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)

METHODS: 195 tumor cells were treated with Dabrafenib for 72 h. Cell growth was detected using the CellTiter-Glo Assay.
RESULTS: Sixteen of the 20 cell lines encoding BRAFV600E were sensitive to Dabrafenib (gIC50<200 nM). Three of the other five mutant BRAF cell lines were sensitive to Dabrafenib (gIC50<30nM), including WM-115 (BRAFV600D) and YUMAC (BRAFV600K). 133 of 152 RAS/RAF wild-types and all 18 mutant RAS cell lines were insensitive to Dabrafenib (gIC50>10µM). gIC50>10µM). [1]
METHODS: Melanoma cell lines LCP (BRAFV600R), WM266 (BRAFV600D) and M257 (BRAFWT) were treated with Dabrafenib (3-100 nM) for 72 h. Target protein expression levels were detected using Western Blot.
RESULTS: Cell lines with the BRAFV600D/R mutation exhibited faster and stronger inhibition of phosphorylated ERK compared to control cells with wild-type BRAF. [2]

METHODS: To assay antitumor activity in vivo, Dabrafenib (3-100 mg/kg, 0.5% hydroxypropylmethylcellulose+0.2% Tween 80 in pH 8.0 distilled water) was administered by gavage to human colorectal cancer tumor-bearing Colo 205 ( BRAFV600E) CD1 nu/nu mice once daily for fourteen days.
RESULTS: Dabrafenib showed dose-dependent inhibition of tumor growth, with four of eight mice showing partial regression at a dose of 100 mg/kg. [1]
METHODS: To test the antitumor activity in vivo, Dabrafenib (0.1-100 mg/kg, 0.5% hydroxypropylmethylcellulose+0.2% Tween 80 in pH 8.0 distilled water) was administered by gavage to mice harboring human malignant melanoma A375P F11 ( BRAFV600E) CD1 nu/nu mice once daily for fourteen days.
RESULTS: Dabrafenib significantly reduced tumor growth in mice bearing B-RafV600E human melanoma tumors. complete tumor regression was observed in 50% of treated animals in the 100 mg/kg group. [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].