Sorafenib Tosylate

 

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Biological Activity

Description	Sorafenib Tosylate (Bay 43-9006) is a multikinase inhibitor of Raf-1, B-Raf and VEGFR-2 with IC50 of 6 nM, 22 nM and 90 nM, respectively.
Targets	Raf-1	B-Raf	VEGFR2	PDGFRβ
IC50	6 nM	22 nM	90 nM	57 nM [1]
In vitro	Sorafenib tosylate inhibits both wild-type and V599E mutant B-Raf activity with IC50 of 22 nM and 38 nM, respectively. Sorafenib tosylate also potently inhibits mVEGFR2 (Flk-1), mVEGFR3, mPDGFRβ, Flt3, and c-Kit with IC50 of 15 nM, 20 nM, 57 nM, 58 nM, and 68 nM, respectively. Sorafenib tosylate weakly inhibits FGFR-1 with IC50 of 580 nM. Sorafenib tosylate is not active against ERK-1, MEK-1, EGFR, HER-2, IGFR-1, c-Met, PKB, PKA, cdk1/cyclinB, PKCα, PKCγ, and pim-1. Sorafenib tosylate markedly inhibits VEGFR2 phosphorylation in NIH 3T3 cells with IC50 of 30 nM, and Flt-3 phosphorylation in HEK-293 cells with IC50 of 20 nM. Sorafenib tosylate potently blocks MEK 1/2 and ERK 1/2 phosphorylation in most cell lines but not in A549 or H460 cells, while having no effect on inhibition of the PKB pathway. Sorafenib tosylate inhibits the proliferation of HAoSMC and MDA-MB-231 cells with IC50 of 0.28 μM and 2.6 μM, respectively. [1] In addition to inhibition of the RAF/MEK/ERK signaling pathway, Sorafenib tosylate significantly inhibits the phosphorylation of eIF4E and down-regulates Mcl-1 levels in hepatocellular carcinoma (HCC) cells in a MEK/ERK-independent manner. Sorafenib tosylate inhibits the proliferation of PLC/PRF/5 and HepG2 cells with IC50 of 6.3 μM and 4.5 μM, respectively, and leads to the significant induction of apoptosis. [2]
In vivo	Oral administration of Sorafenib tosylate (~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]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

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 μg) in assay buffer [20 mM Tris (pH 8.2), 100 mM NaCl, 5 mM MgCl2, and 0.15% β-mercaptoethanol] at a final concentration of 1% DMSO. The Raf kinase assay (final volume of 50 μL) is initiated by adding 25 μL of 10 μM γ[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 β-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 performed in 96-well opaque plates in the time-resolved fluorescence energy transfer format. Final reaction conditions are as follows: 1 to 10 μM 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% β-mercaptoethanol. Reaction volumes are 100 μL and are initiated by addition of enzyme. Plates are read at both 615 and 665 nM on a Perkin-Elmer VictorV Multilabel counter at ~1.5 to 2.0 hours after reaction initiation. Signal is calculated as a ratio: (665 nm/615 nM) × 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 μM to 4.56 nM in 1% DMSO.

Cell Assay: ^[1]

Cell lines	MDA-MB-231, and HAoSMC
Concentrations	Dissolved in DMSO, final concentrations ~10 μM
Incubation Time	72 hours
Method	Cells are exposed to increasing concentrations of Sorafenib tosylate for 72 hours. Cell number is quantitated using the Cell TiterGlo ATP Luminescent assay kit. This assay measures the number of viable cells per well by measurement of luminescent signal based on amount of cellular ATP.

Animal Study: ^[1]

Animal Models	Female NCr-nu/nu mice implanted s.c. with MDA-MB-231, Colo-205, HT-29, H460, or A549 cells
Dosages	~60 mg/kg
Administration	Orally once daily
Solubility	2% Cremophor EL, 2% N,N-dimethylacetamide, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT01988493	Not yet recruiting	Carcinoma, Hepatocellular	Merck KGaA	2013-11	Phase 1|Phase 2
NCT02015728	Active, not recruiting	Recurrent Childhood Brain Tumor	Seattle Childrens Hospital|Cures Within Reach	2013-12
NCT01897610	Active, not recruiting	Advanced Hepatocellular Carcinoma	Green Cross Cell Corporation	2013-12	Phase 2
NCT01982097	Not yet recruiting	Carcinoma, Renal Cell	Bayer	2014-01
NCT01964235	Suspended	Advanced Hepatocellular Carcinoma	Novartis|Novartis Pharmaceuticals	2014-03	Phase 2
NCT01353794	Not yet recruiting	Carcinoma, Renal Cell	Bayer	2014-09	Phase 4

Chemical Information

Download Sorafenib Tosylate SDF

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

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.63703	6.3703	12.7406	19.1109

Customer Reviews (4)

Click to enlarge	Rating
	Source	Christina W Yde/CDM Danish Cancer Society Research Center Denmark. Sorafenib Tosylate purchased from Selleck
	Method	Cell growth assay
	Cell Lines	MCF-7 breast cancer cells
	Concentrations	0-1 μM
	Incubation Time	5 d
	Results	Sorafenib potently inhibited the survival of MCF7 cells in a dose-dependent manner.

Product Citations (29)

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  Differential effects of sorafenib on liver versus tumor fibrosis mediated by SDF1α/CXCR4 axis and Gr-1+ myeloid cell infiltration in mice. [Chen Y, et al. Hepatology 2013;10.1002/hep.26790]

  
  PubMed: 24242874


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  Functional characterization of the novel BRAF complex mutation, BRAFV600delinsYM, identified in papillary thyroid carcinoma. [Matsuse M, et al. Int J Cancer 2012;132(3):738-43]

  
  PubMed: 22752848


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  Semaphorin 6A regulates angiogenesis by modulating VEGF signaling. [Segarra M, et al. Blood 2012;120(19):4104-15]

  
  PubMed: 23007403