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

Yasuhiro Masubuchi, Shigeki Igarashi, Tokuji Suzuki, Toshiharu Horie, Shizuo Narimatsu


35 被引用数 (Scopus)


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.

ジャーナルJournal of Pharmacology and Experimental Therapeutics
出版ステータスPublished - 11月 1 1996

ASJC Scopus subject areas

  • 分子医療
  • 薬理学


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