In vitro-in vivo correlations of human ( s)-nicotine metabolism

Abstract

The profile of ( S)-nicotine metabolism in human liver microsomes was examined at concentrations approaching in vivo conditions (10 μM). At such concentrations, no ( S)-nicotine N-1′-oxygenation was seen, and thus C-oxidation to the ( S)-nicotine Δ 1′,5′-iminium ion was the sole product observed in the metabolic profile in the presence of the human liver microsomes. For simplicity of analysis, the ( S)-nicotine Δ 1′,5′-iminium ion formed was converted to ( S)-cotinine in the presence of exogenously added aldehyde oxidase. To explain the lack of ( S)-nicotine N-1′-oxygenation at low ( S)-nicotine concentrations, inhibition of flavin-containing monooxygenase (FMO) activity by ( S)-cotinine was examined. Although ( S)-cotinine was observed to inhibit pig FMO1 ( K i = 675 μM), partially purified cDNA-expressed adult human liver FMO3 was not inhibited by ( S)-cotinine. We therefore concluded that the kinetic properties of the nicotine N′- and C-oxidases were responsible for the metabolic product profile observed. Kinetic constants were determined for individual human liver microsomal preparations from low (10 μM) and high (500 μM) ( S)-nicotine concentrations by monitoring ( S)-cotinine formation with HPLC. The mean K m app and V max for formation of ( S)-cotinine by the microsomes examined were 39.6 μM and 444.3 pmol· min −1 · (mg protein) −1, respectively. The formation of ( S)-cotinine was strongly dependent on the previous drug administration history of each subject, and among the highest rates for ( S)-cotinine formation were those of the barbiturate-pretreated subjects. The rate of ( S)-cotinine formation at low (10 μM) concentration correlated well with immunoreactivity for cytochrome P450 2A6 ( r = 0.89). In vitro-in vivo correlation of the results suggests that the low amount of ( S)-nicotine N-1′-oxygenation and the large amount of ( S)-cotinine formed in human smokers (i.e. 4 and 30% of a typical dose, respectively) are determined primarily by the kinetic properties of the human monooxygenase enzyme systems. However, additional non-hepatic monooxygenase(s) contributes to ( S)-nicotine metabolism.