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

Y Masubuchi, S Igarashi, T Suzuki, T Horie and S Narimatsu

Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Japan.

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 microM) 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.

Volume 279, Issue 2, pp. 724-731, 11/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				H. Takakusa, H. Masumoto, H. Yukinaga, C. Makino, S. Nakayama, O. Okazaki, and K. Sudo Covalent Binding and Tissue Distribution/Retention Assessment of Drugs Associated with Idiosyncratic Drug Toxicity Drug Metab. Dispos., September 1, 2008; 36(9): 1770 - 1779. [Abstract] [Full Text] [PDF]