METHOTREXATE AND METABOLISM IN HEPATIC CELLS

建议的研究是针对更深层次的理解和新颖的 抗叶酸剂的方法。 我们计划评估几个决定因素， 他们的临床前治疗，其中包括1）的关系 抗叶酸剂对肿瘤细胞毒性和宿主毒性的谷氨酰化 抗叶酸剂发挥细胞毒性的生化机制 通过细胞中的扰动，3）生物化学 以及调节谷氨酰化和水解的药理学过程 和4）叶酸-谷氨酸的相互作用。 胸苷酸合成酶的类似物抑制剂与靶酶。 在接近第一个目标的一系列叶酸和叶酸拮抗剂已被 合成的谷氨酸盐的4个碳中含有氟， 严重考虑谷氨酰化，但不改变其他性质的 分子。 4-氟甲氨蝶呤已被用于显示 氨甲喋呤不能被谷氨酰化时的活性。 4-氟亚叶酸 将用于确定肿瘤细胞和宿主的拯救能力 组织. 本实验设计旨在确定选择性 通过阻止 救援人员。 一种由Priest开发的高灵敏度叶酸辅酶检测方法， 同事们已经在我们的实验室进行了调整和修改， 5，10CH2H4PteGlun，H4PteGlun， H2 PteGlun、10-HCO-H4 PteGlun和10-HCOH 2 PteGlun，它们是关键叶酸 参与嘌呤和嘧啶的生物合成。 我们将测量这些 用甲氨蝶呤和4- 氟甲氨蝶呤和用亚叶酸和4-氟亚叶酸进行拯救。 DHFR抑制剂与炔丙基喹唑啉抑制剂的组合 胸苷酸合成酶和GAR转化酶抑制剂5，10 二脱氮四氢叶酸将用在亚叶酸培养基中培养的细胞进行评价。 酸或5甲基四氢叶酸，以确定是否大于添加剂药物 活动发生。 虽然我们已经显示出协同药物活性 与在含叶酸的培养基中的组合一起，使用还原的 叶酸底物将提示体内活性的潜力。 如果 发现积极的药物相互作用，将进行机制评价， 进行了一项研究，包括对叶酸干扰的彻底分析， 上面描述 此外，还详细分析了 炔丙基喹唑啉的活性将用野生型细胞进行 胸苷酸合成酶扩增70倍。 这些调查 被指导详细确定细胞对这一新类别的反应， 抑制剂。 贯穿所有这些研究的一个主题是理解 调节谷氨酰化和叶酰水解的机制， 抗叶酸聚谷氨酸盐。 这将在酶水平上进行评价， 叶酰聚谷氨酸合成酶和γ-谷氨酰水解酶研究 在细胞水平上，通过检查干扰细胞增殖速率的试剂， 聚谷氨酸的形成和分解。

The proposed studies are directed at a deeper understanding and novel approaches to the antifolates. We plan to evaluate several determinants of their preclinical therapeutics which includes 1) the relationship of glutamylation of antifolates to tumor cytotoxicity and host toxicity 2) the biochemical mechanisms by which antifolates exert their cytotoxicity through perturbations in the cell folylpolyglutamates 3) the biochemical and pharmacological processes which regulate glutamylation and hydrolysis of folyl and antifolylpolyglutamates and 4) the interaction of the folate- analog inhibitors of thymidylate synthase with the target enzyme. In approaching the first goal a series of folates and antifolates have been synthesized containing a fluorine in the 4 carbon of glutamate which severely regards glutamylation but does not alter other properties of the molecule. 4-Fluoromethotrexate has been used to show changes in the activity of methotrexate when it can't be glutamylated. 4-Fluorofolinic will be used to determine the rescue capacity with tumor cells and host tissue. This experimental design is intended to determine if selective host rescue can be achieved more effectively by preventing glutamylation of the rescue agent. A highly sensitive assay for folate coenzymes developed by Priest and coworkers has been adapted and modified in our laboratory to give essentially quantitative recoveries of 5,10CH2H4PteGlun, H4PteGlun, H2PteGlun, 10-HCO-H4PteGlun, and 10-HCOH2PteGlun which are the key folates involved in purine and pyrimidine biosynthesis. We will measure these species following treatment of cells with methotrexate and 4- fluoromethotrexate and rescue with folinic and 4-fluorofolinic acid. Combinations of DHFR inhibitors with propargylquinazoline inhibitors of thymidylate synthase and the GAR transformylase inhibitor 5,10 dideazatetrahydrofolate will be evaluated with cells cultured in folinic acid or 5 methyltetrahydrofolate to determine if greater than additive drug activity occurs. Although we have already shown synergistic drug activity with combinations in folic acid containing medium, the use of the reduced folate substrates will suggest the potential for in vivo activity. If positive drug interactions are found, mechanistic evaluations will be undertaken including a thorough analysis of folate perturbations as described above. In addition a detailed mechanistic analysis of the activity of propargylquinazolines will be undertaken with wild type cells and those amplified 70 fold for thymidylate synthase. These investigations are directed determining in detail the cellular response to this new class of inhibitors. A theme throughout all these studies is understanding the mechanisms which regulate the glutamylation and hydrolysis of folyl and antifolyl polyglutamates. This will be evaluated at the enzymatic level by investigation of folylpolyglutamate synthetase and gamma-glutamylhydrolase and at the cellular level by examining agents which perturb the rate of polyglutamate formation and breakdown.