Metabolism of levo-alpha-acetylmethadol (LAAM) by human liver cytochrome P450: Involvement of CYP3A4 characterized by atypical kinetics with two binding sites

levo-alpha -Acetylmethadol (LAAM) is a long-acting opioid agonist prodrug u sed for preventing opiate withdrawal. LAAM undergoes bioactivation via sequ ential N-demethylation to nor-LAAM and dinor-LAAM, which are more potent an d longer-acting than LAAM. This study examined LAAM and nor-LAAM metabolism using human liver microsomes, cDNA-expressed CYP, CYP isoform-selective ch emical inhibitors, and monoclonal antibody to determine kinetic parameters for predicting in vivo drug interactions, involvement of constitutive CYP i soforms, and mechanistic aspects of sequential N-demethylation. N-Demethyla tion of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadi e-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K-m values wer e 19 and 600 muM for nor-LAAM and 4 and 450 muM for dinor-LAAM formation, r espectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selec tive inhibitors troleandomycin, erythromycin, ketoconazole, and midazolam. Of the cDNA-expressed isoforms examined, CYP2B6 and 3A4 had the highest act ivity toward LAAM and nor-LAAM at both low (2 muM) and high (250 mM) substr ate concentrations. N-Demethylation of LAAM and nor-LAAM by expressed CYP3A 4 was unusual, with hyperbolic velocity curves and Eadie-Hofstee plots and without evidence of positive cooperativity. Using a two-site model, K-m val ues were 6 and 0.2 muM, 1250 and 530 muM, respectively. Monoclonal antibody against CYP2B6 inhibited CYP2B6-catalyzed but not microsomal LAAM or nor-L AAM metabolism, whereas troleandomycin inhibited metabolism in all microsom es studied. The ratio [dinor-LAAM/(nor-LAAM plus dinor-LAAM)] with microsom es 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 ar e predominantly metabolized by CYP3A4 in human liver microsomes, and CYP3A4 exhibits unusual multisite kinetics.