Kerry N L, Somogyi A A, Mikus G, Bochner F
Department of Clinical and Experimental Pharmacology, University of Adelaide, Australia.
Biochem Pharmacol. 1993 Feb 24;45(4):833-9. doi: 10.1016/0006-2952(93)90166-t.
The O-demethylation of dextromethorphan (DM) to dextrorphan (DR) is catalysed by the polymorphic CYP2D6 (cytochrome P4502D6) isozyme in man. DM is commonly used as a probe for phenotyping subjects as either poor or extensive metabolizers for the debrisoquine/sparteine oxidative polymorphism via CYP2D6. The enzyme kinetics of DM O- and N-demethylation, and the N- and O-demethylations of the primary metabolites DR and 3-methoxymorphinan (3MM), respectively, were studied in liver microsomes from female Dark Agouti (DA) rats, the poor metabolizer counterpart, and female Sprague-Dawley (SD) rats, the extensive metabolizer counterpart. The formation of metabolites was quantified by HPLC with fluorescence detection and kinetic parameters were calculated. The intrinsic clearance (Vmax/Km) of the O-demethylation of 3MM to 3-hydroxymorphinan (3OHM) was 180-fold lower in DA rats (0.11 vs 20.77 mL/hr/mg) due to a 60-fold higher Km (108.7 vs 1.76 microM) and 3-fold lower Vmax (11.5 vs 35.95 nmol/mg/hr). The kinetics for DR N-demethylation to 3OHM did not differ between rat strains. The Michaelis-Menten constant (Km) for DM N-demethylation to 3MM was similar between SD and DA rats (85.04 vs 68.99 microM); however, SD rats displayed a 2-fold higher Vmax (83.37 vs 35.49 nmol/mg/hr) and intrinsic clearance (0.96 vs 0.51 mL/hr/mg). The O-demethylation of DM to DR in SD rats showed a high and low affinity enzyme component, with the high affinity intrinsic clearance contributing 98% of the total intrinsic clearance in these rats. DM O-demethylation in DA rats was characterized by a single enzyme system. The high affinity O-demethylating enzyme in SD rats showed a 20-fold lower Km (2.5 vs 55.6 microM) and a three-fold higher Vmax (51.04 vs 16.84 nmol/mg/hr) resulting in a 66-fold higher intrinsic clearance (20.04 vs 0.31 mL/hr/mg) compared to DA rats. Quinine, dextropropoxyphene, (+/-)methadone and (+/-)propafenone were shown to be potent inhibitors of 3MM and DM O-demethylation but did not inhibit DR or DM N-demethylation at similar concentrations. SD and DA rats showed a clear strain difference in 3MM O-demethylation and DM O-demethylation. In contrast, DR N-demethylation and DM N-demethylation do not appear to be under genetic control in the female SD-DA rat model. Kinetic parameters and inhibition studies suggest that 3MM and DM O-demethylation pathways in the rat may be mediated by the same cytochrome P450 isozyme.
右美沙芬（DM）向右啡烷（DR）的O-去甲基化反应由人体中的多态性细胞色素P450 2D6（CYP2D6）同工酶催化。DM通常用作探针，通过CYP2D6对受试者进行表型分析，判断其为异喹胍/鹰爪豆碱氧化多态性的慢代谢者还是快代谢者。分别在雌性暗褐鼠（DA）（慢代谢者对应品系）和雌性斯普拉格-道利大鼠（SD）（快代谢者对应品系）的肝微粒体中研究了DM的O-去甲基化和N-去甲基化以及主要代谢产物DR和3-甲氧基吗啡喃（3MM）的N-去甲基化和O-去甲基化的酶动力学。通过高效液相色谱-荧光检测法定量代谢产物的形成，并计算动力学参数。由于Km高60倍（108.7对1.76 μM）和Vmax低3倍（11.5对35.95 nmol/mg/hr），DA大鼠中3MM向3-羟基吗啡喃（3OHM）的O-去甲基化的内在清除率（Vmax/Km）低180倍（0.11对20.77 mL/hr/mg）。大鼠品系之间DR N-去甲基化生成3OHM的动力学无差异。SD大鼠和DA大鼠中DM N-去甲基化生成3MM的米氏常数（Km）相似（85.04对68.99 μM）；然而，SD大鼠的Vmax高2倍（83.37对35.49 nmol/mg/hr），内在清除率高2倍（0.96对0.51 mL/hr/mg）。SD大鼠中DM向DR的O-去甲基化表现出高亲和力和低亲和力酶组分，高亲和力内在清除率占这些大鼠总内在清除率的98%。DA大鼠中的DM O-去甲基化由单一酶系统表征。与DA大鼠相比，SD大鼠中的高亲和力O-去甲基化酶的Km低20倍（2.5对55.6 μM），Vmax高3倍（51.04对16.84 nmol/mg/hr），导致内在清除率高66倍（20.04对0.31 mL/hr/mg）。奎宁、右丙氧芬、（±）美沙酮和（±）普罗帕酮被证明是3MM和DM O-去甲基化的有效抑制剂，但在相似浓度下不抑制DR或DM N-去甲基化。SD大鼠和DA大鼠在3MM O-去甲基化和DM O-去甲基化方面表现出明显的品系差异。相反，在雌性SD-DA大鼠模型中，DR N-去甲基化和DM N-去甲基化似乎不受遗传控制。动力学参数和抑制研究表明，大鼠中的3MM和DM O-去甲基化途径可能由同一种细胞色素P450同工酶介导。
