Allitinib tosylate

Nude mice bearing SK-OV-3 xenograft tumors and, SK-OV-3FVB-2/Nneu transgenic mouse

2-Propenamide, N-[4-[[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]amino]-6-quinazolinyl]-, 4-methylbenzenesulfonate (1:1)

0.001-1 uM

25 mg/kg, 50 mg/kg and 100 mg/kg

0.5 nM, 0.5 nM, 0.5 nM, 0.5 nM, 0.5 nM, 0.5 nM

Twice daily oral administration of AST-1306 gives rise to a dramatic prevention of tumor growth in SK-OV-3 and Calu-3 xenograft models. In SK-OV-3 models, tumors nearly disappears after treatment with AST-1306 for 7 days. In contrast, AST-1306 only slightly inhibits the growth of tumor in HO-8910 and A549 xenograft models. Therefore, the antitumor efficacy of AST-1306 is greater in ErbB2-overexpressing tumor models than in models expressing low levels of ErbB2. AST-1306 is well tolerated. Lapatinib displays antitumor activity in these ErbB2-overexpressing tumor models, but AST-1306 is more efficacious than lapatinib in the SK-OV-3 xenograft tumor model when given at the same dose and schedule. In addition, oral administration of AST-1306 twice daily for 3 weeks dramatically suppresses the growth of tumor in the FVB-2/Nneu models. After treatment for 11 days, tumors almost completely disappears. The body weights of the mice reduces by less than 20% during treatment. [1]

Tyrosine kinase assays, The tyrosine kinase activities are determined in 96-well ELISA plates precoated with 20 ug/mL Poly (Glu, Tyr)4:1. First, 80 uL of 5 uM ATP solution diluted in kinase reaction buffer (50 mM HEPES pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) is added to each well. Various concentrations of AST-1306 diluted in 10 uL of 1% DMSO (v/v) are then added to each reaction well, with 1% DMSO (v/v) used as the negative control. Subsequently, the kinase reaction is initiated by the addition of purified tyrosine kinase proteins diluted in 10 uL of kinase reaction buffer solution. Experiments at each concentration are performed in duplicate. After incubation for 60 min at 37 C, the plate is washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 uL anti-phosphotyrosine antibody (PY99, 1:500 dilution) diluted in T-PBS containing 5 mg/mL BSA is added. After 30 min incubation at 37 C, the plate is washed three times as before. Horseradish peroxidase-conjugated goat anti-mouse IgG (100 uL) diluted 1:2000 in T-PBS containing 5 mg/mL BSA is added. The plate is reincubated at 37 C for 30 min, and then washed with PBS. Finally, 100 uL of a solution containing 0.03 % H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, is added and samples are incubated at room temperature until color emerged. The reaction is terminated by the addition of 50 uL of 2 M H2SO4, and the plate is read using a multi-well spectrophotometer at 490 nm. The inhibition rate (%) is calculated using the following equation: [1-(A490 treated /A490 control)] x100%. IC50 values are determined from the results of at least three independent tests and calculated by Logit method.

621, 08

DMSO 124 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL