| In Vitro | In vitro activity: PLX7904 potently inhibits pERK in BRAFV600E cells. PLX7904 is also evaluated in the human SCC cell line A431 and the human breast adenocarcinoma cell line SKBR3 as these cells achieve MAPK pathway activation by upstream signals feeding into RAS (through overexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), respectively). PLX7904 shows preferential inhibition of the mutated BRAFV600E over wild-type BRAF and CRAF and a level of kinome selectivity.
Kinase Assay: PLX7904 (also known as PB04) is a novel potent and selective paradox-breaker B-Raf inhibitor with IC50 of ~5 nM against BRAFV600E in mutant RAS expressing cells.
Cell Assay: Cells (WM793, WM115, WM9, WM278, WM1346, WM1366, WM1361A, Sbcl2, 1205Lu, A375, SK-MEL 24, SKMEL-2, and SK-MEL 207 cells) are treated with PB04(PLX7904) at different concentrations (0, 0.05, 0.1, 1, 5 μM) for 24 h. Cells are lysed and analyzed by Western blotting with phospho-MEK1/2, total MEK1/2, phospho-ERK1/2 and total ERK1/2 antibodies. |
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| Animal model | COLO205 tumour cells are cultured in DMEM 10% FBS 1% penicillin/streptomycin supplemented with bovine insulin, at 37°C. Balb/C nude mice, female, 6-8 weeks old, weighing approximately 18-22 g, are inoculated subcutaneously at the right flank with COLO205 tumour cells (5×106) in 0.1 mL of PBS mixed with matrigel (50:50) for tumour development. The treatment is started when mean tumour size reach approximately 100 mm3, with eight mice in each treatment group randomized to balance the average weight and tumour size. B9 cells are expanded in DMEM 10% FBS 1% penicillin/streptomycin. Upon trypsinization the cells are washed three times with 20 mL RPMI, and after the final centrifugation are re-suspended, counted, and adjusted by volume to a final concentration of 5×107 cells per millilitre. B9 xenografts are started by injection of 5×106 cells subcutaneously in 6- to 7-week-old female nude Balb/c mice. Compound dosing starts when the average size of tumours reach 50-70 mm3. Animals are equally distributed over treatment groups (n=10) to balance the average tumour size and body weight. Animals are dosed orally for days 1-14 twice daily and days 15-28 once daily with vehicle, vemurafenib 50 mg per kg, or PLX7904 50 mg per kg. 12-O-tetradecanoylphorbol-13-acetate (TPA) is put on the skin of all mice twice a week during weeks 3 and 4 at a dose of 2 μg in 200 μL acetone. |
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