Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

Abstract

Clopidogrel, a thienopyridine antiplatelet prodrug, is metabolized by oxidation to 2-oxo-clopidogrel, followed by conversion to its pharmacologically active thiol metabolite. After oral administration of clopidogrel to humans, two thiol isomers (H3 and H4) are observed in plasma, with similar concentrations, and only H4 is active in humans. In this work, the mechanism of stereoselectivity in the formation and S-methylation of H3 and H4 was investigated in vitro. The two diastereomers of 2-oxo-clopidogrel were epimerized rapidly at physiologic pH. The intrinsic clearance (CL int ) for H3 formation from 2-oxo-clopidogrel in human liver microsomes (HLMs) was 3.1-fold higher than that for H4 formation, indicating stereoselective metabolism. Kinetic studies using expressed enzymes demonstrated that the contributions of CYP2B6, CYP2C19, and CYP3A4 to the formation of H4 from 2-oxo-clopidogrel were 18.5%, 26.1%, and 53.5%, respectively. The CL int ratios of H3 formation to H4 formation from 2-oxo-clopidogrel by CYP2B6, CYP2C19, and CYP3A4 were 2.2, 1.0, and 1.7, respectively. In HLMs, H3 and H4 were further S-methylated, and the S-methylation was inhibited by 2,3-dichloromethyl benzylamine, indicating the involvement of thiol S-methyltransferase. The CL int value for the S-methylation of H3 in HLMs was 98.1-fold higher than that for H4. The stereoselective formation of H3 from 2-oxo-clopidogrel and the stereoselective S-methylation of H3 may lead to the similar exposure levels of H3 and H4 previously reported in humans. The epimerization of 2-oxoclopidogrel and the variations of thiol S-methyltransferase may affect the exposure to H4 in humans.

Association of PON1 gene promoter DNA methylation with the risk of Clopidogrel resistance in patients with coronary artery disease

“…For instance, ABCA1 DNA methylation was a predictive biomarker for the CAD development and was independent of plasma lipid concentration . Since 2014, several studies have explored the influence of DNA methylation in ABCB1 , P2Y12 , and cytochrome P450 enzymes on CR. This time, we chose PON1 as the target gene and found some significant results.…”

Influence of verapamil on pharmacokinetics of pristimerin in rats

“…These results indicated that, when the rats were pretreated with verapamil, the absorption of pristimerin was significantly increased. As verapamil has been reported to be an inhibitor of P‐gp and CYP3A (Gao et al ., ), we speculated that verapamil might increase the absorption of pristimerin by inhibiting P‐gp‐mediated drug efflux or CYP3A‐mediated metabolism because CYP3A is one of the most abundant drug‐metabolizing P450 isoforms in human liver microsomes, and almost 34.1% of drugs are metabolized by CYP3A (Liu et al ., ).…”

N-Methylation of BI 187004 by Thiol S-Methyltransferase

“…Interestingly, the formation of M1 and M14 was linear during the extended incubation time course, demonstrating that methyltransferase enzymes and SAM were thermally stable at 37°C up to 24 hours. Historically, incubation times for investigating methyltransferase activity in vitro range from 10 minutes to 2 hours (Van Loon et al, 1985;Kazui et al, 2014;Liu et al, 2015). From our work, it is noteworthy that, unlike cytochrome P450 enzymes, methyltransferase enzymes in microsomal or cytosolic preparations can be incubated at 37°C for 24 hours without the loss of enzyme activity.…”

“…In Asians, TPMT activity is high with a unimodal distribution due to the lack of TPMT L allele (Jang et al, 1996). TMT is responsible for the S-methylation of captopril and ziprasidone, and, most recently, the S-methylation of the pharmacologically active metabolites of clopidogrel and prasugrel (Keith et al, 1984;Obach et al, 2012;Kazui et al, 2014;Liu et al, 2015). Although the gene that encodes TMT has yet to be identified, it was reported that TMT activity varies 5-fold among the Caucasian population (Keith et al, 1983b;Glauser et al, 1992).…”

“…In HLC, the M1 formation rate was 1.8-fold lower than in HLM, suggesting that a liver cytosolic methyltransferase may also contribute to BI 187004 methylation. Although TMT is notably a microsomal enzyme, TMT activity in HLC has been previously reported, but only for S-methylation reactions (Glauser et al, 1992;Liu et al, 2015).…”