Mechanism-Based Inhibition of Human Cytochrome P450 2B6 by Ticlopidine, Clopidogrel, and the Thiolactone Metabolite of Prasugrel

Abstract

ABSTRACT:Mechanism-based inhibition of CYP2B6 in human liver microsomes by thienopyridine antiplatelet agents ticlopidine and clopidogrel and the thiolactone metabolites of those two agents plus that of prasugrel were investigated by determining the time-and concentration-dependent inhibition of the activity of bupropion hydroxylase as the typical CYP2B6 activity. By comparing the ratios of k inact (maximal inactivation rate constant)/K I (the inactivator concentration producing a half-maximal rate of inactivation), it was found that the thiolactone metabolite of prasugrel is 10-and 22-fold less potent, respectively, in the mechanism-based inhibition of CYP2B6 than ticlopidine and clopidogrel. The k inact /K I ratio of the thiolactone metabolite of ticlopidine was comparable with that of the parent compound, whereas this ratio for the thiolactone metabolite of clopidogrel was significantly smaller than that of clopidogrel. In conclusion, ticlopidine, its thiolactone metabolite, and clopidogrel were more potent mechanism-based inhibitors of CYP2B6 than the thiolactone metabolite of prasugrel.The thienopyridine antiplatelet agents ticlopidine and clopidogrel ( Fig. 1) prevent thrombogenesis via blocking ADP-dependent activation of platelets through the P2Y 12 receptor, one of the ADP receptors on platelets (Sharis et al., 1998). They have been widely used for the treatment and prevention of cerebrovascular and cardiovascular diseases. Clopidogrel appears to have a relatively faster onset of action and lower incidence of adverse effects such as neutropenia and thrombotic thrombocytopenic purpura compared with ticlopidine (Kam and Nethery, 2003).Prasugrel ( Fig. 1), a novel thienopyridine P2Y 12 antagonist, demonstrated more potent antiplatelet activity and faster onset than ticlopidine and clopidogrel in preclinical and/or clinical studies (Niitsu et al., 2005;Brandt et al., 2007) and efficacy superior to that of clopidogrel in patients with acute coronary syndrome undergoing percutaneous coronary intervention . Prasugrel, also a prodrug, is first hydrolyzed to a thiolactone metabolite (R-95913), which is then converted to the pharmacologically active metabolite (R-138727) through a single, P450-mediated oxidation step (Fig. 1) (Rehmel et al., 2006;Williams et al., 2008).The thiolactone metabolites of ticlopidine and clopidogrel are produced by P450-mediated oxidation (Yoneda et al., 2004;Kurihara et al., 2005), whereas R-95913 is produced by esterase-mediated hydrolysis of prasugrel (Williams et al., 2008). The hydrolysis of prasugrel is very rapid both in vitro and in vivo, such that it is not detected in human plasma even at early time points after oral administration . The thiolactones of each of these agents are converted to the pharmacologically active metabolites, each of which possesses a thiol group, by P450-mediated oxidation (Yoneda et al., 2004;Kurihara et al., 2005;Rehmel et al., 2006). Because of the P450 dependency of the metabolic pathways, ticlopidine, clopidogrel, and the thiolactone metabolites o...

Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Clopidogrel: Involvement of Both Covalent Modification of Cysteinyl Residue 475 and Loss of Heme

“…This gives a k inact /K I ratio of 0.015 min Ϫ1 ⅐ M Ϫ1 . Therefore, 2-oxo-clopidopgrel is approximately 5-fold less efficient than clopidogrel as a mechanism-based inactivator for CYP2B6 WT, consistent with an earlier observation by Nishiya et al (2009a), who reported a decrease of nearly 12-fold in the k inact /K I ratio in human liver microsomes. Similar results were also reported for CYP2C19 that showed clopidogrel was significantly more efficient than its thiolactone metabolite in inactivating the (S)-mephenytoin 4Ј-hydroxylase activity of human liver microsomes (Hagihara et al, 2008;Nishiya et al, 2009b).…”

“…A number of in vitro studies have provided evidence supporting this suggestion. It has been reported that thienopyridine drugs such as clopidogrel and ticlopidine are potent mechanism-based inhibitors of CYP2B6 and -2C19 (Ha-Duong et al, 2001;Richter et al, 2004;Walsky and Obach, 2007;Nishiya et al, 2009a;Nishiya et al, 2009b) and may thereby cause significant adverse drug-drug interactions. In particular, clopidogrel is highly potent and selective for inhibition of CYP2B6 in human liver microsomes, with K I and k inact values of 0.5 M and 0.35 min Ϫ1 , respectively (Richter et al, 2004).…”

Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0-Å Resolution

“…4) and mean binding energies of Ϫ7.67 and Ϫ7.57 kcal/ mol, respectively. These poses are consistent with 2B6-mediated oxidation of ticlopidine (Nishiya et al, 2009), and each 4-CPI contact residue listed in Table 4 falls within a 5-Å radius of the docked inhibitor. In addition, 16 poses showed alternate ligand orientations and mean energies from Ϫ7.57 to Ϫ7.27 kcal/mol.…”

Progress Curve Mechanistic Modeling Approach for Assessing Time-Dependent Inhibition of CYP3A4

“…Although advances in LC-MS/MS have allowed use of lower protein concentration in the predilution step (Nishiya et al, 2009;Rowland Yeo et al, 2011), most of the two-step studies reported in the literature have used a high enzyme concentration in the preincubation to facilitate a significant dilution into the final incubation. Under these conditions, the assumption of EϽ ϽI can be compromised, and rapid metabolite production, which is not accounted for in the data analysis process, may lead to significant bias in parameter estimates.…”