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

and Sprouting by Cyclosporin A and Itraconazole. PLoS ONE 6(9): e24793. doi:10.1371/journal.pone.0024793 Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole Benjamin A. Nacev 0 Jun O. Liu 0 Marc Tjwa, University of Frankfurt - University Hospital Frankfurt, Germany 0 1 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 2 Medical Scientist Training Program, Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 3 Department of Oncology, Johns Hopkins University School of Medicine , Baltimore, Maryland , United States of America 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. - Funding: This work was supported in part by NCI [Grant RO1 CA122814], the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research [Grant UL1 RR 025005], the Flight Attendant Medical Research Institute Fund, the Keck Foundation, the Walsh Prostate Cancer Fund, and the Commonwealth Foundation (J.O.L.), and by the NIH Medical Scientist Training Program [Grant T32GM07309] (B.A.N). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Angiogenesis, the process of new blood vessel growth and development, underlies a number of human diseases including cancer, macular degeneration, psoriasis, rheumatoid arthritis, diabetic retinopathy, and pulmonary hypertension [1]. Inhibitors of angiogenesis such as the anti-VEGF antibody bevacizumab are used clinically to treat cancer. However, the experience with existing therapies has been mixed. While they have shown efficacy, their effects in terms of halting disease progression and improving survival have been modest and often involve side effects including hypertension and increased risk of stroke [2]. Hence, although the promise of angiogenesis inhibitors has been demonstrated, there is a clear need for more effective anti-angiogenic therapies. On average, the development of a clinically viable drug requires an investment of roughly $800 million and takes about 12 years [3]. One strategy to accelerate drug development is to re-purpose existing drugs [4]. Because re-purposed drugs have already been approved for clinical use, their pharmacodynamic and pharmacokinetic properties are well established. In addition, existing drugs have acceptable levels of toxicity and in many cases they have known mechanisms, which makes their pharmacology amenable to detailed molecular study. Thus, by focusing on existing drugs, many hurdles in drug development are already cleared. The end result is a drastically shortened path from bench to bedside when old drugs are discovered to have new applications. We have previously adopted this approach when we assembled and screened the Johns Hopkins Drug Library (JHDL) for inhibitors of angiogenesis and other activities [59]. Presently, the JHDL contains ,3,300 drugs approved by the US Food and Drug Administration or foreign equivalent. The initial screen for angiogenesis inhibitors identified 221 compounds with .50% inhibition of human umbilical vein endothelial cell (HUVEC) proliferation at a 10 mM dose. A number of these hits had IC90 doses above the peak plasma level obtained under clinical dosing regimens or had dose-limiting toxicities. One way to expand the clinical applicability of these hits, we reasoned, was to find synergy between them, thereby reducing the doses needed for those synergistic pairs to inhibit angiogenesis in vivo. We have thus conducted a screen for synergy among 741 binary combinations of 39 clinical drugs that were hits from the initial screen. In addition to lowering the necessary dose of otherwise toxic agents, combination therapy is often used to limit the potential for drug resistance and to achieve synergistic inhibition of multiple independent pathways that converge on a single essential molecular process. For these reasons, the simultaneous use of multiple drugs has been an effective strategy to overcome diseases intractable to single agent therapies. For instance, combination therapy is now the standard of care in treating HIV infection and many neoplasms [10]. Synergy, or superadditivity, is often observed upon the inhibition of multiple pathways that converge to promote a single biological process such as proliferation. Thus, it is hypothetically possible that synergistic inhibition of angiogenesis may be possible given that signaling in angiogenesis is complex and involves multiple pathways including those downstream of growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [11,12]. In fact, the limitations of anti-VEGF therapy have been attributed to the existence of redundant alternative pathways in the in vivo pro-angiogenic signaling network [13]. Thus, a more effective strategy to inhibit angiogenesis may be to simultaneously target multiple pathways. Just as anticancer regimens have evolved to simultaneously utilize drugs with multi (...truncated)