Dive C, Workman P, Watson J V
MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, UK.
Cancer Chemother Pharmacol. 1989;25(3):149-55. doi: 10.1007/BF00689574.
Using flow cytometry and conventional spectrofluorimetry we have previously shown that chloroethylnitrosoureas (CNUs) can exhibit marked inhibition of cellular enzymes catalysing hydrolysis of fluorescein diacetate (FDA). More potent inhibition was seen for the carbamoylating CNUs, whereas alkylating agents were largely inactive. We now report results obtained with the developmental imidazotetrazines mitozolomide and temozolomide in comparison with BCNU, the novel alkylating agents clomesome and cyclodisone, and the active mitozolomide metabonate MCTIC. Inhibition of EMT6 mouse mammary-tumour esterases was seen for mitozolomide and temozolomide, and activity against purified porcine carboxylesterase was demonstrated. Flow cytometric analysis showed that inhibition occurred across the entire EMT6 cell population, with no evidence of a subpopulation resistant to enzyme inhibition. Inhibitory potency for the imidazotetrazines was much weaker than for BCNU. With EMT6 cells, I50 values from flow cytometry were 9.7 x 10(-3) M and 1.5 x 10(-3) M for mitozolomide and temozolomide compared with 3.7 x 10(-4) M for BCNU. These were higher than the ID50 values for in vitro antitumour activity (MTT assay), 8.5 x 10(-6) M in the case of mitozolomide and 1.2 x 10(-5) M for BCNU, but similar to that of 5.6 x 10(-4) M for the less toxic temozolomide. MCTIC and cyclodisone showed very low activity, but significant inhibition was seen for clomesome. The results are consistent with the view that the imidazotetrazines do not exhibit major carbamoylating ability, although significant effects are seen at cytotoxic concentrations of temozolomide. In addition, the potential for the generation of carbamoylating species at the enzyme active site cannot be ruled out.
我们之前使用流式细胞术和传统荧光分光光度法表明，氯乙基亚硝脲（CNUs）可显著抑制催化荧光素二乙酸酯（FDA）水解的细胞酶。氨基甲酰化的CNUs表现出更强的抑制作用，而烷基化剂大多无活性。我们现在报告与卡莫司汀（BCNU）、新型烷基化剂氯美索和环二酮以及活性代谢物MCTIC相比，开发中的咪唑并四嗪类药物米托唑胺和替莫唑胺的研究结果。米托唑胺和替莫唑胺对EMT6小鼠乳腺肿瘤酯酶有抑制作用，并证明了其对纯化猪羧酸酯酶的活性。流式细胞术分析表明，整个EMT6细胞群体均出现抑制，没有证据表明存在对酶抑制有抗性的亚群。咪唑并四嗪类药物的抑制效力比BCNU弱得多。对于EMT6细胞，流式细胞术得到的米托唑胺和替莫唑胺的半数抑制浓度（IC50）值分别为9.7×10⁻³ M和1.5×10⁻³ M，而BCNU为3.7×10⁻⁴ M。这些值高于体外抗肿瘤活性（MTT法）的半数抑制浓度（ID50）值，米托唑胺为8.5×10⁻⁶ M，BCNU为1.2×10⁻⁵ M，但与毒性较小的替莫唑胺的5.6×10⁻⁴ M相似。MCTIC和环二酮活性非常低，但氯美索有显著抑制作用。结果与以下观点一致，即咪唑并四嗪类药物不具有主要的氨基甲酰化能力，尽管在替莫唑胺的细胞毒性浓度下有显著作用。此外，不能排除在酶活性位点生成氨基甲酰化物种的可能性。
