Oda Y, Kharasch E D
Department of Anesthesiology, Osaka City University Medical School, Osaka, Japan.
J Pharmacol Exp Ther. 2001 Apr;297(1):410-22.
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.
左旋 -α- 乙酰美沙多（LAAM）是一种长效阿片类激动剂前体药物，用于预防阿片类药物戒断。LAAM 通过连续的 N - 去甲基化生物活化生成去甲 -LAAM 和双去甲 -LAAM，它们比 LAAM 更有效且作用时间更长。本研究使用人肝微粒体、cDNA 表达的细胞色素 P450（CYP）、CYP 同工酶选择性化学抑制剂和单克隆抗体来研究 LAAM 和去甲 -LAAM 的代谢，以确定预测体内药物相互作用的动力学参数、组成型 CYP 同工酶的参与情况以及连续 N -去甲基化的机制方面。人肝微粒体对 LAAM 和去甲 -LAAM 的 N -去甲基化呈现双相 Eadie - Hofstee 图。使用双酶 Michaelis - Menten 模型，去甲 -LAAM 生成的米氏常数（K(m)）值分别为 19 和 600 μM，双去甲 -LAAM 生成的 K(m)值分别为 4 和 450 μM。LAAM 和去甲 -LAAM 的代谢受到 CYP3A4 选择性抑制剂三乙酰竹桃霉素、红霉素、酮康唑和咪达唑仑的抑制。在所检测的 cDNA 表达同工酶中，CYP2B6 和 3A4 在低（2 μM）和高（250 μM）底物浓度下对 LAAM 和去甲 -LAAM 的活性最高。表达的 CYP3A4 对 LAAM 和去甲 -LAAM 的 N -去甲基化不寻常，具有双曲线速度曲线和 Eadie - Hofstee 图，且无正协同性证据。使用双位点模型，K(m)值分别为 6 和 0.2 μM、1250 和 530 μM。抗 CYP2B6 的单克隆抗体抑制 CYP2B6 催化的代谢，但不抑制微粒体中 LAAM 或去甲 -LAAM 的代谢，而三乙酰竹桃霉素抑制所有研究的微粒体中的代谢。微粒体和 CYP3A4 中[双去甲 -LAAM/(去甲 -LAAM + 双去甲 -LAAM)]的比值随 LAAM 浓度增加而降低，表明大多数双去甲 -LAAM 由释放的去甲 -LAAM 形成，随后去甲 -LAAM 与 CYP3A4 重新结合。基于这些结果，我们得出结论，LAAM 和去甲 -LAAM 在人肝微粒体中主要由 CYP3A4 代谢，且 CYP3A4 表现出不寻常的多位点动力学。
