Description

Cancer

Kinase assays

In vitro assays using recombinant VEGFR2 (murine aa785–aa1367), VEGFR3 (murine aa818–aa1363), PDGFRβ (aa561–aa1106), Raf-1 (aa305–aa648) and BRafV600E (aa409–aa765) kinase domains are performed. Initial in vitro kinase inhibition profiling is performed at a fixed 1 μM Regorafenib concentration. Inhibitory concentration of 50% (IC50) values are determined from selected responding kinases, e.g., VEGFR1 and RET. TIE2 kinase inhibition is measured with a homogeneous time-resolved fluorescence (HTRF) assay using a recombinant fusion protein of glutathione-S-transferase, the intracellular domain of TIE2 and the peptide biotin-Ahx-EPKDDAYPLYSDFG as substrate.

Cell Lines

GIST 882 and TT cells

Concentrations

5 nM-10 μM

Incubation Time

96 hours

Methods

For proliferation assays, GIST 882 and TT cells are grown in RPMI medium containing L-glutamine, and MDA-MB-231, HepG2 and A375 cells in DMEM always containing 10% hiFBS. Cells are trypsinized, plated at 5×104 cells/well in 96-well plates in complete media containing 10% FBS and grown overnight at 37 °C. The next day, vehicle or Regorafenib serially diluted in complete growth media to between 10 μM and 5 nM final concentrations, and 0.2% DMSO, is added and incubation is continued for 96 hours. Cell proliferation is quantified.

Animal Models

Female athymic NCr nu/nu mice with Colo-205, MDA-MB-231 or 786-O

Formulation

PEG400/125 mM aqueous methanesulfonic acid (80/20) or polypropylene glycol/PEG400/Pluronic F68 (42.5/42.5/15 + 20% Aqua)

Doses

3 mg/kg, 10 mg/kg, 30 mg/kg, 100 mg/kg

Administration

Orally