Inactivation of Cytochrome P450 2B6 by Ticlopidine and Clopidogrel
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In this study, the inhibition of cytochrome P450 2B6 (2B6) by ticlopidine and clopidogrel was analyzed. It was determined that ticlopidine and clopidogrel were potent mechanism-based inactivators of2B6 confirming previous research (Nishiya et al., 2009; Richter et al., 2004). The inhibition was determined to be time and concentration dependent as indicated by a decrease in activity of 2B6 on the O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), a substrate of2B6. P450 2B6 metabolizes 7-EFC to 7-hydroxy-4-trifluoromethylcoumarin (HFC) which is fluorescent. The inhibition data were fit to the Michaelis-Menten equation and the Ki and kjnaet were found. The average Ki and kjnact for ticlopidine were 5.48 uM and 0.081 min"1, respectively, and for clopidogrel, 8.59 uM and 0.072 min"1, respectively. The partition ratio was determined to be 8 for clopidogrel and 14 for ticlopidine. According to previous research (Nishiya et al., 2009; Richter et al., 2004), ticlopidine and clopidogrel are oxidized to their active metabolites. Liquid chromatography-mass spectroscopy (LC-MS) analysis showed that the monooxygenated and the dioxygenated forms of the inhibitors were being produced in the presence ofNADPH. Titration of2B6 with each inhibitor yielded Kd values of4.5 uM for ticlopidine and 1.2 uM for clopidogrel. These results suggest that there was inactivation while the inhibitor was still bound to the protein. Possible docking conformations were examined using AutoDock 4. From the docking analysis, binding orientation could be seen that would allow for further oxidation of the inhibitors in the active site of the protein.