Determination of the active and inactive metabolites of prasugrel in human plasma by liquid chromatography/tandem mass spectrometry

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148

ABSTRACT:Prasugrel, a prodrug, is a novel and potent inhibitor of platelet aggregation in vivo. The metabolism of prasugrel and the elimination and pharmacokinetics of its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), three inactive metabolites, and radioactivity were determined in five healthy male subjects after a single 15-mg (100 Ci) Clopidogrel and ticlopidine are thienopyridine prodrugs that are activated in vivo to pharmacologically active metabolites that bind irreversibly to the platelets' P2Y 12 receptor, thus inhibiting platelet aggregation. Studies with [14 C]clopidogrel showed that the major metabolic pathway in humans is hydrolysis of clopidogrel to an inactive acid analog; no further metabolites were reported (Lins et al., 1999). Prasugrel (Fig. 1) is a novel and potent thienopyridine prodrug that also inhibits platelet aggregation in vivo. The structure of the thiol-containing active metabolite of prasugrel, R-138727, was previously reported (Sugidachi et al., 2000(Sugidachi et al., , 2001. Similarly, the structure of the thiol-containing active metabolite of clopidogrel was determined by in vitro studies (Pereillo et al., 2002). The platelet-inhibitory activity of prasugrel and initial pharmacokinetic data were recently reported . 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). Compounds R-95913, R-106583, and R-100932 were measured in plasma as indicators for absorption and exposure to prasugrel and its active metabolite. In this report, the physiologic disposition of prasugrel in healthy subjects following a 15-mg (100 Ci) p.o. dose of [14 C]prasugrel is presented. Materials and MethodsRadiolabeled Drug and Chemicals. Prasugrel hydrochloride was provided by Sankyo Co., Ltd. (Tokyo, Japan). Article, publication date, and citation information can be found at

Section: Methodsmentioning

confidence: 99%

“…Determination of Prasugrel Metabolites in Plasma. Plasma concentrations of R-138727, R-95913, R-106583, and R-119251 were determined by validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods described previously (Farid et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

The Disposition of Prasugrel, a Novel Thienopyridine, in Humans

Farid¹,

Smith²,

Gillespie³

et al. 2007

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148

“…Quantification of R-95913, R-138727, and Glutathione Conjugate by LC-MS/MS. The assays were performed based on the methods described in the preceding section and a previous report by Farid et al, 2007b. Quantification of R-95913, MPBr-derivatized R-138727, and glutathione conjugate was carried out with an Alliance HPLC system, which consisted of 2690 Separations Module (Waters Corporation) coupled to a Quattro LC-MS/MS system (Waters Corporation) with the ESI source in positive ion mode.…”

Section: Mechanism For Prasugrel Active Metabolite Formationmentioning

confidence: 99%

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

Hagihara

Kazui²,

Kurihara³

et al. 2010

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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...

“…Prasugrel ( Fig. 1) has a single dominant metabolic pathway leading to the active metabolite (Farid et al, 2007a). However, clopidogrel has two competing metabolic pathways for the parent compound, with the major pathway leading to the formation of an inactive metabolite, clopidogrel carboxylic acid derivative (Caplain et al, 1999).…”

mentioning

confidence: 99%

“…The clopidogrel carboxylic acid derivative is formed through ester hydrolysis by the human carboxylesterase (hCE) 1 (Tang et al, 2006). The minor pathway in clopidogrel metabolism yielding the active metabolite requires two sequential steps of cytochrome P450 (P450) biotransformation (Kurihara et al, 2005), whereas prasugrel bioactivation requires the hydrolysis of the ester and then oxidation of the formed thiolactone, R-95913 (Farid et al, 2007a) (Fig. 1), to form the active metabolite of prasugrel.…”

mentioning

confidence: 99%

The Biotransformation of Prasugrel, a New Thienopyridine Prodrug, by the Human Carboxylesterases 1 and 2

Williams

Jones²,

Ponsler³

et al. 2008

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119

Platelet aggregation and activation are serious concerns for patients who undergo percutaneous coronary intervention and stent placement. The underlying mechanism of platelet aggregation is mediated through two G protein-coupled P2 receptors, P2Y 1 and P2Y 12 (Gachet, 2001). P2Y 1 activation leads to a transient aggregation, whereas P2Y 12 activation maintains a sustained aggregation. To reduce platelet aggregation, the development of P2Y 12 -selective inhibitors has yielded the thienopyridine prodrugs, which include ticlopidine, clopidogrel (structures available in Farid et al., 2008), and prasugrel ( Fig. 1), a novel thienopyridine currently in clinical development.Although the active metabolites for prasugrel and clopidogrel have equipotency at the P2Y 12 receptor in vitro (Sugidachi et al., 2007), p.o. administered prasugrel is 10 and 100 times more effective on an equal-dose basis in inhibiting platelet aggregation than clopidogrel and ticlopidine, respectively (Niitsu et al., 2005). Clopidogrel and prasugrel differ markedly in the biotransformation pathways leading to their activation. Prasugrel (Fig. 1) has a single dominant metabolic pathway leading to the active metabolite (Farid et al., 2007a). However, clopidogrel has two competing metabolic pathways for the parent compound, with the major pathway leading to the formation of an inactive metabolite, clopidogrel carboxylic acid derivative (Caplain et al., 1999). The clopidogrel carboxylic acid derivative is formed through ester hydrolysis by the human carboxylesterase (hCE) 1 (Tang et al., 2006). The minor pathway in clopidogrel metabolism yielding the active metabolite requires two sequential steps of cytochrome P450 (P450) biotransformation (Kurihara et al., 2005), whereas prasugrel bioactivation requires the hydrolysis of the ester and then oxidation of the formed thiolactone, R-95913 (Farid et al., 2007a) (Fig. 1), to form the active metabolite of prasugrel. The oxidation of R-95913 has been shown to be mediated by several P450 enzymes but primarily by CYP3A and CYP2B6 (Rehmel et al., 2006).The carboxylesterases are a multigene family that hydrolyze compounds containing an ester, amide, or thioester linkage. Carboxylesterases are broadly expressed throughout the body with two major Article, publication date, and citation information can be found at