Crizotinib (PF-02341066) is a small molecule tyrosine kinase inhibitor that inhibits c-MET and ALK receptors (IC50=8/20 nM), is ATP-competitive, and also inhibits ROS1. Crizotinib exhibits antitumor activity and inhibits tumor growth.

ALK:24 nM (cell free), c-Met:11 nM (A498 cells)

方法：HMVEC 内皮细胞用 Crizotinib (0.083-1.33 µmol/L) 处理 7 天，使用显微镜观察形态。
结果：Crizotinib 在纤维蛋白凝胶中抑制血清刺激的 HMVEC 分支小管形成。[1]
方法：9 种肺癌症细胞用 Crizotinib 处理 72 h，使用 Cell Titer-Glo Luminescent Cell Viability Assay 检测细胞活力。
结果：具有 MET 扩增的两种细胞系，EBC-1 和 H1993 均对 Crizotinib 敏感，IC50 值≤10 nM。相反，Crizotinib 没有显著抑制具有 MET 突变 (H2122、H1437 和 H596)、具有 EGFR 突变 (PC9 和 HCC827) 或没有这种基因扩增或突变 (A549 和 H1299) 的癌症细胞的增殖。[2]

方法：为检测体内抗肿瘤活性，将 Crizotinib (3.125-50 mg/kg) 灌胃给药给携带 GTL-16 异种移植物的 nu/nu 小鼠，每天一次，持续十一天。
结果：在 50 mg/kg/天的剂量水平下，100% 的肿瘤生长抑制与持续 24 小时的 GTL-16 肿瘤中 c-Met 磷酸化的完全抑制相关。在 >50mg/kg/天的浓度水平下，未观察到肿瘤生长抑制的进一步改善。[1]

c-Met catalytic activity was quantitated using a continuous-coupled spectrophotometric assay in which the time-dependent production of ADP by c-Met was determined by analysis of the rate of consumption of NADH. NADH consumption was measured by a decrease in absorbance at 340 nm by spectrophotometry at designated time points. To determine Ki values, PF-2341066 was introduced into test wells at various concentrations in the presence of assay reagents and incubated for 10 min at 37°C. The assay was initiated by the addition of the c-Met enzyme [1].

Cells were seeded in 96-well plates in media supplemented with 10% fetal bovine serum (FBS) and transferred to serum-free media (with 0.04% BSA) after 24 h. In experiments investigating ligand-dependent RTK phosphorylation, corresponding growth factors were added for up to 20 min. After incubation of cells with PF-2341066 for 1 h and/or appropriate ligands for the designated times, cells were washed once with HBSS supplemented with 1 mmol/L Na3VO4, and protein lysates were generated from cells. Subsequently, phosphorylation of selected protein kinases was assessed by a sandwich ELISA method using specific capture antibodies used to coat 96-well plates and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates were (a) incubated in the presence of protein lysates at 4°C overnight; (b) washed seven times in 1% Tween 20 in PBS; (c) incubated in a horseradish peroxidase-conjugated anti–total-phosphotyrosine (PY-20) antibody (1:500) for 30 min; (d) washed seven times again; (e) incubated in 3,3′,5,5′-tetramethylbenzidine peroxidase substrate to initiate a colorimetric reaction that was stopped by adding 0.09 N H2SO4; and (f) measured for absorbance in 450 nm using a spectrophotometer [1].

Daily treatment with PF-2341066 given in water by oral gavage was initiated when tumors were 100 to 600 mm^3 in volume. Tumor volume was determined by measurement with electronic Vernier calipers, and tumor volume was calculated as the product of its length × width2 × 0.4. Tumor volume was expressed on indicated days as the median tumor volume ± SE indicated for groups of mice. Percent (%) inhibition values were measured on the final day of study for drug-treated compared with vehicle-treated mice and are calculated as 100 × {1?[(TreatedFinal day ? TreatedDay 1)/(ControlFinal day ? ControlDay 1)]}. Tumor regression values were determined by calculating the ratio of median tumor volumes at the time when treatment was initiated to the median tumor volume on the final day of study for a given treatment group. Significant differences between the treated versus the control groups (P ≤ 0.001) were determined using one-way ANOVA [1].