Allitinib tosylate (AST-1306) is a novel irreversible inhibitor of EGFR and ErbB2 with IC50 of 0.5 nM and 3 nM, respectively.

EGFR:0.5 nM, ErbB2:3.0 nM

Allitinib同时针对 ErB2 和 EGFR T790M/L858R 双突变体。 Allitinib的活性大约是 lapatinib 的 500 倍，并且针对 ErbB 家族激酶相较于其他激酶家族（包括 PDGFR、KDR 和 c-Met）的选择性超过 3000 倍。 Allitinib可能与 EGFR 和 ErbB2 的特定氨基酸残基形成共价结合。 Allitinib以浓度依赖的方式显著抑制 HIH3T3-EGFR T790M/L858R 细胞的生长，并有效抑制这些细胞中 EGFR 的磷酸化。此外， Allitinib在浓度依赖的方式中阻断带有 EGFR T790M/L858R 突变的 NCI-H1975 细胞的生长，并阻断 EGFR 及其下游途径的磷酸化。同时， Allitinib以剂量依赖的方式明显抑制 A549 细胞中 EGF 诱导的 EGFR 磷酸化。 Allitinib抑制 EGFR 和 ErbB2 的磷酸化及包括 A549 细胞、Calu-3 细胞和 SK-OV-3 细胞在内的人类癌症细胞的下游信号传导。[1]

每日两次口服AST-1306能显著防止SK-OV-3和Calu-3异种移植模型中的肿瘤生长。在SK-OV-3模型中，经过7天的AST-1306治疗，肿瘤几乎消失。相反，在HO-8910和A549异种移植模型中，AST-1306仅轻微抑制肿瘤生长。因此，AST-1306在过表达ErbB2的肿瘤模型中的抗肿瘤效能大于在低表达ErbB2的模型中。AST-1306具有良好的耐受性。虽然Lapatinib在这些过表达ErbB2的肿瘤模型中显示出抗肿瘤活性，但与Lapatinib相比，AST-1306在相同剂量和计划下治疗SK-OV-3异种移植肿瘤模型时更加有效。此外，每日两次口服AST-1306连续3周显著抑制了FVB-2/Nneu模型中的肿瘤生长。治疗11天后，肿瘤几乎完全消失。治疗期间，小鼠体重减少不到20%。[1]

Tyrosine kinase assays: The tyrosine kinase activities are determined in 96-well ELISA plates precoated with 20 μg/mL Poly (Glu,Tyr)4:1. First, 80 μL of 5 μM 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 μL 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 μL 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 μL 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 μL) 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 μL of a solution containing 0.03 % Water2 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 μL 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)] ×100%. IC50 values are determined from the results of at least three independent tests and calculated by Logit method.

Cell (including Calu-3, A-549 cell line et al.) proliferation is evaluated using the SRB (Sulforhodamine B) assay. Briefly, cells are seeded into 96-well plates and grown for 24 hours. The cells are then treated with increasing concentrations of AST-1306 and grown for a further 72 hours. The medium remains unchanged until the completion of the experiment. The cells are then fixed with 10% precooled trichloroacetic acid (TCA) for 1 hour at 4 °C and stained for 15 min at room temperature with 100 μL of 4 mg/mL SRB solution in 1% acetic acid. The SRB is then removed, and the cells are quickly rinsed five times with 1% acetic acid. After cells are air-dried, protein-bound dye is dissolved in 150 μL of 10 mM Tris base for 5 min and measured at 515 nm using a multiwell spectrophotometer. The inhibition rate on cell proliferation is calculated as (1 - A515 treated/A515 control) × 100%. The IC50 value is obtained by the Logit method and is determined from the results of at least 3 independent tests.(Only for Reference)