Kinase experiment:

Each GST-fused kinase is incubated under optimized buffer conditions. ATP in a total volume of 30 μL in the presence or absence of a test substance (Vatalanib) for 10 min at ambient temperature. The reaction is stopped by adding 10 μL of 250 mM EDTA[1].

Cell experiment:

Subconfluent HUVECs are seeded into 96-well plates coated with 1.5% gelatin. After 24 h, growth medium is replaced by basal medium containing 1.5% FCS and a constant concentration of VEGF (50 ng/mL), bFGF (0.5 ng/mL), or FCS (5%), in the presence or absence of Vatalanib. As a control, wells without growth factor are also included. After 24 h of incubation, BrdUrd labeling solution is added, and cells incubated an additional 24 h before fixation, blocking, and addition of peroxidase-labeled anti-BrdUrd antibody. Bound antibody is then detected using 3,3' 5,5'-tetramethylbenzidine substrate[1].

Animal experiment:

A porous Teflon chamber (volume, 0.5 mL) is filled with 0.8% w/v agar containing heparin (20 units/mL) with or without growth factor (3 μg/mL human VEGF, 2 μg/mL human PDGF) is implanted s.c. on the dorsal flank of C57/C6 mice. The mice are treated with Vatalanib (12.5, 25 or 50 mg/kg dihydrochloride p.o. once daily) or vehicle (water) starting 1 day before implantation of the chamber and continuing for 5 days after. At the end of treatment, the mice are killed, and the chambers are removed. The vascularized tissue growing around the chamber is carefully removed and weighed, and the blood content is assessed by measuring the hemoglobin content of the tissue[1].

Vatalanib, also known as PTK787, is a potent vascular endothelial growth factor (VEGF) receptor tyrosine kinases inhibitor that inhibits VEGF receptor/KDR, VEGF receptor/Flt-1 and VEGF receptor/Flk with the half maximal inhibition concentration IC50 values of 0.037 μM, 0.077 μM and 0.27 μM respectively [1].

Vatalanib also inhibits other tyrosine kinases belonging to the same family of tyrosine kinase receptors as the VEGF receptors, including the platelet-derived growth factor receptor β tyrosine kinase (PDGFR-β), c-Kit and c-Fms, to a lesser degree with IC50 values of 0.58 μM, 0.73 μM and 1.4 μM respectively [1].

Vatalanib has been found to be potentially therapeutic for the treatment of angiogenesis-related diseases including solid tumors [1].

References:
[1] Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, Persohn E, Rosel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, Schirner M, Thierauch KH, Schneider MR, Drevs J, Martiny-Baron G, Totzke F. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 2000 Apr 15;60(8):2178-89.