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

Abstract

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 P2Y12ADP receptor. R-138727 is composed of 4 stereo-isomers, (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.

Dissecting the Activation of Thienopyridines by Cytochromes P450 Using a Pharmacodynamic Assay In Vitro

“…Whether this is because SR-26334 is a poor substrate for cytochrome P450 oxidation or because the ring-opened version of the carboxylic acid is a poor antagonist for the platelet P2Y 12 receptor is unclear, although recent work suggests the former to be the case (Zahno et al, 2010). Metabolic activation of prasugrel, a relatively new choice for antiplatelet therapy, follows a somewhat similar pathway, which involves cleavage of the ester linkage to form the oxo intermediate [2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone] (R-95913) followed by ring opening to the AM, 2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene acetic acid (R-138727) (Sugidachi et al, 2001;Hasegawa et al, 2005). A number of esterases have been shown to catalyze the ester hydrolysis (Williams et al, 2008) although cytochromes P450 could also potentially catalyze this reaction.…”

The Disposition of Prasugrel, a Novel Thienopyridine, in Humans

“…In vivo, prasugrel is rapidly hydrolyzed to a pharmacologically inactive thiolactone (R-95913), followed by cytochrome P450-dependent ring opening to form the active metabolite R-138727 (Rehmel et al, 2006). The active metabolite of prasugrel possesses two chiral centers, and its four isomers were shown to possess varying degrees of activity toward inhibition of platelet aggregation (Hasegawa et al, 2005).…”

“…In vivo, prasugrel is rapidly hydrolyzed to a pharmacologically inactive thiolactone (R-95913), followed by cytochrome P450-dependent ring opening to form the active metabolite R-138727 (Rehmel et al, 2006). The active metabolite of prasugrel possesses two chiral centers, and its four isomers were shown to possess varying degrees of activity toward inhibition of platelet aggregation (Hasegawa et al, 2005).The prasugrel metabolites measured in human plasma in initial studies (Asai et al, 2006) indicated that after formation of R-95913, two thiol-containing compounds are formed, R-138727 and M4, which are further metabolized by S-methylation to R-106583 and R-100932, or conjugation with cysteine to R-119251 and R-118443 (Fig. 1).…”

Stereoselective Metabolism of Prasugrel in Humans Using a Novel Chiral Liquid Chromatography-Tandem Mass Spectrometry Method

“…The pharmacological action of prasugrel is the result of R-138727 binding irreversibly to the P2Y 12 platelet adenosine diphosphate receptor, thus inhibiting platelet activation and aggregation (Jakubowski et al, 2006). R-138727 possesses two chiral centers, and it has been reported that the four stereoisomers possess differing antiplatelet activity, with the two stereoisomers with the R-configuration at the thiol group (R-125689 and R-125690) being the most potent (Hasegawa et al, 2005).Xenobiotics that contain multiple chiral centers (stereocenters) pose a challenging dimension to the overall drug development process. The chemistry and pharmacological significance of multiple stereocenters have been reviewed (Testa et al, 1993;Tomaszewski and Rumore, 1994;Reist et al, 1995;Van Miert, 2003).…”

“…The pharmacological action of prasugrel is the result of R-138727 binding irreversibly to the P2Y 12 platelet adenosine diphosphate receptor, thus inhibiting platelet activation and aggregation (Jakubowski et al, 2006). R-138727 possesses two chiral centers, and it has been reported that the four stereoisomers possess differing antiplatelet activity, with the two stereoisomers with the R-configuration at the thiol group (R-125689 and R-125690) being the most potent (Hasegawa et al, 2005).…”