The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function

SU5416, a selective inhibitor of the tyrosine kinase activity of the vascul ar endothelial growth factor (VEGF) receptor Flk-1/KDR, is currently in Pha se III clinical trials for the treatment of advanced malignancies, In cellu lar assays, SU5416 inhibits the VEGF-dependent mitogenic/proliferative resp onse of human umbilical vein endothelial cells (HUVECs). In tumor xenograft models, SU5416 inhibits the growth of tumors from a variety of origins by inhibiting tumor angiogenesis. In three different human tumor xenograft mod els, infrequent (once or twice a week) administration of SU5416 is efficaci ous despite the fact that it has a short plasma half-life (30 min), which s uggests that SU5416 has long-lasting inhibitory activity in vivo. The goal of the present study was to determine the basis for the prolonged activity of SU5416, The results indicate that a short (3 h) exposure to 5 muM SU5416 (to mimic plasma levels of the compound as measured in patients who were r eceiving SU5416 therapy) produced long-lasting (at least 72 h) inhibition o f the VEGF-dependent proliferation of HUVECs in culture, which indicate tha t SU5416 has long-lasting inhibitory activity in vitro as well as in vivo. SU5416 treatment of HUVECs did not affect surface expression of Flk-1/KDR o r the affinity of the receptor for VEGF, Instead, the durability of the in vitro activity of SU5416 was shown to be attributable to its long-lasting a bility to specifically inhibit VEGF-dependent phosphorylation of Flk-1/KDR and subsequent downstream signaling, although SU5416 is not an irreversible inhibitor of FII<-1/KDR tyrosine kinase activity. The long-lasting inhibit ion of cellular responses to VEGF was attributable to the accumulation of S U5416 in cells, as shown using radiolabeled compound, such that inhibitory cellular concentrations of SU5416 are maintained long after the removal of the compound from the medium, The long-lasting inhibitory activity of SU541 6 ill vitro is consistent with the finding that SU5416 has demonstrated evi dence of biological activity in clinical studies when administered twice a week despite a short plasma half-life.