Vatalanib dihydrochloride (PTK787 dihydrochloride)(IC50=37 nM) is an inhibitor of VEGFR2/KDR. It exhibits less effective against VEGFR1/Flt-1 and 18-fold against VEGFR3/Flt-4.

VEGFR2/KDR:37 nM, VEGFR1/FLT1:77 nM, VEGFR2/Flk1:270 nM, PDGFRβ:580 nM, VEGFR3/FLT4:660 nM

Vatalanib对Flk、c-Kit和PDGFRβ的抑制作用分别表现在IC50为270 nM、730 nM和580 nM。此外，通过抑制VEGF在HUVECs中诱导的胸腺嘧啶核苷酸的结合，Vatalanib展现了IC50为7.1 nM的抗增殖效果，并且在相同的剂量范围内，剂量依赖性抑制VEGF诱导的内皮细胞的存活和迁移，而对不表达VEGF受体的细胞没有细胞毒性或抗增殖作用。[1] 最近的研究表明，Vatalanib显著抑制肝细胞性癌细胞的生长，并通过增加Bax蛋白水平和降低Bcl-xL与Bcl-2，增强了IFN/5-FU诱导的细胞凋亡。[2]

Vatalanib 通过每日一次口服（25-100 mg/kg）在生长因子植入模型和肿瘤细胞驱动的血管生成模型中对 VEGF 和 PDGF 的血管生成反应产生剂量依赖性抑制。在相同剂量范围内，Vatalanib 还能抑制裸鼠体内多种人类癌症的生长和转移，而对循环血细胞或骨髓白细胞无显著影响。[1]

VEGF Receptor Tyrosine Kinase Assays : The in vitro kinase assays are performed in 96-well plates as a filter binding assay, using the recombinant GST-fused kinase domains expressed in baculovirus and purified over glutathione-Sepharose. γ-[33P]ATP is used as the phosphate donor, and poly-(Glu:Tyr 4:1) peptide is used as the acceptor. Recombinant GST-fusion proteins are diluted in 20 mM Tris·HCl (pH 7.5) containing 1–3 mM MnCl2, 3–10 mM MgCl2, 0.25 mg/mL polyethylene glycol 20000, and 1 mM DTT, according to their specific activity. Each GST-fused kinase is incubated under optimized buffer conditions [20 mM Tris-HCl buffer (pH 7.5), 1–3 mM MnCl2, 3–10 mM MgCl2, 3–8 μg/mL poly-(Glu:Tyr 4:1), 0.25 mg/mL polyethylene glycol 20000, 8 μM ATP, 10 μM sodium vanadate, 1 mM DTT, and 0.2 μCi[γ-33P]ATP in a total volume of 30 μL in the presence or absence of a test substance for 10 minutes at ambient temperature. The reaction is stopped by adding 10 μL of 250 mM EDTA. Using a 96-well filter system, half the volume (20 μL) is transferred onto a Immobilon-polyvinylidene difluoride membrane. The membrane is then washed extensively in 0.5% H3PO4 and then soaked in ethanol. After drying, Microscint cocktail is added, and scintillation counting is performed. IC50s for PTK787/ZK 222584 or SU5416 in these as well as all assays described below are calculated by linear regression analysis of the percentage inhibition.

As a test of the ability of PTK787/ZK 222584 to inhibit a functional response to VEGF, an endothelial cell proliferation assay, based on BrdUrd incorporation is used. Subconfluent HUVECs are seeded into 96-well plates coated with 1.5% gelatin and then incubated at 37 °C and 5% CO2 in growth medium. After 24 hours, 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 PTK787/ZK 222584. As a control, wells without growth factor are also included. After 24 hours of incubation, BrdUrd labeling solution is added, and cells incubated an additional 24 hours before fixation, blocking, and addition of peroxidase-labeled anti-BrdUrd antibody. Bound antibody is then detected using 3,3′5,5′-tetramethylbenzidine substrate, which results in a colored reaction product that is quantified spectrophotometrically at 450 nm. (Only for Reference)