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In Vitro Biotransformation Studies of 2‑Oxo-clopidogrel: Multiple Thiolactone Ring-Opening Pathways Further Attenuate Prodrug Activation Yaoqiu Zhu*,† and Jiang Zhou‡ †

MetabQuest Research Laboratory, 202 Chengfu Road, Beijing 100871, P. R. China Analytical Instrumentation Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China

‡

S Supporting Information *

ABSTRACT: The biotransformation of clopidogrel has been under extensive investigation to address the observed high clinical variability and resistance of its antithrombotic prodrug therapy. Clopidogrel (M0) is first activated to its thiolactone intermediate, 2-oxo-clopidogrel (M2), by hepatic cytochrome P450 (P450) enzymes. Subsequent P450-catalyzed S-oxidation is followed by thioester hydrolysis, which cleaves the thiolactone ring and yields a sulfenic acid intermediate (M12); this intermediate is reduced to the final active metabolite (M13). The aim of the present study is to characterize the metabolic fates of M2 more comprehensively with focus on the thiolactone ring-opening pathways. It was found that the bioactivating S-oxidation confers on the thiolactone moiety not only one electrophilic site at the carbonyl C-atom (Site A), but also a second one at the allylic bridge C-atom (Site B). Both sites can react with H2O or other nucleophiles, like glutathione (GSH), leading to different thiolactone ring-opening pathways. In addition to the pharmacologically desired A-H2O pathway leading to M13 formation, the A-GSH pathway leads to the formation of a glutathione conjugate (GS-3), the B-H2O pathway leads to the formation of a desulfurized hydroxyl metabolite (M17), and the B-GSH pathway leads to the formation of a desulfurized glutathione conjugate (GS-2). These results demonstrate the reactive nature of the electrophilic thiolactone S-oxide intermediate (M11) and suggest that M13 formation from M2 might be accompanied by more attenuating pathways than previously reported. The research presented here may facilitate future studies exploring the clinical antithrombotic response to clopidogrel as well as the susceptibility to the adverse effect of clopidogrel and its close prodrug analogues.

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INTRODUCTION Clopidogrel is a thienopyridine antiplatelet prodrug that has been widely used in the treatment of cardiovascular diseases, including atherothrombosis, unstable angina, and myocardial infarction.1 As shown in Scheme 1, clopidogrel (M0) is activated through a two-step cytochrome P450 (P450) catalyzed process to form its active metabolite (M13).2,3 The first step is thiophene 2-oxidation leading to the formation of 2oxo-clopidogrel (M2). The second step is oxidative cleavage of the thiolactone ring of 2-oxo-clopidogrel to a sulfenic acid intermediate (M12). The pharmacologically active metabolite, M13, is formed after reduction of the sulfenic acid function of M12 to a thiol function. To address the clinically observed high intersubject variability leading to lower or no therapeutic response in 20−40% of the patients,4,5 the chemical mechanism of clopidogrel bioactivation has been under extensive investigation to identify the potential genetic factors that regulate the active metabolite formation. Recent research results have shown that the two steps of the prodrug activation are catalyzed by various P450s, and some genetic polymorphic enzymes like paraoxonase-1 (PON-1) or CYP2C19 do not play deciding roles in catalyzing the active metabolite formation.6−8 The high intersubject variability of © XXXX American Chemical Society

clopidogrel is related to a principle regarding metabolic stability of a “regular” drug: a higher percentage remaining of the parent molecule, the pharmacologically active form, which comes through metabolic attrition, leads to lower intra- and intersubject variability in its therapy.9 Applying this principle to a prodrug, of which a metabolite is the active form, a high percentage conversion of the prodrug to the active metabolite is desired. On the basis of this analogy, it has been speculated that the two-step bioactivation of clopidogrel might be accompanied by heavy metabolic attrition that significantly attenuates active metabolite formation.8,10 Our recent research has revealed that the first step of clopidogrel bioactivation, 2-oxidation of the thiophene motif leading to M2 formation, is significantly attenuated by CYP3A4/5-catalyzed oxidation of the nonactivating piperidine motif and the thiophene 3-position,8 in addition to previously reported methyl ester hydrolysis.10,11 In this study, we investigated the in vitro biotransformation of M2 and identified that the electrophilic nature of the thiolactone S-oxide intermediate leads to not only the pharmacologically desired Received: November 18, 2012

A

dx.doi.org/10.1021/tx300460k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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Scheme 1. Generally Accepted Two-Step Clopidogrel Bioactivation Pathwaysa

a

The O-atom derived from H2O is shown in bold. vortexed and incubated at room temperature for 12 h. The samples were then centrifuged at 10,800 rpm for 2 min before LC-MS/MS analysis. Formation of 2-Oxo-clopidogrel Metabolites in the Presence of H218O. An initial 200 μL incubation mixture containing 2.0 mg/mL HLM, 2.0 mM NADPH, 100 μM 2-oxo-clopidogrel, and 10 mM GSH in potassium phosphate buffer (200 mM, pH 7.4) was evenly divided into two vials (100 μL each). To one vial was added 100 μL of H2O (16O: 100%); to the other was added 100 μL of H218O (16O/18O: 50%/50%)). The two samples were incubated in parallel at 37 °C for 30 or 60 min before being quenched with equal volumes of MeOH/CH3CN (1/4, v/v) followed by consecutive LC-MS/MS analysis. Low Resolution LC-MS/MS Analysis. Chromatographic analysis was conducted on a Surveyor HPLC system consisting of an autosampler, an MS pump, and a photo diode array detector (Thermo Fisher Scientific, Waltham, MA) using a Zorbax SB-Phenyl column (4.6 × 75 mm, 3.5 μm, Agilent Technologies, Santa Clara, CA) at room temperature. The volume of each injection was 10 μL. The mobile phase consisted of H2O (solvent A, containing 0.1% formic acid) and MeOH (solvent B, containing 0.1% formic acid), and was delivered at 750 μL/min. The initial composition of solvent B was maintained at 10% for 2 min and then increased linearly to 50% by 7 min and 90% by 17 min. It was then maintained at 90% solvent B for 1 min before being decreased linearly to 10% by 19 min. The column was allowed to equilibrate at 10% solvent B for 1 min before the end of the 20 min gradient elution program for the next injection. The scan range of the photo diode array detector was set to be 220−400 nm. Mass spectrometric (MS) analysis was performed on a Thermo LCQ ion trap mass spectrometer (Thermo Fisher Scientific), which was interfaced to the above HPLC system. The HPLC eluate was split after the photo diode array detector, and 10% eluate (75 μL/min) was injected onto the mass spectrometer. MS analysis was conducted using a standard electrospray ionization (ESI) source operating under positive ionization mode. Source operating conditions were as follows: a spray voltage of 4.5 kV, heated capillary temperature of 225 °C, capillary voltage of 20 V, and sheath gas flow at 60 (arbitrary unit). The MS experiment parameters including the nitrogen gas flow rate, capillary voltage, and the tube lens voltages were all tuned and optimized using a 2-oxo-clopidogrel standard solution (10 μM in MeOH/H2O = 1/1, v/v). The MS full scans were monitored over a mass range of m/z 300 to 1000. Product ion (MS2) scans were generated via collision-induced dissociation (CID) with helium using a normalized collision energy of 60% and a precursor ion isolation width of m/z 2.0. Data was centroid and processed in Qual Browser

ring-opening pathway but also other pathways that further attenuate active metabolite formation.

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EXPERIMENTAL PROCEDURES

Chemicals and Reagents. 2-Oxo-clopidogrel hydrochloride (mixture of diastereomers) was purchased from Toronto Research Chemicals (North York, Ontario, Canada). β-Nicotinamide adenine dinucleotide 2′-phosphate (reduced, NADPH) was purchased from Roche Applied Sciences (Shanghai, China). L-Glutathione (GSH) was purchased from Acros (Beijing, China). Dithiothreitol (DTT) was purchased from J&K Scientific (Beijing, China). Pooled human liver microsomes (HLM) and cDNA-expressed supersomes (CYP2C19, CYP3A4, and CYP3A5) were purchased from BD Gentest (Woburn, MA). 18O water (H218O, 97% 18O) was purchased from Cambridge Isotope Laboratories (Andover, MA). Other chemicals of the highest available grade were purchased from Sigma-Aldrich (St. Louis, MO). 2-Oxo-clopidogrel Incubation in Pooled HLM. 2-Oxoclopidogrel was initially prepared as a 10 mM stock solution. The in vitro incubation mixture contained 1.0 mg/mL HLM and 1.0 mM NADPH, with 20, 100, or 200 μM 2-oxo-clopidogrel, with or without 5 mM GSH, with or without 100 mM KF, with or without 2 mM CaCl2, and with or without 5 mM ethylenediamine tetraacetate (EDTA), in a final volume of 100 or 200 μL of potassium phosphate (100 mM) buffer (pH 7.4). Control incubation samples were made by replacing NADPH with potassium phosphate buffer. The samples were incubated in a shaking water bath at 37 °C for 30 or 60 min. Each 100 μL incubation mixture was quenched with 15 μL of trichloroacetic acid (10%, w/v) or 100 μL of MeOH/CH3CN (1/4, v/v) and kept on ice for 5 min before being centrifuged at 12,000 rpm for 5 min on a 5415D Centrifuge (Eppendorf AG, Hamburg, Germany) to fully pelletize the precipitated protein. Supernatants were transferred to sealed vials and placed in an autosampler pending LC-MS/MS analysis. 2-Oxo-clopidogrel Incubation in cDNA-Expressed CYP2C19, CYP3A4, and CYP3A5. An incubation mixture of 25 pmol/L P450 supersomes also contained 1.0 mM NADPH, 100 μM 2-oxoclopidogrel, and 5 mM GSH in a final volume of 100 μL of potassium phosphate (100 mM) buffer (pH 7.4). The samples were incubated in a shaking water bath at 37 °C for 60 min before they were quenched with equal volumes of MeOH/CH3CN (1/4, v/v). The mixtures were centrifuged and treated as described above. Postincubation DTT Treatment. To the trichloroacetic acid or the organic solvent quenched supernatant (50 μL) from the above HLM incubation was added 1−2 mg DTT powder. The mixture was B



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Figure 1. Extracted ion chromatogram of 2-oxo-clopidogrel (M2) along with previously reported metabolites (A) and newly identified metabolites (B). Nonlabeled peaks are extracted isotopic signals of other metabolite or background signals.

Table 1. LC-MS Retention Times (Rt), Observed (Obs.) and Calculated (Calcd.) High Resolution Masses, Chemical Formulas, Numbers of 18O (H218O) Incorporation, and Postulated Biotransformation Pathways of 2-Oxo-clopidogrel (M2) Metabolites Identified in HLM Rt (min) M2 M13 M16 M17 GS-1 GS-2 GS-3

13.69 11.94 12.65 11.94 11.10 9.51 10.44 9.56 11.46

chemical formula (MH+) +

Δformula

C16H17CINO3S C16H19CINO4S+

\ +2H+O

C16H17CINO4S+ C16H34CINO5+ C26H34CIN4O10S2+

+0 +2H+2O−S [+2H+O]+GS-H

C26H34CIN4O11S+ C26H34CIN4OSS2+

[+2H+2O-SJ+GS-H +H+GS

Obs. mass (Da)

Calcd.mass (Da)

error (ppm)

338.06137 356.07153 356.07245 354.05616 340.09472 661.14110 661.14095 645.16341 645.14566

338.06122 356.07178

0.5 −0.7 1.9 0.1 0.3 1.7 1.5 1.0 1.0

354.05613 340.09463 661.13994 645.16278 645.14502

18

O (from H218O) # \ 1 1 0 2 1 1 1 0

pathway thiolactone A + H2O N-oxidatbn thiolactone B + H2O thiolactone A + H2O thiolactone B + GSH thiolactone A+ GSH

dry gas flow rate of 12 L/min. Product ion scan (MS/MS) was generated via sustained off-resonance irradiation collision-induced dissociation (SORI-CID) with argon using SORI power of 1.5% and a precursor ion isolation width of m/z 2.0. Data was acquired and processed in DataAnalysis 4.0 (Bruker Daltonics).

(Thermo Fisher Scientific). Fragmentations were proposed based on plausible protonation sites, subsequent isomerization, and even electron species, as well as bond saturation. For quantitative studies, supernatants of the reaction phenotyping studies were injected onto the same LC-MS/MS system as in the metabolite identification experiment with a shorter scan range (m/z = 300−700). The chromatographic peaks of metabolites were extracted and integrated in Qual Browser (Thermo Fisher Scientific). Samples were analyzed in duplicates or triplicates. High Resolution LC-MS/MS Analysis. Chromatographic analysis was conducted on a 1100 HPLC system consisting of a manual injector, a quaternary pump, and a diode array detector (Agilent Technologies, Santa Clara, CA) using the same column, same solvents, and same elution program as described in low resolution LC-MS/MS experiments. High resolution mass spectrometric analysis was conducted on an Apex IV Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS, Bruker Daltonics, Billerica, MA), which was interfaced to the above HPLC system. The HPLC eluate was split after the diode array detector, and 6% eluate (45 μL/min) was injected onto the mass spectrometer. MS analysis was performed using a standard electrospray ionization (ESI) source operating in positive ionization mode. Source operating conditions were as follows: end plate electrode voltage of −3.5 kV, capillary entrance voltage of −4.0 kV, skimmer voltage of 30 V, dry gas temperature of 250 °C, and

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RESULTS Metabolism Studies of 2-Oxo-clopidogrel in HLM. Aside from the prodrug activation pathway, previously reported metabolic pathways of M2 are (1) methyl ester hydrolysis, leading to a carboxylic acid metabolite;10,12 (2) exo-to-endo double bond migration followed by PON-1-catalyzed thioester hydrolysis, leading to the endo isomer of the active metabolite.6,7 To characterize the complete biotransformation of M2, in vitro studies were designed as follows: addition of selective esterase inhibitor potassium fluoride (KF, 100 mM) to block the methyl ester hydrolysis and exemplify the other pathways;2 inclusion of GSH (5 mM) as both the reducing equivalent required for active metabolite formation and the trapping agent for electrophilic metabolites (this more closely mimics the in vivo environment where hepatic cells contain 4− 10 mM of GSH13); optimization of the chromatographic C



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Figure 2. Product ion spectra and fragmentation analyses (insert) of M2 (A) and M16 (B).

Figure 3. Structural elucidation of M17: product ion spectrum (A) with fragmentation analysis (A, insert); observed (B) and calculated high resolution MS spectra (C).

a mono-oxygenated metabolite, which was confirmed by high resolution MS data (Table 1). It is not detected in freshly prepared stock solution, while it is detected as a minor degradation product in a one month old stock solution (data not shown). This suggests that its formation is either nonenzymatic or catalyzed by a non-NADPH-dependent enzyme. The product ion spectrum (MS2) of M16 is shown in Figure 2B along with that of the parent compound (M2) (Figure 2A). Fragmentation analysis suggests that M16 is the N-oxide metabolite of M2. The H218O experiment result shows that the additional O-atom in M16 is not derived from H2O (Table 1; see MS spectra in Supporting Information), which is consistent with the tentative structure. It was previously reported that the piperidine moiety of clopidogrel can undergo nonenzymatic14 or NADPH-independent N-oxidation8 leading to the formation of an iminium metabolite, which resembles the formation of M16 from M2.

conditions to separate the metabolites from their isomers; and confirmation of the known metabolites and profiling of the unknown metabolites at the same time. With these approaches, metabolite profiling experiments were conducted. The previously reported metabolites of M2 (MH+ = 338) are shown in Figure 1A: active metabolites M13 (MH+ = 356), M13-GSH conjugate GS-1 (MH+ = 661), and an active metabolite endo isomer M15 (MH+ = 356). As shown in Figure 1B, the following new metabolites were identified with both the characteristic 35Cl/37Cl isotopic pattern and parent-related fragmentation: M16 (MH+ = 354); M17 (MH+ = 340), GS-2 (MH+ = 645), and GS-3 (MH+ = 645). Structural Elucidation of Metabolite M16. From the HLM incubations of 2-oxo-clopidogrel (M2), a prominent metabolite, M16, was identified with the retention time of 11.94 min (Figure 1B). Its formation was found to be independent of NADPH, KF, or GSH. The +16 Da mass shift suggests that it is D



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Figure 4. Product ion spectra of the two “645 Da” metabolites, GS-2 (A) and GS-3 (B), and their postulated structures with fragmentation analyses (insert). TL, thiolactone fragment; TL*, fragment of the thiolactone moiety; GSH, glutathione fragment; GSH*, fragment of the glutathione moiety; TL-GSH*, “co-fragment” of both moieties.

Figure 5. Comparison of the high resolution MS spectra of the two “645 Da” metabolites: GS-2 (A1, observed; A2, calculated) and GS-3 (B1, observed; B2, calculated).

Structural Elucidation of Metabolite M17. In the HLM incubations fortified with NADPH, a +2 Da metabolite, M17, was identified at the retention time of 11.10 min (Figure 1). The mass shift suggests a net formula change of +2H or +2H +2O−S from M2. The latter was supported by high resolution MS data (Table 1). Fragmentation analysis was conducted based on the MS2 spectrum of M17 (Figure 3A) and supported modification on the thiolactone motif. The prominent -H2O fragmentation patterns (m/z 262, 140, 290) associated with this modified motif implicates the existence of a hydroxyl group,

which is also contradictory to the biotransformation of +2H. The mass value and the isotopic pattern revealed by high resolution MS analysis confirms that M17 is the desulfurized thiolactone ring-opening metabolite (Figure 3B and C). H218O experiment data supports the hypothesis that the two O-atoms (that replace the S-atom) are mainly derived from H2O (Table 1; see MS spectra in Supporting Information). On the basis of these experimental analyses, the structure of M17 was elucidated (Figure 3A, insert). The desulfurized γ-hydroxylE



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Figure 6. Extracted ion chromatogram of GS-1, GS-2, and GS-3 of acid quenched samples before (A) and after (B) 12 h of DTT treatment.

α,β-unsaturated carboxylic acid substructure was proposed to be formed from thiolactone oxidative cleavage. Structural Elucidation of the GSH Conjugating Metabolites GS-2 and GS-3. Besides the M13-GSH disulfide conjugates (GS-1, MH+ = 661, Figure 1A), two other GSH conjugating metabolites, GS-2 (MH+ = 645) and GS-3 (MH+ = 645), were identified with the retention time of 9.56 and 11.46 min, respectively (Figure 1B). Although their molecular masses are the same, they are well separated by liquid chromatography and possess different MS2 spectra (Figure 4), which indicates that they might not be just stereoisomers. Although the preliminary fragmentation analysis suggests that in both GS-2 and GS-3 GSH conjugates with the oxidatively cleaved thiolactone motif high resolution LC-MS data confirm that GS-2 and GS-3 have different formulas (Table 1 and Figure 5), GS-3 contains one less O-atom than GS-1; GS-2 is the desulfurized counterpart of GS-3 with one less S-atom but two more O-atoms. On the basis of the high resolution MS data and in-depth fragmentation analyses, the structures of GS-2 and GS-3 are proposed and are shown in Figure 4 (insert). GS-3 was proposed to be the adduct resulting from conjugation of the thiolactone motif with a molecule of GSH through a carbonyl-S (thioester) bond; GS-2 is proposed to have a structure similar to that of M17 with a glutathionyl replacing the hydroxyl of M17. The S-atom of the cysteinyl moiety in GS-2 was proposed to be in the oxidized form of a sulfoxide. With the oxidized thiolactone moiety conjugated with the fragmentation friendly dipeptidyl GSH, the MS2 spectra of GS2 (Figure 4A) and GS-3 (Figure 4B) contain some similar patterns including the loss of pyroglutamic acid (neutral loss of 129) from the GSH moiety (m/z 516, -H2O, m/z 498). The MS2 spectra of GS-2 and GS-3 also contain major differences, which can provide important insights for their structural elucidation. In GS-3, the GSH moiety and the oxidatively

cleaved thiolactone moiety were proposed to be conjugated via a thioester function, which is relatively fragile and readily undergoes primary fragmentation, forming the thiolactone daughter ion or the GSH daughter ion. The two daughter ions can undergo individual subsequent fragmentation. The MS2 spectrum of GS-3 has limited co-fragmentation daughter ions containing fragments from both the thiolactone and the GSH moieties. This is often observed with phase II metabolites, especially glucuronic acid conjugation metabolites of alcohols, whose MS2 spectrum is often dominated by the fragmentation of the fragile ester conjugation bond.15 As shown in Figure 4B, the collision-induced dissociation of GS-3 yields the two daughter ions of the conjugation moieties. The thiolactone moiety (m/z 338) undergoes subsequent fragmentation to yield an m/z 278 daughter ion, while the GSH moiety (m/z 308) yields its characteristic daughter ions m/z 290 and m/z 233 (see the MS2 spectrum of GSH in Supporting Information). A similar fragmentation pattern was also observed with the M13GSH conjugate (GS-1) containing a fragile disulfide conjugation bond (see Supporting Information). For GS-2, however, the desulfurized thiolactone moiety and GSH were proposed to be conjugated through a more stable sulfoxide function. The collision-induced dissociation of GS-2 yields cofragmentation daughter ions containing fragments from both the thiolactone and the GSH moieties (m/z 305, m/z 287, m/z 269, and m/z 241) (Figure 4A). High resolution MS data also confirm the proposed co-fragmentation daughter ions (see Supporting Information). The H218O experiment result also strongly supports the above structural elucidations: the observed numbers of H2Oderived O-atoms in GS-1, GS-2, and GS-3 are 1, 1, and 0, respectively (Table 1; see MS spectra in Supporting Information), which are consistent with the proposed structures. F



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Postincubation Dithiothreitol (DTT) Treatment of GS1, GS-2, and GS-3. Unlike the extensively investigated M13GSH disulfide conjugate GS-1,12,16,17 GS-2 and GS-3 were proposed to be S−C adducts. The disulfide bond of GS-1 can be reduced, and the active metabolite (M13) can be regenerated; however, the sulfoxide bond of GS-2 and the thioester bond of GS-3 cannot be cleaved through such reduction. This hypothesis was tested by postincubation chemical derivatization experiments. As shown in Figure 6, upon a 12 h incubation with DTT, a known reducing agent that cleaves the disulfide bonds in proteins or in small molecules,16,17 GS-1 (MH+ = 661) in the acid quenched sample diminishes as a result of disulfide bond reduction, forming M13, while GS-2 and GS-3 (in the same sample as GS1) remain almost unchanged. These results support the hypothesis that the two close analogues of GS-1, GS-2, and GS-3, are not disulfide GSH conjugates and that they could not release M13 upon reduction like GS-1 does (Figure 7). Similar results were obtained for organic solvent quenched samples (see Supporting Information).

transferase (GST), M2 was incubated with cDNA-expressed CYP2C19, CYP3A4, and CYP3A5 (devoid of other enzymes like GST, esterase, or PON-1). With P450 incubation fortified with NADPH and GSH, both GS-2 and GS-3 were detected (see Supporting Information), which suggests that the M11 that is formed from P450-catalyzed S-oxidation of M2 can spontaneously conjugate with GSH to form GS-2 and GS-3. The formation of M17 was also detected in these incubations, which supports the hypothesis that the hydrolysis of M11 leading to M17 formation is nonenzymatic as well (see Supporting Information).

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DISCUSSION Clopidogrel has been one of the most prescribed antithrombotic drugs in the past two decades. Recently, more and more attention has been given to its major therapeutic drawback: the high clinical intersubject variability leading to lower or no response in 20−40% of the patients. It is believed that over 80% of the individual therapeutic variance is attributed to genetic factors, which are mostly unidentified. Clopidogrel is a prodrug and needs to be bioactivated to its active metabolite. The general hypothesis for its clinical variability is that the varying levels of prodrug activating enzymes in patients lead to different plasma exposures of the active metabolite and consequently different therapeutic effects. Although some clinical studies show correlations between the antiplatelet responses of clopidogrel and the genotypes of PON-1 or CYP2C19, research results from recent studies suggest that neither enzyme plays a deciding role in catalyzing active metabolite formation.6,8 It has been shown that the two-step prodrug bioactivation is catalyzed by various P450 enzymes.6,8,18 On the basis of these observations, it has been speculated that instead of variation in the prodrug activating enzymes, the high clinical variability of clopidogrel might stem from a low conversion percentage of the active metabolite, whose plasma exposure could be significantly impacted by multicomponent factors, leading to the varying levels of therapeutic responses.8,10 Although the prodrug bioactivation pathway (Scheme 1), the conversion from clopidogrel (M0) to 2-oxo-clopidogrel (M2) and then to the active thiol metabolite (M13), has been extensively studied,3,6,18 other biotransformation pathways have not been comprehensively reported. Our recent study has shown that the first step in prodrug activation, 2-oxidation of the thiophene motif leading to M2 formation, is attenuated by the oxidative attrition of the piperidine motif and thiophene 3oxidation. 8 In the present study, we investigate the biotransformation pathways that might accompany the second step of bioactivation, conversion of M2 to active metabolite M13 (Scheme 1). Because of its conjugation to a double bond and to a S-atom, the thiolactone carbonyl functionality in M2 is not reactive enough for direct hydrolysis. The thiolactone ring-opening required for M13 formation was proposed to be facilitated by P450-catalyzed, NADPH-dependent S-oxidation, leading to the formation of a thiolactone S-oxide intermediate, M11 (Scheme 1). The carbonyl functionality in M11 is more electrophilic and undergoes hydrolysis to form a sulfenic acid intermediate M12 followed by reduction, forming the ultimate thiol active metabolite (M13) (Scheme 1).3 Previous in vitro studies have reported that approximately 48% of the M2 formed from clopidogrel undergoes esterase-catalyzed hydrolysis to form the inactive thiolactone acid metabolite (M14).10 M2 that bears an exo double bond of the piperidine motif was proposed to be in

Figure 7. Postincubation DTT treatment of the three GSH conjugates.

Dependence of M17 Formation on PON-1. The formation of the desulfurized metabolite (M17) implicates an important thiolactone ring-opening pathway that involves both oxidation and hydrolysis. Since the incubation that yields M17 contains selective esterase inhibitor KF, which completely suppresses the methyl ester hydrolysis of M2, the formation of M17 is not dependent on esterase. The hydrolysis might be dependent on PON-1, since it catalyzes the hydrolysis of the endo isomer of M2. The formation of M17 was analyzed in HLM incubation with or without PON-1 antagonist EDTA. The results support the hypothesis that M17 formation is not dependent on PON-1 either (see Supporting Information). Formation of M17, GS-2, and GS-3 in cDNA-Expressed CYP2C19, CYP3A4, and CYP3A5. M17 was proposed to be a hydrolysis product of the oxidized thiolactone intermediate (M11), and GS-2 and GS-3 were proposed to be the GSH conjugates of M11. The thiolactone oxidation of M2, leading to the formation of M11, is known to be catalyzed by P450 enzymes such as CYP2C19 and CYP3A4.18 To test whether the GSH conjugation of M11 leading to GS-2 or GS-3 formation is dependent on liver microsomal enzymes like glutathione SG



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Scheme 2. Proposed Mechanistic Pathways for M17 Formationa

a

The O-atom derived from H2O is shown in bold.

Scheme 3. Proposed Mechanistic Pathway for GS-2 Formationa

a

The O-atom derived from H2O is shown in bold.

equilibrium with its endo isomer, which can be hydrolyzed by Ca2+-dependent PON-1 to form the endo isomer of the active metabolite (M15).6 In addition to these two hydrolytic pathways, an oxidative pathway for M2 that leads to M16 formation was identified in our in vitro studies (Figure 1). On the basis of high resolution MS data (Table 1), extensive fragmentation analysis (Figure 2) and the H218O experiment result (Table 1), M16 is proposed to be the N-oxide metabolite of M2. Its formation is reminiscent of an iminium degradation product of clopidogrel.14 Formation of both piperidine N-

oxidation metabolites was found to be non-NADPH-dependent. A similar N-oxide metabolite has also been reported for another antiplatelet prodrug ticlopidine, a close analogue of clopidogrel.19 With HLM incubation, active metabolite M13 was found to be in equilibrium with its GSH disulfide conjugate GS-1 (Figure 1), which was proposed to be the reductive intermediate of the M12-to-M13 conversion (Scheme 1).3,17 Parallel to the formation of M13, one desulfurized metabolite, M17, and two more GSH conjugates, GS-2 and GS-3, were H



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Scheme 4. Proposed Mechanistic Pathway for GS-3 Formationa

a

GS-3 contains no H2O-derived O-atoms.

Scheme 5. Proposed Biotransformation Pathways of 2-Oxo-clopidogrel (M2)a

a

The red arrows represent the second step prodrug activation pathways; the gray arrows represent the previously reported attrition pathways; the black arrows represent the newly identified attrition pathways.

metabolite (M17). Phenotyping studies, including in (1) HLM incubation with selective esterase inhibitor KF and PON-1 antagonist EDTA and (2) cDNA-expressed CYP2C19, CYP3A4, and CYP3A5, support the notion that the hydrolysis of M11 leading to M17 formation is spontaneous, similar to the hydrolysis of M11 leading to M13 formation (Scheme 1).3 One alternative pathway for M17 formation is the hydrolysis of bioactivation intermediate M12 (Scheme 2, dotted arrow), which might be less favored because the sulfenic acid moiety in M12 might readily undergo reduction to form M13. A metabolite similar to M17 was reported for another thienopyridine antiplatelet prodrug prasugrel.20 Its formation was proposed to be initiated by hydroxylation of the tertiary

identified. The high resolution MS data suggest that the chemical formula of M17 differs from that of M13 by only one hydroxyl-to-sulfhydryl change (Table 1). On this basis, the product ion spectrum of M17 was examined (Figure 3A), and detailed fragmentation analysis was conducted (Figure 3A, insert). The structure of M17 has been elucidated and is supported by data from further high resolution MS analysis (Figure 3B and C). The mechanistic pathway for M17 formation is proposed in Scheme 2. Upon thiolactone Soxidation, the allylic bridge carbon in M11 becomes more electrophilic and is attacked by a molecule of H2O leading to thiolactone ring scission. The resulting acylsulfenic acid becomes hydrolyzed to yield the desulfurized ring-opening I



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bridged carbon and involve a γ-keto-α,β-unsaturated thioacetic acid as the intermediate. This pathway could be an alternative for M17 formation from M2 (Scheme 2, the hydroxylation pathway, gray arrows). However, the H218O experiment data supports the notion that the two acquired O-atoms in M17 (Scheme 2, shown in bold) are mainly derived from H2O, which does not favor the hydroxylation pathway because according to this pathway, one O-atom in M17 is from H2O, and the other is from O2. The H218O experiment data strongly supports the proposed new thiolactone ring-opening pathway involving the electrophilic allylic bridge carbon in M11. The proposed electrophilic nature of the allylic bridge carbon (adjacent to the S-oxide) in M11 is also well supported by numerous literature examples including omeprazole bioactivation and diallyl sulfide S-oxidation followed by glutathionylation.21 The structural elucidation of GS-2 was discussed earlier in this article. The thiolactone moiety of the GSH conjugate was confirmed by high resolution MS analysis to be desulfurized (Table 1). The mechanistic pathway for GS-2 formation is proposed in Scheme 3. Similar to M17 formation (Scheme 2), the sulfhydryl group of a molecule of GSH attacks the electrophilic allylic bridge carbon in M11, and the thiolactone ring opens to yield an intermediate with a sulfide and an acylsulfenic acid functionality. The acylsulfenic acid functionality resembles the structure of a peroxy acid and is expected to be oxidative.22 With a reductive sulfide nearby, a facile intramolecular redox reaction (involving a partially constrained seven-membered ring) is proposed to take place to yield a sulfoxide and a thiocarboxylic acid. Subsequent hydrolysis of the thiocarboxylic acid moiety yields GS-2. This proposed pathway (S-oxidation followed by glutathionylation) is mechanistically parallel to the M17 formation pathway (Soxidation followed by hydrolysis) and is also supported by welldocumented literature examples.21 Although GS-3 possesses the same mass value as GS-2 under low resolution MS analysis (MH+ = 645), the difference in LC retention times (Figure 1) and product ion spectra (Figure 4) suggest that it is not just a stereoisomer of GS-2. While GS-2 was proposed to be a desulfurized thiolactone-GSH conjugate, high resolution MS data support that GS-3 is the S-retaining thiolactone-GSH conjugate (Table 1). Comparison of the high resolution mass spectra of GS-2 and GS-3 confirms their proposed molecular formulas (Figure 5). The molecular formula of GS-3 differs from that of M13 by a glutathionylto-hydroxyl change (Table 1), which suggests that GS-3 might be formed through a pathway similar to that of M13 with GSH replacing H2O as the nucleophile that attacks the electrophilic carbonyl carbon in M11 (Scheme 4). Again, very similar pathways can be found with literature examples like dithiopyr biotransformation.21 The identification of the three additional thiolactone ringopening metabolites, M17, GS-2, and GS-3, in conjunction with the active metabolite M13, demonstrates the comprehensive electrophilic nature of the thiolactone S-oxide intermediate (M11). The P450-catalyzed S-oxidation confers on the thiolactone moiety not only one electrophilic site at the carbonyl C-atom (Site A) but also a second one at the allylic bridge C-atom (Site B) (Scheme 5). Both sites can be attacked by nucleophiles like H2O or GSH: the A-H2O pathway leads to the formation of pharmacologically desired active metabolite M13 (via the GSH redox equilibrium involving the intermediate GS-1); the A-GSH pathway leads to the formation

of GS-3; the B-H2O pathway leads to the formation of desulfurized metabolite M17, and the B-GSH pathway leads to the formation of desulfurized GSH conjugate GS-2. Clopidogrel was reported to be an inhibitor of P450 enzymes such as CYP2B6 and CYP2C19.16,23 Previous studies have shown that clopidogrel is a mechanism-based inhibitor of CYP2B6. The Cys475 residue of CYP2B6 was found to form a disulfide bond with the active metabolite. This inactivation mechanism is the same as the process underlying the pharmacological activity of clopidogrel: active metabolite M13 irreversibly binds with a subtype of adenosine diphosphate (ADP) receptor P2Y12 and inhibits the ADP-induced platelet aggregation.24 The thiol function is believed to be vital to the active metabolite for the desired biological potency. However, it could also give the active metabolite a high potential to form adducts with off-target macromolecules, which might not only decrease the active metabolite plasma exposure but also lead to possible adverse effects. With the formation of GS-1 demonstrating the reactivity of M13 toward cysteine residues, the formation of GS-2 and GS-3 suggests that M11, the precursor of M13, also has sufficient reactivity to form adducts with nucleophilic residues of proteins. M11 might be able to take over some roles of M13 in inactivating CYP2B6, the enzyme that catalyzes its formation,16,18 or binding with P2Y12 for the desired antiplatelet effect, through pathways similar to those of GS-2 (Scheme 3) and GS-3 formation (Scheme 4). Although the M11-protein adducts remain to be investigated, the identification of two electrophilic sites suggests that M11 is chemically reactive and might lead to undesired interactions, causing toxicity, which might partially explain the narrow therapeutic window of clopidogrel.17 In vivo studies have shown that during clopidogrel bioactivation, the plasma concentration of M2, the precursor of M11, is very low.25 The M11 formed from M2 may be readily detoxified by nucleophiles like H2O or GSH to diminish its potential adverse effects. However, for vicagrel, a recently reported antiplatelet analogue of clopidogrel, M2 is readily formed through a facile hydrolysis.12 In animal studies of vicagrel, the plasma concentration of M2 was found to be much higher, which implicates a higher plasma concentration of M11. Evaluation of the toxic profile of M11 will be an important part of the safety testing of vicagrel. The overall in vitro biotransformation pathways of M2 are summarized in Scheme 5. M2 can go through nonactivation metabolic pathways of methyl ester hydrolysis, undergo double bond migration followed by thiolactone hydrolysis, and Noxidation. The remaining M2 undergoes the P450-catalyzed thiolactone S-oxidation to form M11. As revealed by the present study, the desired hydrolytic ring-opening of M11, the A-H2O pathway, leading to M13 formation, is accompanied by multiple other ring-opening pathways. It should be noted that the metabolic pathways (including the semiquantitative information) presented here were proposed on the basis of the in vitro studies of M2, which might have limitations in predicting the biotransformation of M2 in vivo. With the avoidance of making exuberant in vivo assessment, the proposed ring-opening pathways of M11, Site A or B reacting with H2O or GSH (Scheme 5), can provide the following two mechanistic insights on clopidogrel biotransformation: (1) in addition to the thiolactone carbonyl carbon (Site A), the allylic bridge carbon (Site B) in M11 is also reactive toward nucleophiles like H2O to undergo hydrolysis; (2) the hydrolysis of M11 might be in competition with other small molecule J



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nucleophiles like GSH as well as nucleophilic residues of proteins. Because of its reactive nature and the complex environment, the metabolic pathways of M11 in vivo might go beyond what has been proposed here. Although the results from the in vitro phenotyping studies suggest that the proposed ring-opening pathways are all spontaneous, it is possible that some unidentified pathways of M11 in vivo are regulated by genetic polymorphic enzymes. The first step of clopidogrel bioactivation, 2-oxidation of the thiophene motif leading to M2 formation (Scheme 1), was reported to be attenuated by CYP3A4/5-catalyzed piperidine oxidation and thiophene 3-oxidation,8 in addition to methyl ester hydrolysis.10,11 It has been calculated that the formation of M2 only accounts for 2% of the overall clopidogrel conversion.8 As presented here, the second step of bioactivation, the formation of M13 from M2, is accompanied by multiple pathways in addition to the methyl ester hydrolysis. It has been previously proposed that the esterase-mediated hydrolysis of clopidogrel and 2-oxo-clopidogrel significantly attenuate the desired two-step bioactivation pathway, which might contribute to the observed poorer and more variable antiplatelet response to clopidogrel compared with that to prasugrel.10 It was estimated that only 10% of a clopidogrel dose is ultimately converted to its active metabolite.26 On the basis of these more comprehensive biotransformation studies of clopidogrel, the overall conversion percentage of M13 from clopidogrel is expected to be much lower than previously estimated (i.e., 1% or lower). On the one hand, this low bioactivation efficiency of clopidogrel demonstrates the high potency of its active metabolite against the antiplatelet target P2Y12 as a result of covalent modification. On the other hand, this low active metabolite yield is likely to play an important role in the observed clinical variability and high levels of resistance to clopidogrel therapy, as multiple potential biological factors could impact the already-thin active metabolite formation or significantly diminish its plasma exposure.8 In summary, we have studied the in vitro metabolic pathways of 2-oxo-clopidogrel, the intermediate of clopidogrel bioactivation. Along with active metabolite formation, multiple metabolic pathways were identified. The thiolactone S-oxide intermediate was found to undergo hydrolytic ring-opening not only at the desired carbonyl carbon but also at the allylic bridge carbon of the thiolactone moiety. The hydrolysis of the thiolactone S-oxide was also found to be in competition with other nucleophiles like glutathione. The proposed thiolactone ring-opening pathways of clopidogrel bioactivation can provide mechanistic insights for other thienopyridine antiplatelet prodrugs like ticlopidine, prasugrel, and vicagrel, as all of these prodrugs are believed to share a similar bioactivation mechanism.12,26 Characterization of the reactive nature of the thiolactone S-oxide intermediate might also help to understand the adverse effects of clopidogrel and the other thienopyridine prodrugs. The research results presented here have confirmed that not only the first step but also the second step of clopidogrel bioactivation is significantly attenuated by metabolic attrition, leading to possibly a very low overall conversion ratio of the pharmacologically active metabolite. Characterizing the in vitro biotransformation pathways of clopidogrel and understanding the low active metabolite conversion percentage might help in future studies to further explore the biological mechanisms underlying the observed clinical uncertainties of clopidogrel antithrombotic therapy.

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ASSOCIATED CONTENT

S Supporting Information *

MS and MS2 spectra, fragmentation analysis, and extracted ion chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*MetabQuest Research Laboratory, 1720 Oak Avenue, Suite 403 Evanston, IL 60201. Phone: (847) 530-7238. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Richard B. Silverman (Northwestern University) for critical reading and careful revision of the manuscript. We thank Dr. Kristin Jansen Labby (Northwestern University) for editing and proofreading the manuscript.

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ABBREVIATIONS P450, cytochrome P450; PON-1, paraoxonase-1; HLM, human liver microsomes; GSH, L-glutathione; NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate (reduced); EDTA, ethylene diamine tetraacetic acid; DTT, dithiothreitol; GST, glutathione S-transferase; ADP, adenosine diphosphate; LCMS/MS, liquid chromatography−tandem mass spectrometry; CID, collision-induced dissociation; MS2, product ion spectrum

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