Article pubs.acs.org/crt
Metabolic Activation of Prasugrel: Nature of the Two Competitive Pathways Resulting in the Opening of Its Thiophene Ring Patrick M. Dansette,* Julien Rosi, Justine Debernardi, Gildas Bertho, and Daniel Mansuy Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France S Supporting Information *
ABSTRACT: The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, 1c, is its twostep enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate function leading to a thiolactone metabolite 2c. The second step was described as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone ring of 2c, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding active thiol 3c. This article describes a detailed study of the metabolism of 1c by human liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, after derivatization with 3′-methoxy phenacyl bromide. It shows that there are two competing metabolic pathways for the opening of the 2c thiolactone ring. The major one, which was previously described, results from a P450- and NADPH-dependent redox bioactivation of 2c and leads to 3c, two previously reported thiol diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results from a hydrolysis of 2c and leads to an isomer of 3c, 3c endo, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require NADPH. However, it requires Ca2+ and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone 2c exclusively leads to derivatives of the endo thiol isomer 3c endo.
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INTRODUCTION Ticlopidine (Ticlid) 1a, clopidogrel (Plavix, Iscover) 1b, and prasugrel (Effient) 1c (Figure 1) are antithrombotic prodrugs of the tetrahydrothienopyridine series that must be metabolized in vivo into the corresponding pharmacologically active 4mercapto-3-piperidinyliden acetic acid derivatives 3a, 3b, and 3c, respectively, to exert their activity as antagonists of the platelet receptor P2Y12.1−4 Ticlopidine was introduced to the market in 1979 for prevention of thrombotic stroke. Clopidogrel was first launched in 1998 in the USA and in 1999 in Europe, for the reduction of atherosclerotic problems in patients with stroke, myocardial infarction, or peripheral arterial disease. Their metabolic activation occurs in two steps that are catalyzed by cytochromes P450.1,2 The first step is a classical cytochrome P450dependent hydroxylation of the thiophene ring by NADPH and O2 leading to thiolactone metabolites 2a and 2b, respectively5−7 (Figure 1). It is mainly catalyzed by P450 2C19 and P450 2B6 in the case of 1a and by P450 2C19, P450 1A2, and P450 2B6 in the case of 1b.4,8 The second step © 2012 American Chemical Society
leading to the pharmacologically active thiols 3a and 3b also requires the involvement of a cytochrome P450-catalyzed oxidative step with consumption of NADPH and O2 (P450 3A4, P450 2B6, P450 2C9, and P450 2C19 are mainly involved in oxidation of 2b).8 Prasugrel is the newest member of the class of tetrahydrothienopyridine antithrombotic produgs. It is also an irreversible inhibitor of the platelet receptor P2Y12, after metabolic conversion in vivo to pharmacologically active 3c3,9 (Figure 1). Because of the presence of two chiral carbons in 3c, this thiol exists as a mixture of four stereoisomers (two pairs of enantiomers), and the [R,S] isomer is the most active one.9 Metabolic conversion of prasugrel to 3c involves two enzymatic reactions (Figure 1): (i) the hydrolysis of its ester function leading to thiolactone 2c, which seems to be mainly catalyzed by the hCE2 enzyme in humans,10 and (ii) the oxidative cleavage of the thioester bond of 2c with the eventual Received: January 18, 2012 Published: April 6, 2012 1058
dx.doi.org/10.1021/tx3000279 | Chem. Res. Toxicol. 2012, 25, 1058−1065
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Figure 1. Metabolic activation of tetrahydrothienopyridine antithrombotic drugs into pharmacologically active thiols.
Figure 2. Competing pathways involved in the metabolic opening of the thiophene ring of clopidogrel 1b.
microsomes17,18 and is present in much lower amounts than 3b in the sera of 1b-treated patients.19 It has been also shown17,18 that chemical, hydrolytic opening of the thiolactone ring of 2b, using O-nucleophiles such as CH3O−, only led to 3b endo derivatives, suggesting that hydrolytic opening of thiolactone 2b, either chemical or enzymatic (PON-1-catalyzed), only led to endo compounds. It is thus necessary to oxidize 2b (by P450s) to activate its conjugated CO function to produce the active cis thiols, 3b (Figure 2). It was recently proposed that PON-1 was a major determinant of 1b efficacy and that PON-1 Q192R polymorphism might have a key role in the rate of 1b bioactivation and influence platelet response to 1b and the risk of stent thrombosis in 1b-treated patients.20 However, since then, many articles have reported that there is no relationship between PON-1 polymorphism and 1b efficacy.21−29 Metabolites that should derive from 3c endo, the endo isomer of 3c (Figure 4), were detected in the plasma, urine, and feces of patients treated with prasugrel 1c30 and also of mice, rats, and dogs treated with this drug.31 However, the mechanism of formation of 3c endo was not determined so far. Thus, it was interesting to know whether 3c endo could derive from a thioesterase-catalyzed opening of the thiolactone ring of metabolite 2c, in competition with the P450-dependent oxidative opening of 2c leading to 3c, in a manner similar to what was very recently found in the case of clopidogrel. For that purpose, we have looked for the formation of such an endo isomer of 3c during the metabolism of 1c by human liver microsomes or human serum, by using HPLC methods
formation of 3c, which is catalyzed by cytochromes P450 3A4, 2B6, 2C9, or 2C19 in humans.4 Recent data have established a detailed pathway for the formation of active thiols 3 from thiolactones 2 in the metabolism of prodrugs 1.11−14 It involves a P450-catalyzed oxidative opening of thiolactone metabolites 2 leading to the formation of sulfenic acid intermediates such as 5b and a reduction of these sulfenic acids into the corresponding thiols 3 by GSH11−13,15 (see for instance Figure 2 for the case of 1b). Reduction of these sulfenic acids by GSH would occur via the intermediate formation of GSH conjugate disulfides that are reduced to thiols 3 either by GSH itself11,12 or by the glutaredoxin−thioredoxin system.16 These sulfenic acid intermediates have been trapped by dimedone during the oxidative metabolism of thiolactones 2 by rat and human liver microsomes or by several recombinant human liver cytochromes P450.11,12 A new metabolic pathway resulting in the opening of the thiolactone ring of clopidogrel metabolite 2b has been discovered quite recently.17,18 It leads to an isomer of 3b, 3b endo, in which the double bond has migrated into the piperidine ring (Figure 2). This isomer is formed upon treatment of 2b with human liver microsomes and human serum17,18 and is detected in the serum of 1b-treated patients.19 Its formation from 2b seems to be mainly catalyzed by paraoxonase-1 (PON-1) (Figure 2).17,18 It is noteworthy that 3b endo is formed in much lower amounts than 3b upon metabolism of 2b by NADPH-supplemented human liver 1059
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Figure 3. HPLC profiles of incubations of 1c with human liver microsomes with (B) or without (A) NADPH, or with human serum (C), using MS detection after derivatization with 3′-methoxy-phenacyl bromide and MS2 spectra (parent ion at m/z = 498) of the three derivatized thiol metabolites, 3c′ (2 diastereomers, E and F) and 3c′endo (D and G correspond to HPLC profiles A and C, respectively). Incubations of 1c with Human Sera. Human sera were obtained according to a previously published protocol and were given by J. F. Chassé.32 Typical incubations were performed in potassium phosphate buffer (0.1 M, pH 7.4) containing 2 mM CaCl2, human serum (10 mg protein/mL), 1c (100 μM), and a reducing agent (20 mM ascorbic acid or 5 mM GSH), at 37 °C for 30 min. Reactions were stopped and analyzed as described above for microsomal incubations. HPLC-MS Studies. Studies were performed on a Surveyor HPLC instrument coupled to a LCQ Advantage ion trap mass spectrometer (Thermo, Les Ulis, France), using a Gemini C18 column (100 × 2 mm, 3 μm; Phenomenex) and a gradient starting at 40% B for 1 min then increasing linearly to 100% B in 15 min (A = 10 mM ammonium acetate buffer, pH 4.6, and B = CH3CN/CH3OH/H2O (7:2:1)) at 200 μL/min. Mass spectra were obtained by electrospray ionization (ESI) in positive ionization mode detection under the following conditions: source parameters, sheeth gas, 20; auxiliary gas, 5; spray voltage, 4.5 kV; capillary temperature, 200 °C; capillary voltage, 15 V; and m/z range for MS recorded generally between 300 and 700 (except for exploratory experiments with a wider range 300−800). MS2 energy was tested between 20 and 40 eV and was generally 35 eV. For all products, the indicated parent ions corresponded to M + H+. Chemical Experiments on 1c. Reaction of 1c with CH3ONa in CH3OH. A solution of 2 mM 1c in CH3OH was treated with 120 mM CH3ONa in CH3OH at 20 °C for 45 min. After dilution with 10 vol 100 mM phosphate buffer at pH 7.4, the final product was analyzed by HPLC-MS, as such or after derivatization with MPBr, as described above, or after oxidation to 6, the disulfide of 3c endo methyl ester (Figure 5), with 5 mM iodine. Compound 6 was purified by loading on a SepPak C18 column (Waters), washing with H2O, eluting by CH3OH, and analyzing by 1H NMR (in CD2Cl2) after evaporation of CH3OH.
allowing a good separation of the isomers of 3c and trapping of these thiols by 4′-methoxy phenacyl bromide. This article shows that metabolite 3c endo is formed upon thioesterase-dependent opening of thiolactone 2c during the metabolism of 1c by human liver microsomes and human serum. Hydrolytic formation of 3c endo from 2c is in competition with the P450-dependent oxidative opening of the 2c thiolactone ring leading to the cis diastereomers 3c, as previously found in the metabolism of 2b (Figure 2).17,18 This article also shows that chemical opening of thiolactone 2c by sodium methylate only leads to an endo thiol isomer derivative, 3c endo methyl ester.
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EXPERIMENTAL PROCEDURES
Chemicals and Biochemicals. Prasugrel base (racemate), 1c, was obtained from Bepharm (Shangai, China). Oxo-prasugrel base (racemate), 2c, was obtained from Twisun Pharma (Shangai, China). All other products including enzymes were from SigmaAldrich (St. Quentin Fallavier, France). Microsomal Incubations. Human liver microsomes (pool, 10 mg protein/mL) were obtained from BD-Gentest (Le Pont de Claix, France). Typical incubations were performed in 200 μL of potassium phosphate buffer (0.1 M, pH 7.4) containing 2 mM CaCl2, microsomes (1 mg protein/mL), 1c (100 μM), and a reducing agent (20 mM ascorbic acid or 5 mM GSH) with or without the NADPH generating system (1 mM NADP, 15 mM glucose-6phosphate, and 2 unit/mL of glucose-6-phosphate dehydrogenase) at 37 °C for 30 min. Reactions were stopped by adding one-half volume of CH3CN and 3′-methoxy phenacyl bromide (MPBr) (final concentration 4 mM) for thiol derivatization. After 10 min of incubation, 25 μL/mL CH3COOH was added, and proteins were removed by centrifugation at 13,000g. 1060
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Figure 4. Two competing pathways involved in the metabolic opening of the thiophene ring of prasugrel 1c. Because of the presence of 2 chiral carbons (benzylic carbon and carbon bearing the SH function) in metabolite 3c, this metabolite can exist as a mixture of 2 diastereomers (4 optically active isomers [R,S] and [S,R] and [R,R] and [S,S]). NMR Experiments. 1H NMR spectra of 6 were done on a Bruker Avance 400 spectrometer (500 MHz) at 27 °C. Chemical shifts are given in ppm relative to (CH3)4Si.
Table 1. Formation of 3c and 3c endo upon Metabolism of 1c by Human Liver Microsomes
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(nmol/mg protein/30 min)b
RESULTS Metabolism of 1c by Human Liver Microsomes. Incubation of 1c with human liver microsomes was done for 30 min at 37 °C in the presence of NADPH, which is a required cofactor for P450-dependent activities,33 and of a reducing agent (20 mM ascorbic acid or 5 mM GSH) to reduce the sulfenic acid intermediate 5c into the corresponding thiol 3c (Figure 4).11,12 An HPLC-MS study of the incubate, after derivatization of the thiol metabolites by treatment with MPBr, showed the formation of the phenacyl derivatives of three thiol isomers (Figure 3) exhibiting a parent ion (ESI+) corresponding to M + H+ and characterized by a peak at m/z = 498. Two of these thiol isomers exhibited identical MS2 spectra, with major fragments of the parent ion (m/z = 498) at m/z = 348, 318, 206, 177, and 149, as previously described30,34 for the 3′methoxy phenacyl derivatives (3c′) of the two cis-thiol diastereomers 3c (Figure 4). The third derivatized thiol metabolite exhibited a different MS2 spectrum characterized by a major fragment of the parent ion (m/z = 498) at m/z = 206 (Figure 3), which was identical to that previously described for the 3′-methoxy phenacyl derivative of the endo thiol isomer, 3c′endo.30 This major ion at m/z = 206 should correspond to a retro Diels−Alder fragmentation of the dihydropyridine ring of 3c′endo. Such a fragmentation generally occurs in the case of the derivatives of 3b endo and 3c endo thiols, and the appearance of the corresponding very intense fragments seems to be a characteristic of these endo isomers.18,30 Analyses of the reaction mixtures were also done on the thiol metabolites themselves without derivatization. Three thiol metabolites exhibiting the expected parent ion at m/z = 350 could be detected with a ratio almost identical to that found for their phenacyl derivatives. Identical results were obtained in incubations using 2c instead of 1c as a starting product. When identical microsomal incubations of 1c were done in the absence of NADPH, there was no formation of the cis isomers 3c′ and the only isomer that could be detected was 3c′ endo (Table 1). Moreover, the addition of N-benzylimidazole (20 μM), a well-known inhibitor of microsomal P450s,35 to the
conditions
3c′
3c′ endo
complete systema + 20 μM N-benzyl-imidazole − ascorbate − NADPH − NADPH − CaCl2 − NADPH + 500 μM paraoxon − NADPH − CaCl2 + 5 mM EDTA
22 ± 4 1.7 ± 0.5 (7%)
