Regorafenib (BAY 73-4506) is a multi-targeted receptor tyrosine kinase inhibitor that inhibits RET, C-RAF, VEGFR2, c-Kit, VEGFR1, and PDGFRβ and is orally active. Regorafenib has antitumor and anti-angiogenic activity.

VEGFR2:4.2 nM (cell free), VEGFR1:13 nM (cell free), c-Kit:7 nM (cell free), B-Raf (V600E):19 nM (cell free), RET:1.5 nM (cell free), Raf-1:2.5 nM (cell free)

方法：人肝癌细胞 Hep3B、PLC/PRF/5 和 HepG2 用 Regorafenib (0-10 μM) 处理 72 h，使用 MTT 方法检测细胞活力。
结果：Regorafenib 浓度依赖性降低 Hep3B 细胞活力，IC50 约为 5 μM。PLC/PRF/5 细胞的敏感性与 Hep3B细胞相似。但 HepG2 细胞更敏感，IC50 约为 1 μM。[1]
方法：肿瘤细胞 NIH-3T3/VEGFR2、CHO/TIE2、HAoSMC/PDGFR-β 和 MCF-7/FGFR 用 Regorafenib (10-3000 nM) 处理 1 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：Regorafenib 抑制 p-VEGFR2、p-TIE2、p-PDGFR-β 和 p-FGFR。[2]

方法：为检测体内抗肿瘤活性，将 Regorafenib (3-100 mg/kg) 口服给药给携带肿瘤 Colo-205 或 MDA-MB-231 的 NCr nu/nu 小鼠，每天一次，持续九天。
结果：Regorafenib 抑制肿瘤生长。Colo-205 模型中，Regorafenib 在 10 mg/kg 的剂量下，第 14 天的 TGI 达到 75%。MDA-MB-231 模型，Regorafenib 在低至 3 mg/kg 的剂量下是高效的，导致 81% 的显著TGI。[2]
方法：为检测体内抗肿瘤活性，将 Regorafenib (3-10 mg/kg) 口服给药给携带肿瘤 HT-29 或 MDA-MB-231 的 NMRI nu/nu 小鼠，每天一次，持续二十七天。
结果：Regorafenib 剂量依赖性抑制 HT-29 和 MDA-MB-231 肿瘤生长。[3]

In vitro assays using recombinant VEGFR2 (murine aa785–aa1367), VEGFR3 (murine aa818–aa1363), PDGFR-b (aa561–aa1106), RAF-1 (aa305–aa648) and BRAFV600E (aa409–aa765) kinase domains were performed as previously described. Initial in vitro kinase inhibition profiling was performed at a fixed 1 μM compound concentration under Millipore standard conditions [10 μM adenosine-50'- triphosphate (ATP) concentration]. Inhibitory concentration of 50% (IC50) values were determined from selected responding kinases, e.g., VEGFR1 and RET. TIE2 kinase inhibition was measured with a homogeneous time-resolved fluorescence (HTRF) assay using a recombinant fusion protein of glutathione-S-transferase, the intracellular domain of TIE2 and the peptide biotin-Ahx-EPKDDAYPLYSDFG as substrate.

Each cell line was seeded at 0.3×10^5 cells/2ml of DMEM containing 10% FBS in 35 mm tissue culture dishes. The cells were incubated for 24 h to allow attachment, and then the medium was replaced by fresh culture medium containing Regorafenib at increasing concentrations (1 μM, 2.5 μM, 5 μM, 7.5 μM and 10 μM). In these experimental conditions, the cells were allowed to grow for 72 or 96 h. Time-course experiments on Hep3B cells were performed with 7.5 μM of Regorafenib at short (15, 60, 180 min.), middle (24, 48, 72 and 96 h) or long times (up to seven days). When the cells were treated for long times the drug was replaced with a fresh one. Each experiment included a control with the equivalent concentration of DMSO (solvent control) as the one used for adding Regorafenib. Each experiment was performed in triplicate and repeated 3 times. Subsequent analyses were performed at specific Regorafenib concentrations and incubation times [2].

Female athymic NCr nu/nu mice, kept in accordance with Federal guidelines, were subcutaneously inoculated with 5×10^6 Colo-205 or MDAMB-231 cells or implanted with 1 mm^3 786-O tumor fragments. When tumors reached a volume of ~100 mm^3, regorafenib or vehicle control was administered orally qd×21 in the 786-O model, and qd×9 in the Colo-205 and MDA-MB231 models, respectively, at doses of 100, 30, 10, and 3 mg/kg. Paclitaxel was administered intravenously at 10 mg/kg in ethanol/Cremophor ELV/saline (12.5%/12.5%/75%) every 2 days×5. Tumor size (volume) was estimated twice weekly (l×w^2)/2, and the percentage of tumor growth inhibition (TGI) was obtained from terminal tumor weights (1-T/C100). Mice were weighed every other day starting from the first day of treatment. The general health status of the mice was monitored daily [1].