Anti-Angiogenic activity of flunarizine by in ovo, in vitro and in vivo assays.

Background and Objective: The involvement of T-type calcium channels in cell proliferation and the role of sodium channels in cell migration have been extensively studied in angiogenesis. In the present study Flunarizine, a dual sodium /calcium channel blocker; was selected to evaluate its anti-angiogenic potential. This can be therapeutically beneficial in the diseases caused due to pathologically excess angiogenesis. Methods: The anti-angiogenic activity of ion channel blocker was screened by chick chorioallantoic membrane assay (in ovo), rat aortic ring assay,endothelial cell proliferation assay,transwell migration assay, matrigel cord-like morphogenesis assay (in vitro) and sponge implantation method (in vivo). The anti-angiogenic activity of the test drug was compared with the standard anti-angiogenic drug Bevacizumab and in addition, the test responses were compared with the angiogenic factor VEGF, at a maximal concentration of 500pM. Results: All the groups were compared with the control group using one-way ANOVA, followed by post hoc test, the Dunnett’s test to compare the mean of all the groups with the control mean.In the chick chorioallantoic membrane assay, number of branching points and angiogenic score were evaluated and significant results were observed at 10-5M and 10-4M. In the aortic ring assay reduction in the area of sprouts were observed with 5-10μM and a significant reduction in weight of sponges, number of blood vessels formed and hemoglobin content were observed at all the three tested concentrations of Flunarizine in the sponge implantation method. In the studies on human umbilical vein endothelial cellsthe test drug (1-100nM) showed significant inhibition of proliferation, migration and decrease in network length of cord like tubes in a dose dependent manner. Conclusions: Flunarizine has significant anti-angiogenic action byinhibiting the cell proliferation, migration and cord like tube formation, which have resulted by blocking the T-type u co rre cte d p r o f calcium and sodium channels. Further studies on the structural modifications of Flunarizinefor repurposing this ion channel modulator will be able to treat the diseases due to excess angiogenesis from the root cause.