生物活性

Sorafenib is a RAF kinase inhibitor which suppresses ERK phosphorylation. Studies indicate that sorafenib induces c-Raf phosphorylation at both Ser-43 and Ser-259. When combined with vitamin K1, phosphorylation is increased at these serine residues. Sorafenib also induces the phosphorylation of PKA. In addition, sorafenib induces c-Met phosphorylation at Tyr-1349, which consequently induces PI3K-Akt phosphorylation. Studies determined that sorafenib inhibits other kinases such as Flk-1 (VEGFR2), PDGFR (platelet-derived growth factor receptor), Flt-3/Flk-2 (FLT3), Ret, and c-Kit. Sorafenib is an inhibitor of Raf-1, Raf-B, PDGFR-βand Flt-4. Its ability to affect the Raf/Mek/Erk pathway makes it useful in cancer research studies.

Sorafenib blocks RET/PTC3 autophosphorylation with an IC 50 of 47 nM, and inhibits the growth of cells carrying RET/V804L Mutants (IC 50 = 110 nM) or RET/V804M Mutants (IC 50 = 147 nM). [10]

体外研究

体内研究

激酶实验

In vitro Assays with Recombinant Raf-1 (Residues 305–648), BRAF (Residues 409–765), V599E BRAF (Residues 409–765), MEK-1, and Extracellular Signal-Regulated Kinase (ERK)-1. [2]

COOH-terminal kinase domains of Raf-1 (residues 305–648) and BRAF (residues 409–765) were generated by PCR. The BRAF (residues 409–765) V599E mutation was introduced using the Quik Change Site-directed Mutagenesis kit according to the manufacturer’s protocol. Recombinant baculoviruses expressing Raf-1 (residues 305–648), BRAF (residues 409–765), and V599E BRAF (residues 409–765) were purified as fusion proteins. Full-length human MEK-1 was generated by PCR and purified as a fusion protein from Escherichia coli lysates.

To test compound inhibition against various RAF kinase isoforms, BAY 43-9006 was added to a mixture of Raf-1 (80ng), wt BRAF, or V599E BRAF (80ng) with MEK-1 (1μg) in assay buffer [20mmol/L Tris (pH 8.2), 100mmol/L NaCl, 5mmol/L MgCl2, and 0.15%β-mercaptoethanol] at a final concentration of 1% DMSO. The RAF kinase assay (final volume of 50μL) was initiated by adding 25μL of 10μmol/LƳ-[33P] ATP (400Ci/mol) andincubated at 32°C for 25 minutes. Phosphorylated MEK-1 was harvested by filtration onto a phosphocellulose mat, and 1% phosphoric acid was used to wash away unbound radioactivity. After drying by microwave heating, aβ-plate counter was used to quantify filter-bound radioactivity. Activated MEK-1 and ERK-1 were purchased and assayed according to manufacturer’s instructions.

For the in vitro RET autophosphorylation assay, subconfluent NIH3T3 cells stably transfected with RET/PTC3 were solubilized in lysis buffer without phosphatase inhibitors (sodium fluoride, sodium pyrophosphate, and sodium vanadate). Then, 200 μ g of proteins were immune-precipitated with anti-RET; immune-complexes were recovered with protein A – Sepharose beads, washed five times with kinase buffer (20mM HEPES at pH 7.5, 150mM NaCl, 10% glycerol, 0.1% Triton X-100,

15mM MnCl2, and 15mM MgCl2) and incubated 20 minutes at room temperature in kinase buffer containing 2.5 μCi of [ γ-32P]ATP and unlabeled ATP (20μ M). Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were dried and exposed to film for autoradiography. Signal intensity was analyzed using a PhosphorImager (Typhoon 8600) interfaced with the Image Quant software. For phosphorylation of the synthetic substrate, RET immune-complexes were incubated (20 minutes at room temperature) in kinase buffer containing 200μM poly-(L-glutamic acid-L-tyrosine [poly-GT]), 2.5 μCi of [γ-32P] ATP, and unlabeled ATP (20μ M). Samples were spotted on Whatman 3MM paper, and32P incorporation was measured with a beta counter scintillator. Each experiment was performed at least three times.

细胞实验

Preparation of Sorafenib[1]

Sorafenibis dissolved in DMSO for in vitro experiments.

Cell Lines, Reagents, and Western Blot Analysis

The MDA-MB-231 human mammary adenocarcinoma cell line was obtained. Cell lines were maintained in DMEM (GIBCO), supplemented with 1% L-glutamine (GIBCO), 1% HEPES buffer (GIBCO), and 10% heat-inactivated fetal bovine serum (FBS). Cells were plated at 200,000 cells per well in 12-well tissue culture plates in growth media and incubated overnight. Media was removed and replaced with DMEM supplemented with 0.1% BSA containing either various concentrations of sorafenib, U0126, or vehicle (DMSO) for 2 h. Cells were washed with cold PBS containing 0.1 mM vanadate and lysed in a 1% Triton X-100 solution containing protease inhibitors. Lysates were clarified by centrifugation, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blocked for 1 h in TBS containing 5% non-fat dry milk and 1% BSA. Membranes were probed for 1 h with antibodies to pMEK1/2 (ser217/ ser221), MEK1/2, pERK1/2 (thr202/tyr204), ERK1/2, pPKB (ser473), and PKB. The antibodies were used at a dilution of 1:000. Blots were developed with horseradish peroxidase (HRP)–conjugated secondary antibodies and Amersham ECL reagent on Amersham Hyperfilm.

动物实验

Tumoricidal efficacy of sorafenib and S-1 against HCC in animal model[6]

Male NOD/SCID mice (5–6 weeks old) werepurchased and kept in a SPF environment. The mice were exposed to a 12:12-hlight–dark cycle and fed with food and water ad libitum in a barrier facility. NOD/SCIDmice were inoculated subcutaneously at right flank with PLC/PRF/5 or SK-HEP-1cells (1 x 10⁷cells/mouse). Mice were randomized into four groups (6mice per group) when tumors reached up to a volume of 120–150 mm³.Treatment for 4 groups was as follows: daily oral gavage of 10 mg/kg of S-1(thepro-drug of 5-FU) alone (S-1 group); 10 mg/kg ofsorafenib alone (sorafenib group); combination of S-1 and sorafenib (both 10mg/kg) (combined group); 0.2 ml normal saline (control group). Tumor length (L)and width (W) were measured with a caliper twice per week, and tumor volume wascalculated according to the formula 0.52 x L x W². Body weight ofmice was measured simultaneously.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Adnane L, Trail PA, Taylor I, Wilhelm SM. Sorafenib (BAY 43‐9006, Nexavar®), a Dual‐Action Inhibitor That Targets RAF/MEK/ERK Pathway in Tumor Cells and Tyrosine Kinases VEGFR/PDGFR in Tumor Vasculature. 2006;407:597-612.
[2] Wilhelm SM CC, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004 64(19):7099-7109.
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体内溶解度

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

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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