Cytochromes P450 Catalyze Both Steps of the Major Pathway of Clopidogrel Bioactivation, whereas Paraoxonase Catalyzes the Formation of a Minor Thiol Metabolite Isomer

Abstract

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, clopidogrel, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is a classical cytochrome P450 (P450)-dependent monooxygenation of its thiophene ring leading to 2-oxo-clopidogrel, a thiolactone metabolite. The second step was described as a P450-dependent oxidative opening of the thiolactone ring of 2-oxo-clopidogrel, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding thiol <b>4b</b>. A very recent paper published in <i>Nat. Med.</i> (Bouman et al., (2011) <i>17</i>, 110) reported that the second step of clopidogrel bioactivation was not catalyzed by P450 enzymes but by paraoxonase-1­(PON-1) and that PON-1 was a major determinant of clopidogrel efficacy. The results described in the present article show that there are two metabolic pathways for the opening of the thiolactone ring of 2-oxo-clopidogrel. The major one, that was previously described, results from a P450-dependent redox bioactivation of 2-oxo-clopidogrel and leads to <b>4b cis</b>, two previously reported thiol diastereomers bearing an exocyclic double bond. The second, minor one, results from a hydrolysis of 2-oxo-clopidogrel, which seems to be dependent on PON-1, and leads to an isomer of <b>4b cis, 4b "endo"</b>, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. These results were obtained from a detailed study of the metabolism of 2-oxo-clopidogrel by human liver microsomes and human sera and analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, either as such or after derivatization with 3′-methoxy phenacyl bromide or <i>N</i>-ethyl maleimide (NEM). These results also show that the major bioactive thiol isomer found in the plasma of clopidogrel-treated patients derives from 2-oxo-clopidogrel by the P450-dependent pathway. Finally, chemical experiments on 2-oxo-clopidogrel showed that this thiolactone is in equilibrium with its tautomer having a double bond inside the piperidine ring and that nucleophiles such as CH₃O^– preferentially react on the thioester function of this tautomer. This allowed us to understand why <b>4b cis</b> has to be formed via an oxidative opening of 2-oxo-clopidogrel thiolactone, whereas a hydrolytic opening of this thiolactone ring leads to the "endo" thiol isomer <b>4b "endo"</b>