Differential N-demethylation of l-alpha-acetylmethadol (LAAM) and norLAAM by Cytochrome P450s 2B6, 2C18, and 3A4

Incubation of l-alpha -acetylmethadol (LAAM) or norLAAM with cDNA-expressed P450s 3A4, 2B6, and 2C18 produced significant N-demethylation products. P4 50s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced de tectable product. Coexpression of cytochrome b(5) enhanced LAAM N-demethyla tion, most dramatically for 3A4, but had marginal effects on norLAAM N-deme thylation. Modeling total liver metabolism using immunoquantification and r elative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative acti vity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b(5) coexpression d ependent. This ratio decreased with concentration with 3A4, but was relativ ely constant for 2B6 and 2C18. The human liver microsomes substrate-concent ration response was similar to cDNA-expressed 3A4, but the ratio was higher . Changes in the environment of cDNA-expressed 3A4 also effected the magnit ude of the ratio, but not the concentration-dependent decrease. These studi es show that the N-demethylation of LAAM and norLAAM is not restricted to P 450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18. (C) 2001 Academic Press.