Schmider J, Greenblatt D J, Fogelman S M, von Moltke L L, Shader R I
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.
Biopharm Drug Dispos. 1997 Apr;18(3):227-40. doi: 10.1002/(sici)1099-081x(199704)18:3<227::aid-bdd18>3.0.co;2-l.
Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxymorphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of CYP2d6 (quinidine), CYP1A1/2 (alpha-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.
右美沙芬（DMO）是一种具有止咳作用的可待因合成类似物，在人体内会同时发生O - 去甲基化生成右啡烷（DOP）以及N - 去甲基化生成3 - 甲氧基吗啡喃（MEM）。3 - 羟基吗啡喃是一种双去甲基代谢产物，是次要生成的。O - 去甲基化活性已被充分确立为CYP2D6的指标反应。然而，该途径在体外似乎由至少两种不同的酶介导。最近有人提出N - 去甲基化活性可反映CYP3A3/4的活性。我们用来自三个人类肝脏的微粒体制剂在体外研究了这两条途径，以评估DMO作为CYP2D6和CYP3A3/4探针药物的价值。DMO的O - 去甲基化呈现双相模式，其中高亲和力位点反映CYP2D6活性（奎尼丁的平均Ki为0.1±0.13 microM）。两种介导O - 去甲基化的酶的动力学参数预测，通过高亲和力酶介导的O - 去甲基化总量的平均相对内在清除率（Vmax/K(m)比值）为96%。因此，体外数据证实了DMO的O - 去甲基化作为监测CYP2D6活性的指标反应的有用性。DMO N - 去甲基化的伊迪 - 霍夫斯泰因图与单酶米氏动力学一致（Vmax在3.3至6.8 nmol mg-1 min-1之间，K(m)在231至322 microM之间）。然而，CYP3A3/4抑制剂酮康唑仅使N - 去甲基化降低了60%，并且与其他纯CYP3A3/4介导的反应相比，其平均Ki高一个数量级（0.37 microM）。基于机制的CYP3A3/4抑制剂醋竹桃霉素平均最大抑制MEM形成46%，IC50在1至2.6 microM之间变化。一种多克隆大鼠肝脏CYP3A1抗体仅使MEM形成抑制约50%。基于机制的CYP2E1抑制剂二乙二硫代氨基甲酸盐（DDC）在浓度高达150 microM时使MEM形成降低33%至43%。CYP2d6（奎尼丁）、CYP1A1/2（α - 萘黄酮）和CYP2C9（磺胺苯吡唑）的化学抑制剂，以及一种山羊大鼠肝脏CYP2C11多克隆抗体（对人CYP2C9和CYP2C19有抑制作用）对MEM形成速率的影响极小，因此排除了这些酶中的任何一种参与。DMO的N - 去甲基化仅部分由CYP3A3/4介导，因此无论是在体外还是可能在体内，它都不是CYP3A3/4活性的可靠指标反应。
