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Research Area
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Description
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Cancer |
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Protocol
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Kinase Assay
[1]
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| 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. |
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Cell Assay
[1]
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| 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. |
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Animal Study
[1]
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| 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 |
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References |
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[1] Wilhelm SM, et al. Int J Cancer, 2011, 129(1), 245-255.
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[2] Heng DY, et al. Ther Adv Med Oncol, 2010, 2(1), 39-49.
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[3] Carr BI, et al. J Cell Physiol, 2013, 228(2), 292-297.
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