In vitro activity: Tivozanib (AV-951) is a novel quinoline-urea derivative. AV-951 blocks VEGF-dependent activation of mitogen-activated protein kinases and proliferation of endothelial cells. Tivozanib potently inhibits VEGF-induced VEGFR2 phosphorylation in endothelial cells with IC50 of 0.16 nM. It also inhibits ligand-induced phosphorylation of PDGFRβ and c-Kit with IC50 of 1.72 and 1.63 nM, respectively. Tivozanib inhibits VEGF-mediated migration of human umbilical vein endothelial cells

Kinase Assay: Cell-free kinase assays are done in quadruplicate with 1 μM ATP to determine the IC50 values of AV-951 against a variety of recombinant receptor and nonreceptor tyrosine kinases including VEGFR1, VEGFR2, VEGFR3, c-Kit, PDGFRβ, Flt-3 and FGFR1.

Cell Assay: Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts-based assays are done to determine the ability of AV-951 to inhibit ligand-dependent phosphorylation of tyrosine kinase receptors. The cells are starved overnight in appropriate basic medium containing 0.5% fetal bovine serum (FBS). The cells are incubated for 1 hour following the addition of AV-951 or 0.1% DMSO, and then stimulated with the cognate ligand at 37 °C. Receptor phosphorylation is induced for 5 minutes except for VEGFR3 (10 minutes), c-Met (10 minutes), and c-Kit (15 minutes). All the ligands used in the assays are human recombinant proteins, except for VEGF-C, a rat recombinant protein. Following cell lysis, receptors are immunoprecipitated with appropriate antibodies and subjected to immunoblotting with phosphotyrosine. Quantification of the blots and calculation of IC50 values are carried out.