Canertinib (CI-1033) is a pan-erbB tyrosine kinase inhibitor which work against esophageal squamous cell carcinoma in vitro and in vivo. Canertinib treatment significantly affects tumour metabolism, proliferation and hypoxia as determined by PET.

ErbB2:9.0 nM, EGFR:1.5 nM

CI-1033在MDA-MB 453细胞中展现出针对erbB2自磷酸化的出色不可逆抑制作用。该化合物在Caco-2细胞中也显示出高渗透性，并抑制了长春碱的分泌运输，表明CI-1033可能是P-gp的抑制剂。[1] CI-1033单独使用时，显著抑制了持续激活的Akt和MAP激酶。与吉西他滨联合使用时，CI-1033可以抑制Akt并防止MAPK磷酸化水平的增加。CI-1033在MDA-MB-453细胞中促进p27表达和p38磷酸化。[2] CI-1033对erbB受体家族具有高度特异性，即使在50 μM的浓度下也不对PGFR、FGFR或IR敏感。在表达EGFR的A431细胞中，CI-1033显示出高水平的抑制活性，IC50为7.4 nM。CI-1033可抑制由heregulin刺激的erbB2、erbB3和erbB4的酪氨酸磷酸化，其IC50分别为5、14和10 nM，并且还能抑制对heregulin的响应中pp62c-fos的表达。[3] CI-1033预计能在HER2激酶ATP结合位点内的Cys773上共价修饰，并增强成熟和未成熟ErbB-2分子的破坏。[4] CI-1033显著降低了EGFR酪氨酸残基845和1068的可测量磷酸化，这些残基分别负责Src和Ras/MAPK信号传导。在3μM或更高浓度下，CI-1033能显著去磷酸化Her-2的酪氨酸残基877和1248。CI可以阻断EGFR的内吞作用并在初代骨肉瘤细胞中通过可调节的方式增加凋亡率。[5] 此外，CI-1033在0.1 nM的浓度下显著抑制了TT、TE2、TE6和TE10细胞的增殖。[6]

Canertinib在裸鼠体内对A431异种移植瘤的作用显著，剂量为体重的5 mg/kg。[1] 在H125异种移植模型中，Canertinib（每天20至80 mg/kg）能显著促进肿瘤退缩。[3] 通过口服给予Canertinib，能在不造成动物死亡和体重减轻<10%的情况下，显著抑制裸鼠中TT、TE6和TE10异种移植瘤的生长。[6]

Tyrosine Kinase Assays: Enzyme assays for determination of IC50 are performed in 96-well filter plates in a total volume of 0.1 mL, containing 20 mM Hepes, pH 7.4, 50 mM sodium vanadate, 40 mM magnesium chloride, 10 μM adenosine triphosphate (ATP) containing 0.5 mCi of [32P]ATP, 20 mg of polyglutamic acid/tyrosine, 10 ng of EGFR tyrosine kinase, and appropriate dilutions of CI-1033. All components except the ATP are added to the well and the plate is incubated with shaking for 10 min at 25 °C. The reaction is started by adding [32P]ATP, and the plate is incubated at 25 °C for another 10 min. The reaction is terminated by addition of 0.1 mL of 20% trichloroacetic acid (TCA). The plate is kept at 4 °C for at least 15 min to allow the substrate to precipitate. The wells are then washed five times with 0.2 mL of 10% TCA and 32P incorporation determined with a Wallac β plate counter.

Cells (1 × 104) are seeded in each well of a 24-well plastic culture plate and left overnight in DMEM or RPMI-1640 supplemented with 10% FBS. The next morning, the cells are treated with the indicated concentrations of CI-1033 (0.1-5.0 nM) for varying periods (1, 3, 5 and 7 days). After treatment, the cells are counted using a Coulter counter. The percent of cell proliferation is calculated by this formula: treatment cell number/control cell number × 100 for each time period.(Only for Reference)