Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole

Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.

病理性血管生成（pathological angiogenesis）与多种疾病密切相关，涵盖癌症与黄斑变性。尽管临床中已有血管生成抑制剂（angiogenesis inhibitor），但由于替代及代偿性信号通路的存在，其对多数癌症的疗效仍较为有限。鉴于血管生成在体内依赖于多种生长因子与复杂的信号网络，我们旨在探索多药联合方案用于血管生成抑制的潜力。我们针对内皮细胞增殖的协同抑制效应，筛选了741种临床药物组合。我们特别聚焦于已上市的临床药物，因为与全新药物实体相比，临床药物的重定位可实现更快速且更具成本效益的临床研究转化。本次筛选发现，免疫抑制剂环孢素A（cyclosporin A，CsA）与抗真菌药物伊曲康唑（itraconazole）可协同抑制内皮细胞增殖。联合使用时，两种药物的半数抑制浓度（IC50）均降低3至9倍。我们还测试了该组合对内皮细胞管形成与出芽的抑制能力——这两个过程是血管生成中内皮细胞迁移与分化的核心环节。结果显示，环孢素A与伊曲康唑可协同抑制管网络规模与出芽形成。最后，我们在人包皮成纤维细胞活力、Jurkat T细胞及HeLa细胞增殖实验中验证了该组合的效应，发现其可选择性靶向内皮细胞。综上，联合使用已上市临床药物可协同抑制体外血管生成模型，该策略或可用于下一代抗血管生成治疗的开发。