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

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-nitro5-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 European Union], 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-138727) from the thiolactone intermediate (R-95913) (Fig. 1). Materials and Methods Materials. R-138727 (prasugrel active metabolite), glutathione conjugate of R-138727, and R-95913 (prasugrel thiolactone metabolite) were synthesized by Ube Industries, Ltd. (Ube, Japan). 5,5 -Dithio-bis(2-nitrobenzoic acid) (DTNB) and tris(carboxyethyl)phosphine (TCEP), used as a reagent to reduce DTNB to 2-nitro-5-thiobenzoic acid (TNB), were purchased from SigmaAldrich (St. Louis, MO). Microsomes from insect cells that expressed CYP3A4 (cDNA-expressed CYP3A4) were purchased from Gentest Corporation (Woburn, MA). All other chemicals and reagents were commercially available and were of the highest grade. Water was purified by using a Milli-Q purification system (Millipore Corporation, Billerica, MA). Preparation of TNB-R-138727 Disulfide. A mixed disulfide of R-138727 with TNB (TNB-R-138727 disulfide) was prepared by reacting R-138727 with equimolar DTNB in an aqueous solution (Fig. 2) because the synthesis using the chemically unstable sulfenic acid-derivative of R-138727 and TNB was difficult to perform. After dilution of the reaction mixture with acetonitrile and after centrifugation, a 20l aliquot of the supernatant fraction was directly injected onto a YMC-Pack ODS-A (A-312) column (150 mm 6.0 mm i.d., 5 m; YMC Co., Ltd., Kyoto, Japan), maintained at ambient temperature. A high-performance liquid chromatography (HPLC) analysis was performed by using a low-pressure gradient elution system that consisted of an L-7100 Intelligent Pump, a D-7500 Chromato-Integrator, and an L-7400S UV Detector (Hitachi, Ltd., Tokyo, Japan). The mobile phase, 30% (v/v) acetonitrile in water that contained 0.01% (v/v) trifluoroacetic acid, was used at a flow rate of 1.0 ml/min. Profiling of the reaction products was performed with UV absorption detection at 220 nm. Two peaks in the chromatogram, which corresponded to the two diastereoisomers of TNB-R-138727 disulfide [retention times 19.18 (Peak A) and 21.57 (Peak B) min], were collected separately and lyophilized for structure elucidation by liquid chromatogN.A.F. has retired from Eli Lilly and Company. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.110.032086. ABBREVIATIONS: prasugrel, 2-acetoxy-5-( -cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine; DTNB, 5,5 -dithiobis(2-nitrobenzoic acid); TCEP, tris(carboxyethyl)phosphine; TNB, 2-nitro-5-thiobenzoic acid; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; ESI, electrospray ionization; MS, mass spectrometry; MPBr, m-methoxyphenacybromide; MS/MS, tandem mass spectrometry. 0090-9556/10/3806-898–904$20.00 DRUG METABOLISM AND DISPOSITION Vol. 38, No. 6 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 32086/3589665 DMD 38:898–904, 2010 Printed in U.S.A. 898 at A PE T Jornals on A ril 0, 2017 dm d.aspurnals.org D ow nladed from