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

Abstract

Objectives: 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 diseases caused by pathologically excessive angiogenesis. Materials and 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 vascular endothelial growth factor at a maximal concentration of 500 pM. Results: All the groups were compared with the control group using one-way ANOVA, followed by a post hoc test, Dunnett’s test, to compare the mean of all the groups with the control mean. In the chick chorioallantoic membrane assay, the number of branching points and angiogenic score were evaluated and significant results were observed at 10-5 M and 10-4 M. In the aortic ring assay a reduction in the area of sprouts was observed with 5-10 μM and significant reductions in the weight of sponges, number of blood vessels formed, and hemoglobin content were observed at all three tested concentrations of flunarizine in the sponge implantation method. In the studies on human umbilical vein endothelial cells the test drug (1-100 nM) showed significant inhibition of proliferation and migration and a decrease in the network length of cord-like tubes in a dose-dependent manner. Conclusion: Flunarizine has significant anti-angiogenic action by inhibiting cell proliferation, migration, and cord-like tube formation, which resulted from blocking of the T-type calcium and sodium channels. Further studies on the structural modifications of flunarizine for repurposing this ion channel modulator will lead to treatment of the diseases due to excessive angiogenesis from the root cause.