Metabolism of methadone and levo-alpha-acetylmethadol (LAAM) by human intestinal cytochrome P450 3A4 (CYP3A4): Potential contribution of intestinal metabolism to presysternic clearance and bioactivation

Methadone and levo-alpha -acetylmethadol (LAAM) are opioid agonists used fo r analgesia and preventing opiate withdrawal. Methadone is sequentially N-d emethylated to the inactive metabolites 2-ethylidene-1,5-dimethyl-3,3-diphe nylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP). LAA M is essentially a prodrug that undergoes bioactivation via sequential N-de methylation to levo-alpha -acetyl-N-normethadol (nor-LAAM) and levo-alpha - acetyl-N,N-dinormethadol (dinor-LAAM). Methadone and LAAM are metabolized b y CYP3A4 in human liver. Since they are administered orally, and CYP3A4 is expressed in human intestine, we tested the hypotheses that human intestine can metabolize methadone and LAAM, and evaluated the participation of CYP3 A4. Intestinal microsomal methadone N-demethylation exhibited hyperbolic no ncooperative kinetics and biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K-m values were 11 and 1200 muM for EDDP and 23 an d 930 muM for EMDP formation, respectively. CYP3A4 inhibitors (troleandomyc in and ketoconazole) inhibited EDDP and EMDP formation by >70%. Methadone N -demethylation by CYP3A4 showed biphasic Eadie-Hofstee plots without eviden ce of positive cooperativity; K-m values were 10 and 1100 muM for EDDP and 20 and 1000 muM for EMDP formation. Intestinal microsomal LAAM and nor-LAAM N-demethylation also exhibited hyperbolic kinetics and biphasic Eadie-Hofs tee plots. K-m values were 21 and 980 muM for nor-LAAM from LAAM and 18 and 1200 muM for dinor-LAAM from nor-LAAM. Troleandomycin and ketoconazole inh ibited N-demethylation by >70%. LAAM and nor-LAAM metabolism by CYP3A4 show ed biphasic Eadie-Hofstee plots without evidence of positive cooperativity; K-m values were 8 and 1300 muM, 6 and 950 muM, respectively. Predicted in vivo intestinal extraction of methadone and LAAM is 21 and 33%, respectivel y. We conclude that methadone, LAAM, and nor-LAAM are metabolized by human intestinal microsomes; CYP3A4 is the predominant cytochrome P450 isoform; C YP3A4-catalyzed methadone, LAAM, and nor-LAAM metabolism is characterized b y noncooperative, multisite kinetics; and intestinal metabolism may contrib ute to presystemic methadone inactivation and LAAM bioactivation.