N-oxygenation of amphetamine and methamphetamine by the human flavin-containing monooxygenase (Form 3): Role in bioactivation and detoxication

(+)- And (-)-amphetamine and methamphetamine were N-oxygenated by the cDNA expressed adult human flavin-containing monooxygenase form 3 (FMO3), their corresponding hydroxylamines. Two major polymorphic forms of human FMO3 wer e studied, and the results suggested preferential N-oxygenation by only one of the two enzymes. Chemically synthesized (+/-)-amphetamine hydroxylamine was also a substrate for the human FMO3 and it was converted to phenylprop anone oxime with a stereoselectivity ratio of trans/cis of 5:1. Human FMO3 also N-oxygenated methamphetamine to produce methamphetamine hydroxylamine. Methamphetamine hydroxylamine was also N-oxygenated by human FMO3, and the ultimate product observed was phenylpropanone. For amphetamine hydroxylami ne, studies of the biochemical mechanism of product formation were consiste nt with the production of an N,N-dioxygenated intermediate that lead to phe nylpropanone oxime. This was supported by the observation that alpha-deuter o (+/-)-amphetamine hydroxylamine gave an inverse kinetic isotope effect on product formation in the presence of human FMO3. For methamphetamine, the data were consistent with a mechanism of human FMO3-mediated N,N-dioxygenat ion but the immediate product, a nitrone, rapidly hydrolyzed to phenylpropa none. The pharmacological activity of amphetamine hydroxylamine, phenylprop anone oxime, and methamphetamine hydroxylamine were examined for effects at the human dopamine, serotonin, and norepinephrine transporters. Amphetamin e hydroxylamine and methamphetamine hydroxylamine were apparent substrates for the human biogenic amine transporters but phenylpropanone oxime was not . Presumably, phenylpropanone oxime or nitrone formation from amphetamine a nd methamphetamine, respectively, represents a detoxication process. Becaus e of the potential toxic nature of amphetamine hydroxylamine and methamphet amine hydroxylamine metabolites and the polymorphic nature of N-oxygenation , human FMO3-mediated metabolism of amphetamine or methamphetamine may have clinical consequences.