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. P450s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced detectable product. Coexpression of cytochrome b(5) enhanced LAAM N-demethylation, most dramatically for 3A4, but had marginal effects on norLAAM N-demethylation. Modeling total liver metabolism using immunoquantification and relative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative activity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b(5) coexpression dependent. This ratio decreased with concentration with 3A4, but was relatively constant for 2B6 and 2C18. The human liver microsomes substrate-concentration response was similar to cDNA-expressed 3A4, but the ratio was higher. Changes in the environment of cDNA-expressed 3A4 also effected the magnitude of the ratio, but not the concentration-dependent decrease. These studies show that the N-demethylation of LAAM and norLAAM is not restricted to P450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18.
Neff
J A
JA
Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112-9457, USA.
Moody
D E
DE
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008692:Methadyl Acetate / Q000031:analogs & derivatives* / Q000378:metabolism*; D008745:Methylation; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000502:physiology*; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
C020783:paracymethadol; C020700:1 alpha-acetyldinormethadol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; C082185:CYP2C18 protein, human; C104464:CYP3A protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D008692:Methadyl Acetate