Walton M I, Workman P
MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, U.K.
Biochem Pharmacol. 1990 Jun 1;39(11):1735-42. doi: 10.1016/0006-2952(90)90119-6.
SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel benzotriazine di-N-oxide which shows unusually high selective toxicity towards hypoxic cells, probably as a result of reductive bioactivation. Using an HPLC assay for the parent drug and its 2- and 4-electron reduction products (SR 4317 and SR 4330, respectively), we have examined the enzymology of SR 4233 reductive metabolism in vitro using a variety of different enzyme preparations. SR 4233 was converted extremely rapidly to SR 4317 under N2 by mouse liver microsomes, and showed a marked preference for NADPH over NADH as a reduced cofactor. The reaction was inhibited completely in air and boiled preparations. It was also inhibited by 78-86% in carbon monoxide (CO), implicating cytochrome P-450 as the major microsomal SR 4233 reductase. The kinetics of reductive metabolism of SR 4233 to SR 4317 by mouse liver microsomes conformed to Michaelis-Menten kinetics, with a Km of 1.4 mM and a Vmax of 950 nmol/min/mg protein. SR 4233 reduction was also catalysed by mouse liver cytosol under N2. However, rates were markedly slower than for microsomes and showed an equal dependency on NADH and NADPH. The cytosolic enzymes aldehyde oxidase and xanthine oxidase both catalysed SR 4233 reduction to SR 4317 under N2. Purified buttermilk xanthine oxidase also catalysed this reaction. In contrast to other enzyme preparations, DT-diaphorase from Walker 256 tumour cells reduced SR 4233 predominantly to SR 4330, and this reaction occurred under aerobic conditions. These data illustrate that SR 4233 is reduced rapidly by a wide variety of reductases. We propose that the therapeutic selectivity of SR 4233 will be controlled by the relative expression of reductases in tumour versus normal tissues, and in particular by the differential participation of putative activating versus detoxifying enzymes.
SR 4233（3 - 氨基 - 1,2,4 - 苯并三嗪 - 1,4 - 二氧化物）是一种新型的苯并三嗪二 - N - 氧化物，对缺氧细胞表现出异常高的选择性毒性，这可能是还原生物活化的结果。我们使用高效液相色谱法测定母体药物及其2 - 电子和4 - 电子还原产物（分别为SR 4317和SR 4330），利用多种不同的酶制剂在体外研究了SR 4233还原代谢的酶学性质。在氮气环境下，小鼠肝微粒体可将SR 4233极快速地转化为SR 4317，并且相对于NADH，其对作为还原辅因子的NADPH表现出明显的偏好。该反应在空气和煮沸的制剂中完全被抑制。在一氧化碳（CO）中也被抑制78 - 86%，这表明细胞色素P - 450是主要的微粒体SR 4233还原酶。小鼠肝微粒体将SR 4233还原为SR 4317的代谢动力学符合米氏动力学，Km为1.4 mM，Vmax为950 nmol/min/mg蛋白质。在氮气环境下，小鼠肝细胞溶胶也能催化SR 4233的还原反应。然而，其反应速率明显慢于微粒体，并且对NADH和NADPH表现出同等的依赖性。胞质酶醛氧化酶和黄嘌呤氧化酶在氮气环境下均能催化SR 4233还原为SR 4317。纯化的酪乳黄嘌呤氧化酶也能催化此反应。与其他酶制剂不同，来自Walker 256肿瘤细胞的DT - 黄递酶主要将SR 4233还原为SR 4330，并且该反应在有氧条件下发生。这些数据表明，SR 4233能被多种还原酶快速还原。我们推测，SR 4233的治疗选择性将由肿瘤组织与正常组织中还原酶的相对表达所控制，特别是由假定的活化酶与解毒酶的差异参与所控制。
