Inhibition Assay (Testosterone Hydroxylase): To gain insight into the selectivity of the synthetic compounds, nicotine, nicotine related alkaloids and nicotine metabolites for inhibition of other CYPs, we examined the major CYP present in human liver (i.e., CYP 3A4). That the CYP2A6 inhibitors showed low or no inhibitory activity against CYP3A4 suggests that the inhibitors examined selectively inhibited CYP2A6.
Inhibition Assay: The inhibition of human CYP2A6-mediated 7-hydroxy coumarin formation was evaluated in the presence of 95 selected test compounds in a standard assay (Greenlee et al., J Pharmacol Exp Ther, 1978). Our first studies were with highly purified human CYP2A6 that provided a convenient and relatively high-throughput measure of CYP2A6 inhibition.
Selectivity index, ratio of IC50 for inhibition of human CYP3A4 assessed as reduction in enzyme-mediated testosterone 6-hydroxylation to IC50 for inhibition of human CYP2A6 assessed as reduction in enzyme-mediated coumarin 7-hydroxylation
Inhibition of CYP3A4 in human liver microsomes assessed as reduction in enzyme-mediated testosterone 6-hydroxylation in presence of NADPH-generating system, G6P and G6P dehydrogenase
Inhibition of human CYP2C19 assessed as reduction in specific vivid-blue substrate O-dealkylation by modified PanVera vivid assay protocol based method
Inhibition of human CYP2A6 expressed in insect cell membranes assessed as reduction in enzyme-mediated coumarin 7-hydroxylation in presence of glucose-6-phosphate, glucose-6-phosphate dehydrogenase and NADPH-generating system by fluorometry