Wienkers L C, Steenwyk R C, Mizsak S A, Pearson P G
Upjohn Laboratories, Kalamazoo, MI 49001, USA.
Drug Metab Dispos. 1995 Mar;23(3):383-92.
Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage and head injury. In rat, tirilazad seems to be highly extracted and is cleared almost exclusively via hepatic elimination. The biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats. Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites: M1 and M2. In incubations with female rat liver microsomes, M2 was the only primary metabolite detected. Structural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the delta 4-double bond in the steroid A-ring, whereas M1 arose from oxidative desaturation of one pyrrolidine ring. Further structural analysis of M2 by proton NMR demonstrated that reduction at C-5 had occurred by addition of hydrogen in the alpha-configuration. Using metabolic probes and antibodies specific to individual hepatic microsomal enzymes, CYP2C11 and 3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively. The formation of M1 was inhibited by testosterone, nicotine, cimetidine, and anti-CYP2C11 IgG. The formation of M2 was inhibited by finasteride, a potent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-mediated M1 formation in male rat liver microsomal incubations revealed that M1 formation occurred through a low-affinity/low-capacity process (KM = 16.67 microM, Vmax = 0.978 nmol/mg microsomal protein/min); the formation of M2 was mediated by 5 alpha-reductase in a high-affinity/low-capacity process (KM = 3.07 microM, Vmax = 1.06 nmol/mg microsomal protein/min). In contrast, the formation of M2 in female rat liver microsomes was mediated by 5 alpha-reductase in a high-affinity/high-capacity process (KM = 2.72 microM, Vmax = 4.11 nmol/mg microsomal protein/min). Comparison of calculated intrinsic formation clearances (Vmax/KM) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the male rat. Therefore, the clearance of tirilazad mesylate in the rat is mediated primarily by rat liver 5 alpha-reductase, and the capacity in the female rat is 5-fold the capacity in the male. These observations correlate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.
甲磺替拉扎特是一种在体外能有效抑制膜脂质过氧化的药物，目前正处于治疗蛛网膜下腔出血和头部损伤的临床研发阶段。在大鼠体内，替拉扎特似乎具有很高的提取率，几乎完全通过肝脏消除。已在成年雄性和雌性斯普拉格-道利大鼠的肝微粒体制剂中研究了替拉扎特的生物转化。雄性大鼠肝微粒体中的替拉扎特代谢产生了两种主要代谢物：M1和M2。在与雌性大鼠肝微粒体的孵育中，M2是唯一检测到的主要代谢物。通过质谱对M1和M2进行结构表征表明，M2是由甾体A环中δ4-双键的还原形成的，而M1则来自一个吡咯烷环的氧化去饱和。通过质子核磁共振对M2进行进一步结构分析表明，C-5处的还原是通过α-构型的氢加成发生的。使用针对个别肝微粒体酶的代谢探针和抗体，CYP2C11和3-氧代-5α-甾体：NADP+δ4-氧化还原酶（5α-还原酶）分别被确定为负责M1和M2的形成。睾酮、尼古丁、西咪替丁和抗CYP2C11 IgG抑制了M1的形成。5α-还原酶的强效抑制剂非那雄胺抑制了M2的形成。对雄性大鼠肝微粒体孵育中CYP2C11介导的M1形成进行动力学分析表明，M1的形成是通过低亲和力/低容量过程发生的（KM = 毫克微粒体蛋白/分钟）；M2的形成由5α-还原酶以高亲和力/低容量过程介导（KM = 3.07 microM，Vmax = 1.06 nmol/mg微粒体蛋白/分钟）。相比之下，雌性大鼠肝微粒体中M2的形成由5α-还原酶以高亲和力/高容量过程介导（KM = 2.72 microM，Vmax = 4.11 nmol/mg微粒体蛋白/分钟）。对M1和M2计算的内在形成清除率（Vmax/KM）的比较表明，雌性大鼠对替拉扎特生物转化的体外代谢能力比雄性大鼠更强。因此，大鼠中甲磺替拉扎特的清除主要由大鼠肝脏5α-还原酶介导，雌性大鼠的能力是雄性大鼠的5倍。这些观察结果与记录的5α-还原酶活性差异相关，并预测了替拉扎特体内肝脏清除的性别差异。 16.67 microM，Vmax = 0.978 nmol/mg微粒体蛋白/分钟）；M2的形成由5α-还原酶以高亲和力/低容量过程介导（KM = 3.07 microM，Vmax = 1.06 nmol/mg微粒体蛋白/分钟）。相比之下，雌性大鼠肝微粒体中M2的形成由5α-还原酶以高亲和力/高容量过程介导（KM = 2.72 microM，Vmax = 4.11 nmol/mg微粒体蛋白/分钟）。对M1和M2计算的内在形成清除率（Vmax/KM）的比较表明，雌性大鼠对替拉扎特生物转化的体外代谢能力比雄性大鼠更强。因此，大鼠中甲磺替拉扎特的清除主要由大鼠肝脏5α-还原酶介导，雌性大鼠的能力是雄性大鼠的5倍。这些观察结果与记录的5α-还原酶活性差异相关，并预测了替拉扎特体内肝脏清除的性别差异。
