Lenvatinib (E7080) is a multi-target receptor tyrosine kinase inhibitor that inhibits VEGFR1-3, FGFR1-4, KIT, PDGFR, and RET, and has oral activity. Lenvatinib has the strongest inhibitory activity on VEGFR2 and VEGFR3 (IC50=4/5.2 nM). Lenvatinib has strong anti-tumor activity.

FGFR1:46 nM, PDGFRβ:39 nM, VEGFR2/KDR:4.0 nM, VEGFR3/FLT4:5.2 nM, VEGFR1/FLT1:22 nM

方法：六种人肿瘤细胞 A375、DU145、DX3、KM12C、SK23 和 U2OS 用 Lenvatinib (1-100 μM) 处理 72 h，使用 MTT 检测细胞活力。
结果：大多数细胞系中，Lenvatinib 仅在高浓度下抑制增殖 (IC50 为 23.6-44.17 μM)，而 KM12C 细胞系中的 IC50 为 9.54 μM。[1]
方法：人脐静脉内皮细胞 HUVECs 用 Lenvatinib (0.16-20 nM) 处理 1 h，随后用 SCF 或 VEGF (20 ng/mL) 刺激 5 min，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：配体诱导的 KIT 和 KDR 的磷酸化均被 Lenvatinib 抑制。[2]

方法：为检测体内抗肿瘤活性，将 Lenvatinib (30-100 mg/kg in 0.5% methylcellulose) 口服给药给携带人小细胞肺癌肿瘤 H146 的 BALB/c nude 小鼠，每天两次，持续二十一天。
结果：口服 Lenvatinib 以剂量依赖性方式抑制 H146 肿瘤的生长，并在 100 mg/kg 时引起肿瘤消退。[2]
方法：为检测体内抗肿瘤活性，将 Lenvatinib (100 mg/kg) 口服给药给携带人乳腺癌肿瘤 MDA-MB-231 的 nude 小鼠，每天一次，持续八周。
结果：Lenvatinib 在 MDA-MB-231 异种移植物模型中抑制向区域淋巴结和远处肺的转移。Lenvatinib 降低了淋巴结中 MDA-MB-231 肿瘤的已建立转移结节的血管生成和淋巴管生成。[3]

In vitro kinase assay [1]: Tyrosine kinase assays are performed by HTRF (KDR, VEGFR1, FGFR1, c-Met, EGFR) and ELISA (PDGFRβ), using the recombinant kinase domains of receptors. In both assays, 4 μL of serial dilutions of E7080 are mixed in a 96-well round plate with 10 μL of enzyme, 16 μL of poly (GT) solution (250 ng) and 10 μL of ATP solution (1 μM ATP) (final concentration of DMSO is 0.1%). In wells for blanks, no enzyme is added. In control wells no test article is added. The kinase reaction is initiated by adding ATP solution to each well. After 30-minute incubation at 30°C, the reaction is stopped by adding 0.5 M EDTA (10 μL/well) to the reaction mixture in each well. Dilution buffer adequate to each kinase assay is added to the reaction mixture. In the HTRF assay, 50 μL of the reaction mixture is transferred to a 96-well 1/2 area black EIA/RIA plate, HTRF solution (50 μL/well) is added to the reaction mixture, and then kinase activity is determined by measurement of fluorescence with a time-resolved fluorescence detector at an excitation wavelength of 337 nm and an emission wavelengths of 620 and 665 nm. In the ELISA, 50 μL of the reaction mixture is incubated in avidin coated 96-well polystyrene plates at room temperature for 30 minutes. After washing with wash buffer, PY20-HRP solution (70 μL/well) is added and the reaction mixture is incubated at room temperature for 30 minutes. After washing with wash buffer, TMB reagent (100 μL/well) is added to each well. After several minutes (10–30 minutes), 1 M H3PO4 (100 μL/well) is added to each well. Kinase activity is determined by measurement of absorbance at 450 nm with a microplate reader.

HUVECs (1,000 cells in each well in serum-free medium containing 2% fetal bovine serum) and L6 rat skeletal muscle myoblasts (5,000 cells in each well in serum-free DMEM) are dispensed in a 96-well plate and incubated overnight. E7080 and either VEGF (20 ng/mL) or FGF-2 (20 ng/mL) containing 2% fetal bovine serum and PDGFβ (40 ng/mL) are added to each well. Cells are incubated for 3 days and then the ratios of surviving cells are measured by WST-1 reagent. For proliferation assay, samples are duplicated and three separate experiments are done. (Only for Reference)