Refametinib  (Synonyms: 瑞法替尼; BAY 869766; RDEA119)

MEK1 kinase activity is determined using mERK2 K52A T183A as the substrate. Recombinant MEK1 enzyme (5 nM) is first activated by 0.02 unit or 1.5 nM of RAF1 in the presence of 25 mM HEPES (pH 7.8), 1 mM MgCl₂, 50 mM NaCl, 0.2 mM EDTA, and 50 μM ATP for 30 min at 25°C. The reactions are initiated by adding 2 μM of mERK2 K52A T183A and 2.5 μCi [γ-³³P]ATP in a total volume of 20 μL. The MEK2 kinase activity is determined similarly except that activation by RAF1 is not needed and 11 nM of MEK2 enzyme (active) are used in the assays. Kinase profiling is performed. The Z'-LYTE biochemical assay is used. Refametinib is assayed in quadruplicate at 10 μM against 205 kinases^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For anchorage-dependent growth inhibition experiments, A375, SK-Mel-28, A431, Colo205, HT-29, MDA-MB-231, and BxPC3 cell lines are plated in white 384-well plates at 1,000/20 μL/well or white 96-well microplates at 4,000/100 μL/well. After 24-h incubation at 37°C, 5% CO₂, and 100% humidity, Refametinib is incubated for 48 h at 37°C and assayed using CellTiter-Glo. For the 96-well anchorage-independent growth assay, wells of an “ultralow binding” plate are filled with 60 μL of a 0.15% agarose solution in complete RPMI 1640. Then, 60 μL of complete RPMI 1640 containing 9,000 cells in 0.15% agarose are added per well. After 24 h, 60 μL of a 3× drug solution in agarose-free complete RPMI 1640 are added. After 7 d, 36 μL of 6× MTS reagent are added per well. After 2 h at 37°C, absorbance at 490 nm is determined on the M5 plate reader. Nonlinear curve fitting is performed using GraphPad Prism 4^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Female athymic nude mice are used for all the efficacy studies except for the Colo205 study 2, which used male mice. Mice are injected s.c. in the flank with 1×10⁶ tumor cells (Colo205 and A431) or ~1 mm³ tumor fragments (A375 and HT-29). Tumor volumes are monitored by caliper measurements. For efficacy analysis, treatment is initiated when tumors are 80 to 185 mm³. Refametinib (25 and 50 mg/kg/d) is administered by oral gavage once daily or twice daily for 14 d or once every 2 d for 14 doses^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Iverson C, et al. RDEA119/BAY 869766: a potent, selective, allosteric inhibitor of MEK1/2 for the treatment of cancer. Cancer Res. 2009 Sep 1;69(17):6839-47.  [Content Brief]