Dabrafenib (GSK2118436A) is a Raf inhibitor that inhibits C-Raf and B-RafV600E (IC50=5/0.6 nM) and is ATP-competitive. Dabrafenib exhibits antitumor activity for the treatment of B-RafV600E-mutated melanoma.

B-Raf (V600E):0.7 nM (cell free), B-Raf:5.2 nM (cell free), C-Raf:6.3 nM (cell free)

方法：195 种肿瘤细胞用 Dabrafenib 处理 72 h，使用 CellTiter-Glo Assay 检测细胞生长。
结果：编码 BRAFV600E 的 20 个细胞系中有 16 个对 Dabrafenib 敏感 (gIC50<200 nM)。其他 5 个突变 BRAF 细胞系中的 3 个对 Dabrafenib 敏感 (gIC50<30nM)，包括 WM-115(BRAFV600D) 和 YUMAC(BRAFV600K)。152 个 RAS/RAF 野生型中的133个和所有 18 个突变 RAS 细胞系对 Dabrafenib 不敏感 (gIC50>10µM)。[1]
方法：黑色素瘤细胞株 LCP(BRAFV600R)、WM266 (BRAFV600D) 和 M257 (BRAFWT) 用 Dabrafenib (3-100 nM) 处理 72 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：与具有野生型 BRAF 的对照细胞相比，具有 BRAFV600D/R 突变的细胞系对磷酸化 ERK 表现出更快更强的抑制作用。[2]

方法：为检测体内抗肿瘤活性，将 Dabrafenib (3-100 mg/kg，0.5% hydroxypropylmethylcellulose+0.2% Tween 80 in pH 8.0 distilled water) 灌胃给药给携带人结直肠癌肿瘤 Colo 205 (BRAFV600E) 的 CD1 nu/nu 小鼠，每天一次，持续十四天。
结果：Dabrafenib 显示出剂量依赖性的肿瘤生长抑制作用，8 只小鼠中有 4 只在 100 mg/kg 剂量下显示出部分消退。[1]
方法：为检测体内抗肿瘤活性，将 Dabrafenib (0.1-100 mg/kg，0.5% hydroxypropylmethylcellulose+0.2% Tween 80 in pH 8.0 distilled water) 灌胃给药给携带人恶性黑色素瘤 A375P F11 (BRAFV600E) 的 CD1 nu/nu 小鼠，每天一次，持续十四天。
结果：Dabrafenib 可显著减少携带 B-RafV600E 人类黑色素瘤肿瘤的小鼠的肿瘤生长。100 mg/kg 组中，50% 的治疗动物观察到肿瘤完全消退。[3]

A375P-F11 assay: A375P cells were plated in 96-well plates by limiting dilution and single cell-derived clones were harvested and tested for sensitivity to B-Raf inhibitors. The F11 clone was selected for future studies and was named A375P-F11. Cellular pSmad Assay to Measure Anti-TGF-β Activity: Activity of compounds was tested in a mechanistic assay in HepG2 cells. Cells were seeded in 12-well plates at a density of 500,000 cells/well and allowed to adhere overnight at 37℃/5% CO2. Media (BME+10% serum) was removed and compound in serum-free media was added to the cells for 45 minutes at 37℃/5% CO2. Cells were stimulated with 1 ng/ml TGF-β for 60 minutes. Cells were lysed in buffer (25 mM Tris-HCl ph: 7.5, 2 mM EDTA, 2 mM EGTA,1% Triton X-100, 0.1 % SDS, 50 mM sodium-β-glycerophosphate, 2 mM sodium orthovanadate, 12.5 mM sodium pyrophosphate, protease and phosphatase inhibitor cocktails) for 30 minutes, scraped, collected, clarified by centrifugation and prepared for western blots in LDS/reducing reagent. Samples were resolved on 4-12% Bis-Tris gels, transferred to PVDF, and probed for total and phospho-Smad2 using antibodies. Gels were imaged using the odyssey blot scanner and quantified. Phospho:total Smad2 ratios were determined and the IC50 was defined as the concentration of compound which decreased the phospho:total ratio by 50% [1].

Cells were implanted in nude mice and grown as tumor xenografts. Dosing began when tumors achieved ~150-200mm^3 volume. GSK2118436 was administered by oral gavage at a dose volume of 0.2 mL/20 gram body weight in 0.5% hydroxypropylmethylcellulose and 0.2% Tween-80 in distilled water pH 8.0. Dosing was daily for duration stipulated. Results are reported as mean tumor volume for n=7-8 mice/group. Tumors were measured twice weekly with Vernier calipers, and tumor volume was estimated from two-dimensional measurements using a prolate ellipsoid equation (Tumor volume mm^3 = (length x width^2) x 0.5). Complete tumor regression was defined as a >93% decrease in an individual tumor volume for at least 1 week [1].