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 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 sequential N-demethylation. N-Demethylation of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K(m) values were 19 and 600 microM for nor-LAAM and 4 and 450 microM 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 microM) and high (250 microM) 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, K(m) values were 6 and 0.2 microM, 1250 and 530 microM, 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.
Oda
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Department of Anesthesiology, Osaka City University Medical School, Osaka, Japan.
Kharasch
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D000911:Antibodies, Monoclonal / Q000276:immunology; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000502:physiology*; D003640:Dealkylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008692:Methadyl Acetate / Q000031:analogs & derivatives* / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000502:physiology*; D009294:Narcotics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000502:physiology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009294:Narcotics; C020783:paracymethadol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; C104464:CYP3A protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D008692:Methadyl Acetate