Sorafenib is a potent multikinase inhibitor (IC50s: 6/20/22 nM for Raf-1/VEGFR-4/B-Raf).
Besides Raf-1, Sorafenib also inhibits VEGFR-3 (IC50: 20 nM), BRAF wt (IC50: 22 nM), B-RAF V599E (IC50: 38 nM), VEGFR-2 (IC50: 90 nM), PDGFR-β (IC50: 57 nM), c-KIT (IC50: 68 nM), and Flt3 (IC50: 58 nM) in biochemical assays [1]. Sorafenib-induced phosphorylation of c-Met, p70S6K and 4EBP1 is significantly reduced when 10-0505 cells are co-treated with anti-human anti-HGF antibody, suggesting that treatment with Sorafenib leads to increased HGF secretion and activation of c-Met and mTOR targets [2].
Sorafenib Tosylate (10, 30, 50 and 100 mg/kg, p.o.) inhibits the tumor growth of 06-0606 and 10-0505 xenografts in a dose-dependent manner (P<0.01). The growth rate of 06-0606 and 10-0505 xenografts is also significantly reduced by Sorafenib. The weights of 06-0606 tumors in mice that are treated with Sorafenib (50/100 mg/kg) are approximately 13% and 5% of the controls, respectively. 50 mg dose of Sorafenib significantly inhibits tumor growth in mice with lines 5-1318, 26-1004 and 10-0505 (P<0.01). For 50 mg dose, the T/C ratio, where T and C are the median weight (mg) of Sorafenib- and vehicle-treated tumors at the end of the treatment, respectively, for 06-0606, 26-1004, 5-1318, and 10-0505 xenografts is 0.13, 0.10, 0.12 and 0.49, respectively [2]. The survival rate is 73.3 % in Diethylnitrosamine (DENA) group and 83.3 % in the Sorafenib group compared to 100 % in the normal control group. DENA group shows a significant increase in the liver index (1.51-fold increase, p<0.05) compared to normal control group, while treatment with Sorafenib shows a significant decrease (p<0.05) in the liver index when compared to DENA group. The liver index in Sorafenib group significantly decreases to lower than its value in the normal control [3].
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 the 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.
Tumor cell lines were plated at 2 × 105 cells per well in 12-well tissue culture plates in DMEM growth media (10% heat-inactivated FCS) overnight. Cells were washed once with serum-free media and incubated in DMEM supplemented with 0.1% fatty acid-free BSA containing various concentrations of BAY 43-9006 in 0.1% DMSO for 120 minutes to measure changes in basal pMEK 1/2, pERK 1/2, or pPKB. Cells were washed with cold PBS (PBS containing 0.1 mmol/L vanadate) and lysed in a 1% (v/v) Triton X-100 solution containing protease inhibitors. Lysates were clarified by centrifugation, subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in TBS-BSA, and probed with anti-pMEK 1/2 (Ser217/Ser221; 1:1000), anti-MEK 1/2, anti-pERK 1/2 (Thr202/Tyr204; 1:1000), anti-ERK 1/2, anti-pPKB (Ser473; 1:1000), or anti-PKB primary antibodies. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and developed with Amersham ECL reagent on Amersham Hyperfilm [1].
Female NCr-nu/nu mice (Taconic Farms, Germantown, NY) were used for all studies. Three to five million cells were injected s.c. into the right flank of each mouse. DLD-1 tumors were established and maintained as a serial in vivo passage of s.c. fragments (3 × 3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Treatment was initiated when tumors in all mice in each experiment ranged in size from 75 to 144 mg for antitumor efficacy studies and from 100 to 250 mg for studies of microvessel density and ERK phosphorylation. All treatment was administered orally once daily for the duration indicated in each experiment.
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