Biomaterial Database

Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: in relation to covalent binding of its reactive intermediate. 1996

Y Masubuchi, and S Igarashi, and T Suzuki, and 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.

UI	MeSH Term	Description	Entries
D007099	Imipramine	The prototypical tricyclic antidepressant. It has been used in major depression, dysthymia, bipolar depression, attention-deficit disorders, agoraphobia, and panic disorders. It has less sedative effect than some other members of this therapeutic group.	Imidobenzyle,Imizin,4,4'-Methylenebis(3-hydroxy-2-naphthoic acid)-3-(10,11-dihydro-5H-dibenzo(b,f)azepin-5-yl)-N,N-dimethyl-1-propanamine (1:2),Imipramine Hydrochloride,Imipramine Monohydrochloride,Imipramine Pamoate,Janimine,Melipramine,Norchlorimipramine,Pryleugan,Tofranil
D008297	Male		Males
D008862	Microsomes, Liver	Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough.	Liver Microsomes,Liver Microsome,Microsome, Liver
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011233	Precipitin Tests	Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate.	Precipitin Test,Test, Precipitin,Tests, Precipitin
D011412	Propanolamines	AMINO ALCOHOLS containing the propanolamine (NH2CH2CHOHCH2) group and its derivatives.	Aminopropanols
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D005260	Female		Females
D006900	Hydroxylation	Placing of a hydroxyl group on a compound in a position where one did not exist before. (Stedman, 26th ed)	Hydroxylations
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein