In Vitro Biotransformation Studies of 2‑Oxo-clopidogrel: Multiple Thiolactone Ring-Opening Pathways Further Attenuate Prodrug Activation

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 <i>S</i>-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 <i>S</i>-oxidation confers on the thiolactone moiety not only one electrophilic site at the carbonyl <i>C</i>-atom (Site A), but also a second one at the allylic bridge <i>C</i>-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 <i>S</i>-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

Identification of the Significant Involvement and Mechanistic Role of CYP3A4/5 in Clopidogrel Bioactivation

The clinical response to the antiplatelet prodrug clopidogrel is associated with high intersubject variability and a certain level of therapeutic resistance. Previous studies have suggested that genetic polymorphism of CYP2C19 might be one determinant of clopidogrel efficacy and led to the CYP2C19 genotype-tailored antithrombotic therapy. However, evidence against the role of CYP2C19 from multiple studies implied the involvement of other factors. Here, we report that prodrug activation of the thiophene motif in clopidogrel is attenuated by heavy metabolic attrition of the piperidine motif. CYP3A4/5 was identified to be the enzyme metabolizing the piperidine motif. Inhibiting CYP3A4/5-mediated attrition was shown to potentiate active metabolite formation, which was found to be catalyzed by multiple CYP enzymes. Identifying the significant involvement of CYP3A4/5 and characterizing its mechanistic role in clopidogrel bioactivation might assist future pharmacogenomic studies in exploring the full mechanism underlying clopidogrel efficacy

Deuterated Clopidogrel Analogues as a New Generation of Antiplatelet Agents

Clopidogrel (CPG) is an antithrombotic prodrug that needs hepatic cytochrome P450 (CYP) enzymes for its bioactivation. The clinical effects of CPG have been associated with high intersubject variability and a certain level of resistance. Recently, comprehensive biotransformation studies of CPG support that the observed clinical uncertainty stems from the low bioactivation efficiency, which is attributed to extensive attritional metabolism (e.g., hydrolysis of the methyl ester functionality and oxidation of the piperidine moiety). With the goal of potentiating the desired thiophene 2-oxidation through minimal structural modification, we have adopted the strategy of <i>targeted metabolism shift</i> and have designed and synthesized deuterated piperidine analogues of CPG. In vitro studies showed that the prodrug activation percentages have been significantly increased for the deuterated analogues as a result of stability enhancement of the piperidine moiety. In a pharmacological study with a rat model, oral administration of the deuterated analogues also demonstrated higher inhibitory activity than that of CPG against adenosine diphosphate (ADP) induced platelet aggregation. These deuterated analogues represent a new generation of antiplatelet agents with the potential to overcome the major clinical drawbacks of CPG

Overcoming Clopidogrel Resistance: Discovery of Vicagrel as a Highly Potent and Orally Bioavailable Antiplatelet Agent

A series of optically active 2-hydroxytetrahydrothienopyridine derivatives were designed and synthesized as prodrugs of clopidogrel thiolactone in order to overcome clopidogrel resistance. The final compounds were evaluated for their inhibitory effect on ADP-induced platelet aggregation in rats. Compound <b>9a</b> was selected for further in vitro and in vivo metabolism studies, since its potency was comparable to that of prasugrel and was much higher than that of clopidogrel. Preliminary pharmacokinetic study results showed that the bioavailability of clopidogrel thiolactone generated from <b>9a</b> was 6-fold higher than that generated from clopidogrel, implying a much lower clinically effective dose for <b>9a</b> in comparison with clopidogrel. In summary, <b>9a</b> (vicagrel) holds great promise as a more potent and a safer antiplatelet agent that might have the following advantages over clopidogrel: (1) no drug resistance for CYP2C19 poor metabolizers; (2) lower dose-related toxicity due to a much lower effective dose; (3) faster onset of action