Biotransformation of Prasugrel, a Novel Thienopyridine Antiplatelet Agent, to the Pharmacologically Active Metabolite

Abstract

ABSTRACT:Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically active metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the active thiol metabolite in vitro. A mixed disulfide form of R-138727 with glutathione was found to be a possible precursor of R-138727 in vitro when glutathione was present. The rate constant for the reduction of the glutathione conjugate of R-138727 to R-138727 was increased by addition of human liver cytosol to the human liver microsomes. Thus, one possible mechanism for the ultimate formation of R-138727 in vitro can be through formation of a sulfenic acid mediated by P450s followed possibly by a glutathione conjugation to a mixed disulfide and reduction of the disulfide to the active metabolite R-138727. Prasugrel [Effient (Eli Lilly and Company, Indianapolis, IN) in the United States and Efient (Eli Lilly and Company) in the EuropeanUnion], clopidogrel [Plavix (Sanofi-Aventis, Paris, France)/Iscover (Bristol-Myers Squibb, New York, NY)], and ticlopidine (Ticlid; Sanofi-Aventis) are thienopyridine antiplatelet agents. Prasugrel has been shown to reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients with acute coronary syndrome that are undergoing percutaneous coronary intervention (Wiviott et al., 2007) (Effient package insert). The thienopyridines are prodrugs that are converted in vivo to their pharmacologically active metabolites that possess a thiol group via a corresponding thiolactone metabolite (Farid et al., 2010). However, with the exception of an oxidation step catalyzed by cytochrome P450 (Savi et al., 1994;Rehmel et al., 2006), the mechanism for the active metabolite formation from thienopyridines remained unknown until recently reported for ticlopidine and clopidogrel (Dansette et al., 2009). In the case of prasugrel, the active metabolite R-138727 was not detected when the thiolactone metabolite R-95913 was incubated with liver homogenates or microsomes in the absence of cofactors, but it was detected when NADPH and a reducing agent such as glutathione were added to the system (Kazui et al., 2000). A sulfenic acid, which is a likely intermediate for the activation of prasugrel, reacts readily with a thiol compound, typically glutathione in vivo, to yield a disulfide metabolite (Decker et al., 1991;Kassahun et al., 2001;Reddy et al., 2005;Dansette et al., 2009). The formed disulfide can be further reduced to provide a thiolcontaining compound. The objective of this study is to investigate the involvement of a sulfenic acid and a glutathione conjugate of R-138727 in the in vitro production of prasugrel's active metabolite (R-1...

Formation and Fate of a Sulfenic Acid Intermediate in the Metabolic Activation of the Antithrombotic Prodrug Prasugrel

“…During the preparation of this manuscript, data reporting the formation of mixed disulfides between thiol 7 and 2-nitro-5-thiobenzoic acid or GSH upon the metabolism of thiolactone 6 by recombinant P450 3A4 in the presence of NADPH and 2-nitro-5-thiobenzoic acid or GSH have appeared in the online version of an article (14). Our data are in agreement with these results.…”

“…Our data from microsomal oxidation of 5 using 18 O 2 show that this is not the case, which rules out this mechanism. (14). Their proposition was based on the formation of mixed disulfides between thiol 7 and 2-nitro-5-thiobenzoic acid or GSH, upon the metabolism of thiolactone 6 by recombinant P450 3A4 in the presence of NADPH and either 2-nitro-5-thiobenzoic acid or GSH (14).…”

Glutaredoxin and Thioredoxin Can Be Involved in Producing the Pharmacologically Active Metabolite of a Thienopyridine Antiplatelet Agent, Prasugrel

“…Addition of cytosolic fraction to liver microsomes has been shown to decrease the formation of R-133490 from R-95913 with an increase in the formation rate of R-138727. This result indicated that the glutathione conjugate generated could be immediately reduced to R-138727 in the presence of cytosol (Hagihara et al, 2010). In this study, we isolated and identified the enzymes in human liver cytosols catalyzing the in vitro glutathione-mediated reduction of R-133490 to R-138727 as glutaredoxin and thioredoxin.…”

“…R-135766 was used as the internal standard. The assays of R-138727 and R-133490 were performed following methods reported previously (Hagihara et al, 2010). Separation of the analytes by high-performance liquid chromatography (HPLC) was conducted using an Alliance2690 Separations Module (Waters, Milford, MA).…”

“…This intermediate could then be converted to a disulfide-type glutathione conjugate (R-133490) (Hagihara et al, 2009) and subsequently reduced to the pharmacologically active metabolite (R-138727) in the presence of another glutathione molecule ( Fig. 1) (Hagihara et al, 2010). Addition of cytosolic fraction to liver microsomes has been shown to decrease the formation of R-133490 from R-95913 with an increase in the formation rate of R-138727.…”