ArticlesParallel Pathway Interactions in Imipramine Metabolism in Rats
Abstract
The in vitro metabolic inhibitions between imipramine and its metabolites were investigated in rat liver microsomes. A type of precursor-metabolite interaction similar to that shown with lidocaine was observed in imipramine metabolism. Desipramine competitively inhibited the formation of 2-hydroxyimipramine from imipramine. Similarly, imipramine inhibited the formation of 2-hydroxydesipramine from desipramine. As in the cases of those 2-hydroxylations, a competitive inhibitory relationship also existed in the N-demethylation pathways of imipramine and 2-hydroxyimipramine. Studies on age-associated alterations of the metabolic rates of imipramine and its metabolites in rats demonstrated that N-demethylation activities of imipramine and of 2-hydroxyimipramine, which showed a large sex difference (male > female) in young rats, decreased markedly only in old male rats, while 2-hydroxylation activities of imipramine and desipramine, with no sex difference at any age, did not show a marked alteration in either sex. These data strongly suggest that the hydroxylation pathways of imipramine and desipramine and the demethylation pathways of imipramine and 2-hydroxyimipramine are each sharing the same species of cytochrome P-450. The in vivo metabolic inhibition between imipramine and desipramine was examined by simultaneous intraportal infusion of imipramine (25 nmol/min) and desipramine (175 nmol/min). The steady-state concentration of imipramine after simultaneous infusion was increased twofold over that after infusion of imipramine alone, without any change in the free fraction in blood.
References and Notes (23)
- JacobsonM. et al.
Biochem. Pharmacol.
(1969) - SuzukiT. et al.
J. Pharm. Sci.
(1984) - ChibaM. et al.
Biochem. Pharmacol.
(1985) - ChibaM. et al.
Biochem. Pharmacol.
(1986) - OmuraT. et al.
J. Biol. Chem.
(1964) - LowryO.H. et al.
J. Biol. Chem.
(1951) - BorondyP. et al.
Pharmacologist.
(1972) - BastA. et al.
Biochem. Pharmacol.
(1980) - JahnehenE. et al.
Proc. Soc. Exp. Biol. Med.
(1972) - AsleyJ.J. et al.
Res. Comm. Chem. Pathol.
(1973)
Eur. J. Clin. Pharmacol.
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