADP receptors- targets for developing antithrombotic agents.. Curr Pharmaceut Design 2003; 9: 2303-16.
• 19 Yoneda K, Iwamura R, Kishi H. et al. Identification of the active metabolite of ticlopidine from rat in vitro metabolites.. Br J Pharmacol 2004; 142: 551-7.
• 20 Reist M, Roy-de Vos M, Montseny JP. et al. Very slow chiral inversion of clopidogrel in rats: a pharmacokinetic and mechanistic investigation.. Drug Metab Dispos 2000; 28: 1405-10.
• 21 Pereillo JM, Maftouh M, Andrieu A. et al. Structure and stereochemistry of the active metabolite of clopidogrel.. Drug Metab Dispos 2002; 30: 1288-95.
• 22 Savi P, Labouret C, Delesque N. et al. P2Y₁₂, a new platelet ADP receptor, target of clopidogrel.. Biochem Biophys Res Commun 2001; 283: 379-83.
• 23 Ding Z, Kim S, Dorsam RT. et al. Inactivation of the human P2Y₁₂ receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270.. Blood 2003; 101: 3908-14.
• 24 Copeland RA, Williams JM, Giannaras J. et al. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.. Proc Natl Acad Sci 1994; 91: 11202-6.
• 25 Kijima H, Isobe Y, Muramatsu M. et al. Structureactivity characterization of an H2-receptor antagonist, 3-amino-4-[4-[4-(1-piperidinomethyl)-2-pyridyloxy]- cis-2-butenylamino]-3-cyclobutene-1,2-dione hydrochloride (IT-066), involved in the insurmountable antagonism against histamine-induced positive chronotropic action in guinea pig atria.. Biochem Pharmacol 1998; 55: 151-7.
• 26 Winkler JD, Sung CM, Chabot-Flecher M. et al. β-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase.. Mol Pharmacol 1998; 53: 322-9.