Therapeutic Targeting of Novel Antifolates to Solid Tumors via Folate Receptor

抗叶酸剂如甲氨蝶呤最初是作为治疗造血恶性肿瘤的有价值的化疗药物而开发的。最近，新的抗叶酸药物被开发用于治疗实体肿瘤。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。虽然Pmx具有抗肿瘤活性，但该药物缺乏肿瘤选择性并诱导毒性。后者归因于Pmx通过还原性叶酸载体（RFC）在细胞膜内运输。RFC是一种普遍表达的叶酸促进转运蛋白，存在于肿瘤细胞和骨髓等正常细胞中。叶酸受体（FRs）是叶酸内化（抗）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，在肿瘤中暴露于循环血液中，而在正常组织中则定位于根尖。与肿瘤中这种异常的细胞结构相结合，FR¿也在多种恶性肿瘤中过度表达，包括卵巢、子宫和大脑的恶性肿瘤。据报道，大约90%的上皮性卵巢癌表现出FR¿的高表达，受体密度与肿瘤分级和分期相关。我假设FR¿在实体（卵巢）肿瘤细胞中的差异表达使得新的细胞毒性抗叶酸盐靶向肿瘤，反映了FR¿对细胞摄取的选择性。我进一步假设，抑制新嘌呤核苷酸生物合成的新型FR¿抗叶酸底物由于缺乏嘌呤回收和/或间接靶向下游AMPK和mTOR信号通路而表现出肿瘤选择性。针对FR和特定叶酸依赖的细胞内靶点的抗叶酸治疗将诱导肿瘤细胞的选择性杀伤

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.