In vitro effects of vandetanib on VEGFR2, EGFR and RET phosphorylation, endothelial cell tube formation, and tumor cell growth and survival

C189 Vandetanib (ZACTIMA™) is a small molecule tyrosine kinase inhibitor targeting VEGF-dependent angiogenesis and EGFR- and RET-dependent tumor cell proliferation and survival. In the present study, we have investigated the pharmacological activity of vandetanib in vitro using both vascular and tumor cell models. Using Western blot assays, vandetanib (at submicromolar concentrations) inhibited ligand-stimulated phosphorylation of both VEGFR2 and EGFR in human umbilical vein endothelial cells (HUVECs). The selective EGFR inhibitor gefitinib (IRESSA™) inhibited phosphorylation of EGFR but not VEGFR2. In addition, the activity of vandetanib was assessed against RET using TT (human thyroid medullary carcinoma) cells cultured under basal growth conditions, where phosphorylation of RET was also inhibited at submicromolar concentrations. Using an in vitro co-culture tubule formation model consisting of HUVECs and human fibroblasts, we showed that vandetanib potently inhibited each of the tubule growth parameters measured (number of branch points [IC50 = 33.23 nM], total vessel length [IC50 = 60.97 nM], tubule area [IC50 = 92.70 nM]), at potencies consistent with the potency of vandetanib for inhibition of VEGF-stimulated proliferation of HUVECs (Wedge et al. , 2002). The effects of vandetanib on tumor cells were compared with gefitinib across three panels of human tumor cell lines using a viable cell number endpoint (MTS assay). Sensitivity to vandetanib was highly correlated with sensitivity to gefitinib in each of the human tumor cell panels examined (NSCLC [R 2 =0.93], colorectal [R 2 =0.88], head and neck [R 2 =0.8], suggesting that sensitivity of these tumor cell lines to vandetanib is primarily due to inhibition of EGFR signaling. Using flow cytometry, we examined the phenotypic effects of vandetanib on cell cycle and cell death in NSCLC cells (PC9, IC50=138 nM; H1975, IC50=11.17 μM) and head and neck cells (OE21, IC50=70 nM; Hep-2, IC50=8.38 μM) either sensitive or resistant to the effects of vandetanib in the MTS assay. Compared with controls, vandetanib increased both the proportion of cells in G1 and the proportion of cells undergoing apoptosis. These effects were most marked in the more sensitive cell lines (PC9, OE21). Similar phenotypic effects in these cells lines were seen with gefitinib. These data demonstrate that vandetanib inhibits VEGFR2, EGFR and RET activity in endothelial cells and/or tumor cells in culture at submicromolar concentrations. In the human tumor cell lines examined, the effects of vandetanib were most likely attributable to inhibition of EGFR since sensitivity to vandetanib paralleled sensitivity to gefitinib both in the MTS cell viability assay and in phenotypic assays of cell proliferation and cell death. Targeting both VEGFR2-dependent angiogenesis and EGFR tumor growth and survival may offer an attractive therapeutic approach in disease settings where agents targeting each of these pathways alone has shown clinical benefit, such as in NSCLC. Vandetanib is currently in Phase III development.