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系统恶性肿瘤的有价值的化疗药物开发的。最近，开发了新的抗叶酸剂用于治疗实体瘤。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。虽然Pmx显示出抗肿瘤活性，但该药剂缺乏肿瘤选择性并诱导毒性。后者已被归因于通过还原叶酸载体（RFC）的Pmx的细胞内膜转运。RFC是一种广泛表达的叶酸促进转运蛋白，存在于肿瘤和正常细胞如骨髓中。叶酸受体（FR）是内化（抗）叶酸的替代机制。FR？与其他叶酸转运蛋白不同，它只在肿瘤的基底外侧表达，并暴露于循环血液中，而在正常组织中则位于顶端。与肿瘤中的这种异常细胞结构相结合，FR在各种恶性肿瘤中也过表达，包括卵巢、子宫和脑的恶性肿瘤。据报道，大约90%的上皮性卵巢癌表现出FR的表达升高，受体密度与肿瘤分级和分期相关。我假设FR <$在实体（卵巢）肿瘤细胞中的差异表达使新型细胞毒性抗叶酸剂能够靶向肿瘤，反映了FR <$对RFC细胞摄取的选择性。我进一步假设，由于缺乏嘌呤补救和/或间接靶向下游AMPK和mTOR信号通路，抑制从头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和特异性叶酸依赖性细胞内靶点的抗叶酸治疗剂将诱导选择性杀死肿瘤细胞， 降低毒性。为了研究这一假设，我提出了以下具体目标：（目标1）开发新的实体瘤靶向抗叶酸剂治疗剂，其具有通过FR而不是RFC的选择性膜转运，靶向从头嘌呤核苷酸生物合成和下游信号传导途径（例如，AMPK，mTOR）;和（目的2）在体外和体内模型系统中鉴定上述实体瘤靶向剂的细胞毒性活性和选择性的决定因素。为了完成这些目标，我将筛选一系列合理设计的叶酸类似物，并确定其抗增殖和细胞毒性的效力，通过嘌呤核苷酸消耗和膜转运的首选机制。我将建立他们的详细机制，包括细胞代谢，细胞内的目标，并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，包括嘌呤补救和其他叶酸转运系统对药物活性的存在或不存在的影响。最后，我将在这些细胞决定因素的更广泛背景下建立体内功效。目前晚期卵巢癌的治疗策略往往无效，并伴有毒副作用。因此，有一个令人信服的理由开发新的FR靶向治疗卵巢癌。

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.