TY - JOUR T1 - Metabolism of <em>levo</em>-α-Acetylmethadol (LAAM) by Human Liver Cytochrome P450: Involvement of CYP3A4 Characterized by Atypical Kinetics with Two Binding Sites JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 410 LP - 422 VL - 297 IS - 1 AU - Yutaka Oda AU - Evan D. Kharasch Y1 - 2001/04/01 UR - http://jpet.aspetjournals.org/content/297/1/410.abstract N2 - levo-α-Acetylmethadol (LAAM) is a long-acting opioid agonist prodrug used for preventing opiate withdrawal. LAAM undergoes bioactivation via sequential N-demethylation to nor-LAAM and dinor-LAAM, which are more potent and longer-acting than LAAM. This study examined LAAM and nor-LAAM metabolism using human liver microsomes, cDNA-expressed CYP, CYP isoform-selective chemical inhibitors, and monoclonal antibody to determine kinetic parameters for predicting in vivo drug interactions, involvement of constitutive CYP isoforms, and mechanistic aspects of sequentialN-demethylation. N-Demethylation of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model,Km values were 19 and 600 μM for nor-LAAM and 4 and 450 μM for dinor-LAAM formation, respectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selective inhibitors troleandomycin, erythromycin, ketoconazole, and midazolam. Of the cDNA-expressed isoforms examined, CYP2B6 and 3A4 had the highest activity toward LAAM and nor-LAAM at both low (2 μM) and high (250 μM) substrate concentrations. N-Demethylation of LAAM and nor-LAAM by expressed CYP3A4 was unusual, with hyperbolic velocity curves and Eadie-Hofstee plots and without evidence of positive cooperativity. Using a two-site model, Kmvalues were 6 and 0.2 μM, 1250 and 530 μM, respectively. Monoclonal antibody against CYP2B6 inhibited CYP2B6-catalyzed but not microsomal LAAM or nor-LAAM metabolism, whereas troleandomycin inhibited metabolism in all microsomes studied. The ratio [dinor-LAAM/(nor-LAAM plus dinor-LAAM)] with microsomes and CYP3A4 decreased with increasing LAAM concentration, suggesting most dinor-LAAM is formed from released nor-LAAM that subsequently reassociates with CYP3A4. Based on these results, we conclude that LAAM and nor-LAAM are predominantly metabolized by CYP3A4 in human liver microsomes, and CYP3A4 exhibits unusual multisite kinetics. The American Society for Pharmacology and Experimental Therapeutics ER -