Trametinib (GSK1120212)

Trametinib (GSK1120212) inhibits the phosphorylation of MBP regardless of the isotype of Raf and MEK, with IC50 ranging from 0.92 nM to 3.4 nM. It demonstrates no inhibition of the kinase activities of c-Raf, B-Raf, ERK1 and ERK2. In addition, this compound does not show drastic inhibitory activity against the other 98 kinases. It displays potent inhibitory activity against human colorectal cancer cell lines. HT-29 and COLO205 cells, which are known to have a constitutively active B-Raf mutant, are most sensitive to it with IC50 0.48 nM and 0.52 nM, respectively. The cell lines bearing a K-Ras mutation show a wide range of sensitivity to it with IC50 of 2.2-174 nM. In contrast, COLO320 DM cells, bearing the wild-type gene in both B-Raf and K-Ras, are found to be resistant even at 10 μM. Treatment for 24 hours induces cell-cycle arrest at the G1 phase in all sensitive cell lines. Consistently, it leads to upregulation of p15^INK4b and/or p27^KIP1 in most of the colorectal cancer cell lines. It inhibits constitutive ERK phosphorylation in all sensitive cell lines. This compound induces apoptosis both in HT-29 and COLO205 cells, but that COLO205 cells are more sensitive than HT-29 cells in terms of apoptosis induction. ^[1] It blocks tumor necrosis factor-α and interleukin-6 production from peripheral blood mononuclear cells (PBMCs). ^[2]

Oral administration of Trametinib (GSK1120212) at 0.3 mg/kg or 1 mg/kg once daily for 14 days is effective in inhibiting the HT-29 xenograft growth, and 1 mg/kg of this compound almost completely blocks the tumor increase. The phosphorylation of ERK1/2 is completely inhibited in the established tumor tissues by single oral dose of 1 mg/kg, and both p15^INK4b and p27^KIP1 protein levels are upregulated after 14 days of treatment. In the COLO205 xenograft model, tumor regression is observed even at a dose of 0.3 mg/kg. At a dose of 1 mg/kg, a complete regression is obtained in 4 out of 6 mice in which the tumor degenerates to the point that tumor volume is not measurable. ^[1] Administration at 0.1 mg/kg almost completely suppresses adjuvant-induced arthritis (AIA) and type II collagen-induced arthritis (CIA) in Lewis rats or DBA1/J mice, respectively. ^[2]

• Raf-MEK-ERK cascade kinase assay

  Non-phosphorylated myelin basic protein (MBP) is coated onto an ELISA plate, and the active form of B-Raf/c-Raf is mixed with unphosphorylated MEK1/MEK2 and ERERK2 in 10 μM ATP and 12.5 mM MgCl₂ containing MOPS buffer in the presence of various concentrations of Trametinib (GSK1120212). The phosphorylation of MBP is detected by the anti-phospho-MBP antibody.

• Cell lines

  HT-29, HCT-15, HCT116, COLO205, LS-174T, SW480, SW620, T84, LoVo and COLO320

• Concentrations

  Dissolved in DMSO, final concentrations ~10 μM

• Incubation Time

  3 or 4 days

• Method

  Exponentially growing cells are precultured in 96-well tissue culture plates for 24 hours and then exposed to Trametinib (GSK1120212). Cell growth is determined by an in vitro toxicology assay kit, sulforhodamine B based. For apoptosis assay, both floating and adherent cells are collected and fixed with 70% ethanol. After washing with PBS, the cells are suspended in 100 μg/mL RNase and 25 μg/mL propidium iodide (PI) and incubated at 37 °C for 30 minutes in the dark. The DNA content of each single cell is determined using the flow cytometer Cytomics FC500 or Guava EasyCyte plus.

• Animal Models

  Female BALB/c-nu/nu mice inoculated subcutaneously with HT-29 or COLO205 cells

• Dosages

  ~1 mg/kg/day

• Administration

  Orally