Regorafenib Monohydrate is a novel oral multikinase inhibitor with IC50 values of 13, 4.2, 46, 22, 7, 1.5, 2.5, 28, 19 nM for VEGFR1, murine VEGFR2, murine VEGFR3, PDGFR-β, KIT, RET, RAF-1, B-RAF and B-RAF(V600E) respectively.

VEGFR1:13 nM, c-Kit:7 nM, RET kinase:1.5 nM

Regorafenib有效抑制了包括血管生成和基质RTKs VEGFR1-3、TIE2、FGFR1及PDGFR-b在内的一系列特定激酶（IC50s：4-311 nM），以及癌变RTKs KIT和RET，还有细胞内信号激酶c-RAF/RAF-1、BRAF及其V600E变异体（IC50s：1.5-28 nM）。此外，Regorafenib在NIH-3T3/VEGFR2细胞中强效抑制VEGFR2自磷酸化（IC50：3 nM）。在HAoSMCs中，Regorafenib 经PDGF-BB刺激后抑制PDGFR-b自磷酸化（IC50：90 nM）[1]。Regorafenib导致Hep3B细胞生长浓度依赖性下降（IC50：5 μM）。PLC/PRF/5细胞对Regorafenib的响应与Hep3B细胞相似，但HepG2细胞更敏感（IC50：1 μM）[2]。

给带瘤大鼠单次口服regorafenib 10 mg/kg可显著降低肿瘤灌注和对比剂的渗出。regorafenib处理后10小时，归一化的IAUC360显著降低，并持续至2天，与对照组相比。regorafenib通过口服每日一次（qd）的剂量依赖方式抑制了在多种异种移植模型中的肿瘤生长，包括来自CRC（Colo-205）、BC（MDA-MB-231）和RCC（786-O）肿瘤的模型。regorafenib (10–100 mg/kg)有效抑制了Colo-205异种移植体的生长，在10 mg/kg剂量时，第14天的肿瘤生长抑制（TGI）达到约75%[1]。在小鼠异种移植模型中，口服regorafenib、M-2和M-5与对照相比显著抑制了肿瘤的生长。小鼠反复口服regorafenib 10 mg/kg/day后，M-2和M-5在小鼠体内的总峰值血浆化合物浓度和暴露程度与人类相当[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].