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 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.
Masubuchi
Y
Y
Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Igarashi
S
S
Suzuki
T
T
Horie
T
T
Narimatsu
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000929:Antidepressive Agents, Tricyclic / Q000494:pharmacology*; D001711:Biotransformation; D065607:Cytochrome P-450 Enzyme Inhibitors; D005260:Female; D006900:Hydroxylation; D007099:Imipramine / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011412:Propanolamines / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
D000929:Antidepressive Agents, Tricyclic; D065607:Cytochrome P-450 Enzyme Inhibitors; D011412:Propanolamines; C084811:bunitrolol; D007099:Imipramine