Sorafenib tosylate

Female NCr-nu/nu mice implanted s.c. with MDA-MB-231, Colo-205, HT-29, H460, or A549 cells

2-Pyridinecarboxamide, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-, 4-methylbenzenesulfonate (1:1)

Dissolved in DMSO, final concentrations ca.10 uM

ca.60 mg/kg

6 nM, 6 nM, 6 nM, 6 nM, 6 nM, 6 nM

Oral administration of Sorafenib tosylate (ca.60 mg/kg) demonstrates broad spectrum, dose-dependent anti-tumor activity against a variety of human tumor xenograft models including MDA-MB-231, Colo-205, HT-29, DLD-1, NCI-H460, and A549, with no evidence of toxicity. In association with the anti-tumor efficacy, Sorafenib tosylatetreatment potently inhibits MEK 1/2 phosphorylation and pERK 1/2 levels in HT-29 and MDA-MB-231 xenografts but not in Colo-205 xenografts, and significantly suppresses tumor microvessel area (MVA) and microvessel density (MVD) in MDA MB-231, HT-29 and Colo-205 tumor xenografts. [1] Sorafenib tosylate treatment produces dose-dependent growth inhibition of PLC/PRF/5 tumor xenografts in SCID mice with TGIs of 49% and 78% at 10 mg/kg and 30 mg/kg, respectively, consistent with the inhibition of ERK and eIF4E phosphorylation, reduction of the microvessel area, and induction of tumor cell apoptosis. [2]

Biochemical assays, Recombinant baculoviruses expressing Raf-1 (residues 305–648) and B-Raf (residues 409–765) are purified as fusion proteins. Full-length human MEK-1 is generated by PCR and purified as a fusion protein from Escherichia coli lysates. Sorafenib tosylate is added to a mixture of Raf-1 (80 ng), or B-Raf (80 ng) with MEK-1 (1 ug) in assay buffer [20 mM Tris (pH 8.2), 100 mM NaCl, 5 mM MgCl2, and 0.15% beta-mercaptoethanol] at a final concentration of 1% DMSO. The Raf kinase assay (final volume of 50 uL) is initiated by adding 25 uL of 10 uM gamma[33P]ATP (400 Ci/mol) and incubated at 32 C for 25 minutes. Phosphorylated MEK-1 is harvested by filtration onto a phosphocellulose mat, and 1% phosphoric acid is used to wash away unbound radioactivity. After drying by microwave heating, a beta-plate counter is used to quantify filter-bound radioactivity. Human VEGFR2 (KDR) kinase domain is expressed and purified from Sf9 lysates. Time-resolved fluorescence energy transfer assays for VEGFR2 are pemed in 96-well opaque plates in the time-resolved fluorescence energy transfer format. Final reaction conditions are as follows: 1 to 10 uM ATP, 25 nM poly GT-biotin, 2 nM Europium-labeled phospho (p)-Tyr antibody (PY20), 10 nM APC, 1 to 7 nM cytoplasmic kinase domain in final concentrations of 1% DMSO, 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0.015% Brij-35, 0.1 mg/mL BSA, and 0.1% beta-mercaptoethanol. Reaction volumes are 100 uL and are initiated by addition of enzyme. Plates are read at both 615 and 665 nM on a Perkin-Elmer VictorV Multilabel counter at ca.1.5 to 2.0 hours after reaction initiation. Signal is calculated as a ratio: (665 nm/615 nM) x 10, 000 for each well. For IC50 generation, Sorafenib tosylate is added before the enzyme initiation. A 50-fold stock plate is made with Sorafenib tosylate serially diluted 1:3 in a 50% DMSO/50% distilled water solution. Final Sorafenib tosylate concentrations range from 10 uM to 4.56 nM in 1% DMSO.

637, 03

, , Christina W Yde/CDM Danish Cancer Society Research Center Denmark, Sorafenib (Nexavar)purchased from Selleck, Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

DMSO 127 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL