Kinase experiment:

Enzyme reactions are performed in 50 mM TrisHCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20 and 2 mM DTT, containing 10 μM ATP, 0.1 μg/mL biotinylated polyGluTyr (4:1) and 0.1 nM of VEGFR2. Prior to catalytic initiation with ATP, compound (TAK-593) and enzyme are incubated for 5 min at room temperature (preincubation). The reactions are quenched by the addition of 25 μL of 100 mM EDTA, 10 μg/mL AlphaScreen streptavidine donor beads and 10 μg/mL acceptor beads in 62.5 mM HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA. Plates are incubated in the dark overnight and then read by plate reader[1].

Cell experiment:

HUVECs are seeded into a 96-well plate at 3000 cells/well in Human Endothelial-SFM Growth Medium (Invitrogen) containing 3% fetal bovine serum (FBS) and are incubated overnight at 37 C in a 5% CO2 incubator. Various concentrations of the test compounds (TAK-593) are added in the presence of 60 ng/mL VEGF, and the cells are cultured for a further 5 days. Cellular proliferation is determined by the WST-8 formazan assay using Cell Counting Kit-8[1].

Animal experiment:

Rats: The iv administration in rats is conducted under anesthesia with diethyl ether. At 5, 10 (only for iv dosing), 15, 30 min, and 1, 2, 3, 4, 6, 8, 12, 24, 32 (only for monkeys) and 48 h (only for monkeys) after dosing, blood is taken from the tail vein in rats or from the femoral vein in monkeys. Then, the blood is centrifuged to obtain the plasma fraction. The plasma is kept frozen at 20℃ until analysis. The concentration of TAK-593 in plasma is determined by the high-performance liquid chromatography with a fluorescence detector. The excitation and emission are 346 and 420 nm, respectively. Mice: Test compounds are administered at a dose of 10 mg/kg as a cassette dosing to nonfasted mice (BALB/cAJcl; female). After oral administration, blood samples are collected. The blood samples are centrifuged to obtain the plasma fraction. The plasma samples are deproteinized with acetonitrile containing an internal standard. After centrifugation, the supernatant is diluted with a mixture of 0.01 M ammonium formate solution and acetonitrile (9:1, v/v) and centrifuged again. The compound concentrations in the supernatant are measured by LC/MS/MS[1].

TAK-593 is a highly potent inhibitor of VEGFR2 with IC50 value of 0.95nM [1].

TAK-593 is an imidazo [1, 2-b] pyridazine derivative. It is found to inhibit VEGFR2 with a highly potent effect in the non-RI assay using the AlphaScreen system. And in the cell proliferation assay, TAK-593 suppresses VEGF-stimulated cell growth of HUVEC with IC50 value of 0.3nM. TAK-593 is a slow-binding inhibitor and it has a long residence time on VEGFR2. Besides that, TAK-593 also has efficacy on other receptor kinases. It gives the IC50 values of 3.2nM, 1.1nM, 4.3nM and 13nM for VEGFR1, VEGFR3, PDGFRα and PDGFRβ, respectively. Moreover, TAK-593 shows significant anti-tumor efficacy in the mouse xenograft model using A549 human lung adenocarcinoma epithelial cells. Oral administration of TAK-593 twice daily at doses of 0.25mg/kg for two weeks potently inhibits tumor growth with T/C value of 34% [1].

References:
[1] Miyamoto N, Sakai N, Hirayama T, Miwa K, Oguro Y, Oki H, Okada K, Takagi T, Iwata H, Awazu Y, Yamasaki S, Takeuchi T, Miki H, Hori A, Imamura S. Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor. Bioorg Med Chem. 2013 Apr 15;21(8):2333-45.