Kinase experiment:

Human VEGFR-2-transfected CHO cells are seeded into 6-well plates and grown to about 80% confluency. NVP-ACC789 is added in serial dilutions and the cells incubated for 2 h at 37℃ in medium without fetal calf serum (FCS). VEGF (20 ng/mL) is then added. After a 5-min incubation at 37℃, the cells are washed twice with ice-cold phosphate-buffered saline and lysed. Nuclei are removed by centrifugation for 10 min at 4℃. Protein concentrations of the lysates are determined[1].

Cell experiment:

HUVE cell proliferation is determined. Cells are seeded into 1.5% gelatin-coated 96-well plates (5×103 cells per well) and incubated in endothelial cell growth medium containing 5% fetal calf serum (FCS) for 24 h. The medium is replaced with essential basic medium (1.5% FCS), and the cells are incubated for another 24 h. Essential basic medium is then replaced with fresh medium containing either 50 ng/mL VEGF or 0.5 ng/mL bFGF. NVP-ACC789 is added just before addition of growth factors. The cells are incubated for a further 24 h before adding the BrdU labeling solution. Twenty four hours later, the labeling solution is removed, the cells are fixed, and the incorporated BrdU is visualized with a peroxidase-labeled anti-BrdU antibody and TMB substrate[1].

Animal experiment:

Porous Teflon chambers (volume, 0.5 mL) filled with 0.8% w/v agar-containing heparin (20 U/mL) with or without VEGF (2 μg/mL) or bFGF (0.3 μg/mL) are implanted subcutaneously on the dorsal flank of female mice. The mice are treated with NVP-ACC789 (p.o. once daily) or vehicle (5% dimethyl sulfoxide, 1% Tween 80 in water) starting 1 day before implantation of the chamber and continuing for 5 days thereafter. At the end of the treatment period, the mice are killed, and the chambers are removed. The vascularized tissue growing around the chamber is removed carefully and weighed, and the blood content is assessed by measuring hemoglobin levels. The percentage inhibition of the angiogenic response (increase in tissue weight or total blood) is calculated. EC50 values are estimated from the dose response curves (% inhibition versus dose). Each experiment is performed with six animals per dose group and each dose is tested in at least two independent experiments[1].

NVP-ACC789 is an inhibitor of human VEGFR-1, VEGFR-2 (mouse VEGFR-2), VEGFR-3 and PDGFR-β with IC50s of 0.38, 0.02 (0.23), 0.18, 1.4 μM, respectively.

The enzymatic kinase assays demonstrate that NVP-ACC789 is an inhibitor of human VEGFR-1, VEGFR-2 (mouse VEGFR-2), VEGFR-3 and PDGFR-β with IC50s of 0.38, 0.02 (0.23), 0.18, 1.4 μM, respectively. In VEGF-treated cultures, addition of the VEGFR-2 inhibitor NVP-ACC789 reduces BME cell number to baseline levels from 1 μM. Likewise, bFGF-induced BME cell proliferation is reduced markedly by NVP-ACC789 from 1 to 10 μM, without however reaching basal levels. NVP-ACC789 is found to be a potent inhibitor of VEGF-induced HUVE cell proliferation with an IC50 of 1.6 nM. NVP-ACC789 also completely inhibits VEGF-induced BME and BAE cell invasion and VEGF-C-induced BAE cell invasion. The inhibition is dose-dependent in both cell types with a maximal effect from 1 μM[1].

NVP-ACC789 which is given in daily oral doses for 6 days blocks VEGF-induced angiogenesis in a dose-dependent manner. NVP-ACC789 also inhibits the response to bFGF to some extent, but the dose-response curve is not linear for NVP-ACC789[1].

[1]. Tille JC, et al. Vascular endothelial growth factor (VEGF) receptor-2 antagonists inhibit VEGF- and basicfibroblast growth factor-induced angiogenesis in vivo and in vitro. J Pharmacol Exp Ther. 2001 Dec;299(3):1073-85.