Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: In relation to covalent binding of its reactive intermediate

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4- hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [³H]IMI in the presence of NADPH resulted in covalent binding of a ³H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis- Menten kinetics, and the K(m) value (1.1 αM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [³H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti < Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2- hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [³H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [³H]IMI metabolites covalently bound were immunoprecipitated with the anti- CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2- hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.