Liver metabolic reactions: Tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation : I. Tricyclic tertiary amine drugs

Abstract

N-oxide reduction and N-oxide demethylation, shown to occur in homogenates of various rat tissues with imipramine- N-oxide, were also observed with the N-oxides of chlorpromazine and amitriptyline. With imipramine- N-oxide the rates of N-oxide reduction and N-oxide demethylation were higher in liver homogenates of pigs and horses than of rats. The subcellular sites of the two reactions could not be clearly localized in rat liver fractions, however, microsomes, mitochondria, and probably nuclei, alone and in combination with cytosol or NADPH, displayed negligible activity. Next to the total homogenate the crude nuclear fraction was the most active. N-oxide reduction and N-oxide demethylation are clearly differentiated from tertiary amine N-demethylation and N-oxidation by oxygen, nitrogen, heat pretreatment, ferricytochrome c, and neotetrazolium chloride. The microsomal reactions, tertiary amine demethylation and N-oxidation, differ in that the former but not the latter reaction is inhibited by CO, SKF 525-A, and excess substrate. N-oxide reduction and N-oxide demethylation showed identical responses to the procedures mentioned, except for a higher degree of heat stability of the latter reaction. Experiments with labeled imipramine or imipramine- N-oxide and unlabeled pools of one of the compounds unequivocally confirmed the presence or absence of the following metabolic reactions in rat liver preparations: Tertiary amine demethylation and N-oxidation occur in 9000 g supernatant fraction or microsomes + NADPH but not in homogenate without added NADPH; N-oxide reduction and N-oxide demethylation occur in the latter system but not in the former. All four reactions are simultaneously operative in homogenate fortified by an NADPH-generating system, i.e., the combination of the above systems. The kinetics of the four individual reactions have been estimated by incubating labeled imipramine and a pool of unlabeled imipramine- N-oxide in rat liver homogenate with an NADPH-generating system and determination of amounts, absolute and specific radioactivities of these compounds as well as desmethylimipramine as a function of time. In the steady state the reaction constants could be arranged in the following order: N-oxide reduction ⪢ N-oxide demethylation ⩾ tertiary amine demethylation > tertiary amine- N-oxidation. The results demonstrate that the N-oxide can be ruled out as an intermediate in the oxidative N-demethylation of imipramine in liver microsomal systems and is negligible even in NADPH-fortified liver homogenate. The oxidative N-dealkylation of this tertiary amine is thus likely to represent a C-oxygenation proceeding independently from an N-oxygenation.