Therapeutic Targeting of Novel Antifolates to Solid Tumors via Folate Receptor

抗叶酸，如甲氨蝶呤，最初被开发为治疗血液系统恶性肿瘤的有价值的化疗药物。最近，更新的抗叶酸被开发用于治疗实体瘤。培美曲塞(PMX，Alimta)于2004年被批准用于治疗非小细胞肺癌和间皮瘤。尽管PMX具有抗肿瘤活性，但该药缺乏肿瘤选择性，且具有毒性。后者归因于还原型叶酸载体(RFC)对PMX的细胞膜转运。RFC是一种普遍表达的叶酸促进运输蛋白，存在于肿瘤和正常细胞中，如骨髓。叶酸受体(FRs)是内化(抗)叶酸的另一种机制。与其他叶酸转运蛋白不同，FR？仅在基底侧表达，并暴露在肿瘤中的循环血液中，与其在正常组织中的顶端定位相反。与肿瘤中这种异常的细胞结构相结合，FR‘在包括卵巢、子宫和脑在内的各种恶性肿瘤中也过度表达。据报道，大约90%的上皮性卵巢癌显示出FR的高表达，其受体密度与肿瘤分级和分期相关。我推测，FR在实体(卵巢)肿瘤细胞中的差异表达使肿瘤靶向新型细胞毒性抗叶酸，反映了FR对细胞摄取的选择性高于RFC。我进一步假设，由于没有嘌呤挽救和/或间接靶向下游的AMPK和mTOR信号通路，抑制从头开始的嘌呤核苷酸生物合成的新型FR？抗叶酸底物显示出肿瘤选择性。针对FR和特定叶酸依赖细胞内靶点的抗叶酸疗法将诱导对肿瘤细胞的选择性杀伤和 降低毒性。为了研究这一假说，我提出了以下具体目标：(1)开发新型固体肿瘤靶向抗叶酸治疗药物，通过FR通过RFC选择性地膜转运，靶向从头开始嘌呤核苷酸的生物合成和下游信号通路(例如，AMPK，mTOR)；(2)在体外和体内模型系统中确定上述实体肿瘤靶向药物的细胞毒活性和选择性的决定因素。为了实现这些目标，我将筛选一系列设计合理的叶酸类似物，并通过嘌呤核苷酸耗尽和它们首选的膜转运机制来确定它们的抗增殖和细胞毒性潜力。我将建立它们的详细机制，包括细胞代谢、细胞内靶点以及对下游信号通路的影响。我将确定药物活性的细胞决定因素，包括嘌呤回收和其他叶酸转运系统的存在或不存在对药物活性的影响。最后，我将在这些细胞决定因素的更广泛的背景下建立体内的有效性。目前对晚期卵巢癌的治疗策略往往无效，且与毒副作用有关。因此，有一个令人信服的理由来开发新的FR靶向疗法来治疗卵巢癌。

Antifolates such as methotrexate were originally developed as valuable chemotherapy agents to treat hematopoietic malignancies. More recently, newer antifolates were developed for treating solid tumors. Pemetrexed (Pmx, Alimta) was approved in 2004 for treating non-small cell lung cancer and mesothelioma. While Pmx shows antitumor activity, this agent lacks tumor selectivity and induces toxicity. The latter has been attributed to intracellular membrane transport of Pmx by Reduced Folate Carrier (RFC). RFC is a ubiquitously expressed folate facilitative transport protein that is present in both tumor and normal cells such as bone marrow. Folate Receptors (FRs) are an alternative mechanism for internalizing (anti)folates. FR¿, unlike other folate transport proteins is only expressed basolaterally and is exposed to circulating blood in tumors, in contrast to its apical localizationin normal tissues. In conjunction with this abnormal cellular architecture in tumors, FR¿ is also overexpressed in a variety of malignancies including those of the ovaries, uterus and brain. Approximately 90% of epithelial ovarian cancers have been reported to exhibit elevated expression of FR¿ with receptor densities correlating to tumor grade and stage. I hypothesize that the differential expression of FR¿ in solid (ovarian) tumor cells enables tumor targeting of novel cytotoxic antifolates, reflecting selectivity for cellular uptake by FR¿ over RFC. I further hypothesize that novel FR¿ antifolate substrates that inhibit de novo purine nucleotide biosynthesis show tumor selectivity due to the absence of purine salvage and/or indirectly target downstream AMPK and mTOR signaling pathways. Antifolate therapeutics directed to FR¿ and to specific folate-dependent intracellular targets will induce selective killing of tumor cells and reduce toxicity. To investigate this hypothesis, I propose the following specific aims: (Aim 1) to develop novel solid tumor-targeted antifolate therapeutics with selective membrane transport by FR¿ over RFC that target de novo purine nucleotide biosynthesis and downstream signaling pathways (e.g., AMPK, mTOR); and (Aim 2) to identify determinants of cytotoxic activity and selectivity for the aforementioned solid tumor targeted agents in in vitro and in vivo model systems. To complete these aims, I will screen a series of rationally designed folate analogs and determine their antiproliferative and cytotoxic potencies via purine nucleotide depletion and their preferred mechanisms of membrane transport. I will establish their detailed mechanisms including cellular metabolism, intracellular targets, and impact on downstream signaling pathways. I will determine cellular determinants of drug activity including the impact of purine salvage and the presence or absence of other folate transport systems on drug activity. Finally I will establish in vivo efficacies within the broader context of these cellular determinants. Curren treatment strategies for late stage ovarian cancer are often ineffective and are associated with toxic side effects. Hence, there is a compelling rationale for developing newer FR-targeted therapies for treating ovarian carcinomas.