SB590885

Cell experiment:

For proliferation assays, cells are treated with compounds in 0.1% DMSO and incubated for 72 hours at 37°C, 5% CO2. Viable cells are quantified using CellTiter-Glo reagent and luminescence detection on a Victor 2V plate reader. Cells are prepared for cell cycle analysis on a Becton Dickinson FACScan, according to the manufacturer's instructions. Data is acquired and analyzed using CellQuest v3.3 software. Anchorage-independent growth assays are done as described elsewhere, with inhibitors or DMSO vehicle included in the agar layer. Cultures are re-fed with media and inhibitor or DMSO every 5 to 7 days for a total of 28 days. Colonies are visualized and photographed by conventional light microscopy and quantified by counting on a grid in triplicate.

Animal experiment:

The pharmacokinetic properties and safety of SB-590885, following i.p. injection, are determined and 50 mg/kg daily injections are found to give therapeutic levels with minimal body weight changes. Tumors are initiated in 8- to 12-week-old female nude mice by s.c. injection of 5×106 A375P cells in Matrigel suspension, and 3 weeks after tumor induction when the tumors had reached a volume of 150 to 250 mm3, mice are randomized into groups of eight prior to treatment. Animals are treated with vehicle [2% N,N-dimethylacetamide, 2% Cremophor EL, and 96% acidified water (pH 4-5)], or vehicle containing 50 mg/kg of SB-590885 daily for 21 days. A cohort of mice treated with SB-590885 are then observed an additional 14 days following cessation of treatment. Tumor volume is measured for 55 days by calipers twice weekly.

References:

[1]. King AJ, et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res, 2006, 66(23), 11100-11105.
[2]. Takle AK, et al. The identification of potent and selective imidazole-based inhibitors of B-Raf kinase. Bioorg Med Chem Lett, 2006, 16(2), 378-381.
[3]. Smalley KS, et al. Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas. Mol Cancer Ther, 2008, 7(9), 2876-2883.