CN102406641A

CN102406641A - Application of sorafenib to treatment of fibrosis

Info

Publication number: CN102406641A
Application number: CN2010102886466A
Authority: CN
Inventors: 丁小燕; 刘平; 陈跃磊; 刘成海; 徐沁; 孙明瑜; 杨科
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS; Shuguang Hospital Affiliated to Shanghai University of TCM
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS; Shuguang Hospital Affiliated to Shanghai University of TCM
Priority date: 2010-09-20
Filing date: 2010-09-20
Publication date: 2012-04-11

Abstract

The invention relates to a novel purpose of sorafenib, in particular to an application of the sorafenib to treatment of transforming growth factor (TGF)-beta mediated diseases, and particularly to the application of the sorafenib to treatment of fibrosis. The invention also provides an application of the sorafenib or pharmaceutically acceptable salt of the sorafenib to the preparation of medicine for treating TGF-beta mediated diseases.

Description

The application of Sorafenib in the treatment fibrotic disease

Technical field

The present invention relates to a kind of new purposes of Sorafenib (sorafenib); Be specifically related to the application of Sorafenib in the beta mediated disease of treatment TGF-; More specifically relate to the application of Sorafenib in the treatment fibrotic disease, more specifically relate to the application of Sorafenib in treatment hepatic fibrosis or pulmonary fibrosis.

Background technology

China is " hepatopathy big country ", and the sickness rate of multiple hepatopathy is high always.Hepatic fibrosis is as the key character (Friedman, 2003) of multiple chronic hepatopathy pathology generating process, is one of important topic of being paid close attention to of hepatopathy research and treatment field always.Hepatic fibrosis is meant that hepatocyte necroses and during inflammatory stimulus, the pathological process that liver inner cell epimatrix (particularly collagen) increases unusually and piles up.The accumulation of extracellular matrix constitutes the fiber cicatrix, destroys the structure of liver, and the tuberosity of concurrent neonatal liver parenchyma causes liver function and metabolism to be destroyed, and (Gines etc., 2004) such as liver cirrhosis and hepatocarcinoma finally occur.The hepatic fibrosis complex genesis, TGF-β is present known most important one type of short hepatic fibrosis factor, has the activation hepatic stellate cell, promotes the liver collagen gene expression, promotes synthetic grade of extracellular matrix to act on (Gressner etc., 2002).TGF-β protein family comprises members such as TGF-β 1, TGF-β 2 and TGF-β 3, and they have close function.Wherein TGF-β 1 is most important member, it be one contain 112 ammonia aminoacid, molecular weight is the glycoprotein of 25kD, and between species high conservative, for example TGF-β 1 sequence of people and rat, mice has more than 99% identical.The gene of people TGF-β 1, TGF-β 2 and TGF-β 3 is positioned chromosome 19q3,1q41 and 14q24 respectively; All contain 7 exons; Its nucleotide sequence height homology; Coded precursor molecule C end person has 9 conservative Cys, and prompting TGF-β 1, TGF-β 2 and TGF-β 3 genes maybe be from common ancestral genes.In addition, TGF-β also is these three kinds of proteic general names.In view of important representative status and TGF-'beta ' family member's the high homology of TGF-β 1 albumen in the TGF-'beta ' family; Many researcheres adopt TGF-β 1 to study the characteristic of TGF-β protein family; In the product description of some company in addition with TGF-β 1 albumen directly write TGF-β (referring to; For example; Visit of the introduction of the website of dimension Shengong department (Biovision), http://www.biovision.com/tgf-betal-human-recombinant-1765.html) to TGF-β 1.

Residing microenvironment when cell stimulates the reaction that is produced down to depend on cell type and stimulate for TGF-β.For example TGF-β can cause epidermal growth to stop and cell death inducing.In addition, the epithelium that TGF-β also can inducing cell--mesenchyme transforms (EMT).80% cell is a hepatic parenchymal cells in the liver, and when sustaining damage with inflammation, hepatic parenchymal cells produces TGF-β through autocrine and paracrine action.Vivo and vitro experiment has proved that TGF-β can stimulate hepatic parenchymal cells that EMT takes place, and then forms activatory myofibroblast, increases the synthetic of extracellular matrix such as collagen, thereby promotes fibrotic disease that (Zeisberg etc., 2007 take place; Henderson and Forbes, 2008).

Although obtained many progress in the treatment field of hepatic fibrosis in recent years, many therapeutic schemes still can't obtain gratifying curative effect.At world wide, especially Chinese, the treatment of hepatic fibrosis remains one of problem that needs to be resolved hurrily clinically.Therefore, this area is starved of a kind of improved therapeutic scheme and/or medicine is treated hepatic fibrosis.

At present, be used to suppress TGF-β in this area, select very limited thereby suppress the Fibrotic treatment of EMT regulating liver-QI.Therefore, the inventor is to screen at tens kinds of small-molecule drugs of clinical practice at present.Surprisingly, find that the cancer therapy drug Sorafenib can obviously suppress the TGF-signal beta, and then suppress the EMT process of the beta induced hepatic parenchymal cells of TGF-, thus the treatment hepatic fibrosis.

Sorafenib is 4 [4-[[4-chloro-3-(trifluoromethyl) phenyl] carbamoyl is amino] phenoxy group]-N-methyl-pyridine-2-carboxylic acid amides with formula:

Its toluene fulfonate has another name called Nexavar (Nexavar), is the inhibitors of kinases (Sebolt-Leopold and English, 2006) of target spot more than.Verified its action target spot is the tyrosine kinase receptor on tumor cell and the tumor vessel; Comprise kinases Raf-1 and B-Raf, vascular endothelial growth factor receptor VEGFR, platelet derived growth factor receptor PDGFR and kinases FLT3 and c-Kit etc.; Suppress receptor tyrosine kinase activity through specificity; The propagation and tumor-blood-vessel growth (Shimizu etc., 1999 that suppress tumor cell; Gilliland and Griffin, 2002; Heinrich etc., 2002).In December, 2005, Sorafenib be as the medicine of renal carcinoma in late period, obtains the approval of FDA (Food and Drug Adminstration), and this is the medicine of first treatment renal carcinoma of ratifying for over ten years of U.S. FDA, also is first oral many target spots inhibitors of kinases.Afterwards, Sorafenib is used for the treatment of secondary liver cancer again by the FDA approval.Before this, this medicine has been got permission to go on the market in China.Discover that Sorafenib has anti-tumor activity widely, also has better curative effect (Llovet etc., 2008) to colon cancer, nonsmall-cell lung cancer and melanoma etc.Yet, do not see as yet that up to now relevant Sorafenib suppresses the report of TGF-β, also nobody's proof Sorafenib can suppress EMT or hepatic fibrosis.

Summary of the invention

In one aspect, the present invention provides Sorafenib or the application of its pharmaceutically acceptable salt in the medicine of the beta mediated disease of preparation treatment TGF-.In yet another aspect, the present invention provides Sorafenib or the application of its pharmaceutically acceptable salt in the medicine of the disease of preparation treatment TGF-β and STAT3 mediation, and wherein, STAT3 can be the downstream albumen of TGF-β.Aspect another, the disease that the present invention is directed to is the disease of EMT mediation.More specifically, the disease that the present invention is directed to can be a fibrotic disease, preferred hepatic fibrosis or pulmonary fibrosis.

On the other hand, the present invention provides the method for treating the beta mediated disease of TGF-with Sorafenib or its pharmaceutically acceptable salt.In yet another aspect, the present invention provides the method with the disease of Sorafenib or its pharmaceutically acceptable salt treatment TGF-β and STAT3 mediation, and wherein, STAT3 can be the downstream albumen of TGF-β.Aspect another, the disease that the present invention is directed to is the disease of EMT mediation.More specifically, the disease that the present invention is directed to can be a fibrotic disease, preferred hepatic fibrosis or pulmonary fibrosis.

On the other hand, the present invention provides with Sorafenib or its salt method at vitro inhibition TGF-β.In yet another aspect, the present invention provides with Sorafenib or its salt method at vitro inhibition TGF-β and STAT3, and wherein, STAT3 can be the downstream albumen of TGF-β.Aspect another, the present invention provides with Sorafenib or its salt method in vitro inhibition EMT process.

On the other hand; The present invention provides a kind of Sorafenib or its pharmaceutically acceptable salt of comprising; And the pharmaceutical composition of pharmaceutically acceptable excipient or carrier, said pharmaceutical composition can be used for treating the disease of the beta mediated disease of TGF-, TGF-β and STAT3 mediation, disease, fibrotic disease and hepatic fibrosis or the pulmonary fibrosis of EMT mediation.

In one type of embodiment, TGF-β can be TGF-β 1.

The present invention also considers the multiple analog and the derivant of Sorafenib; Such as but not limited to: N-(2-methoxyl group-5-(trifluoromethyl) phenyl)-N '-(4 (2-(N-methylamino formoxyl)-4-pyridyloxy) phenyl) urea, N-(4-chloro-3-(trifluoromethyl) phenyl)-N '-(4 (2-(N-carbamoyl)-4-pyridyloxy) phenyl) urea, N-(4-chloro-3-(trifluoromethyl) phenyl)-N '-(4 (2-(N-methylamino formoxyl)-4-pyridyloxy) phenyl) urea, N-(2-methoxyl group-4-chloro-5-(trifluoromethyl) phenyl)-N '-(3 (2-(N-methylamino formoxyl)-4-pyridyloxy) phenyl) urea, N-(4-chloro-3-(trifluoromethyl) phenyl)-N '-(2 carbamoyls-4-pyridyloxy) phenyl-) urea, N-(4-chloro-3-(trifluoromethyl) phenyl)-N '-(urea (can be referring to U.S. Pat 7235576 for 4 (2-(N-carbamoyl-4-pyridyloxy) phenyl); It is for referencial use to include it in this paper in full); Because they can have the biological action similar with Sorafenib,, they use so also can having above-mentioned each item identical with Sorafenib.

The present invention also provides a kind of method of screening the medicine of treatment hepatic fibrosis or pulmonary fibrosis; Said method comprises waiting to sieve the step that medicine contacts with TGF-β and measures the TGF-'beta ' activity, if contact back TGF-'beta ' activity reduces this is waited to sieve the medicine of drug identification for treatment hepatic fibrosis or pulmonary fibrosis.In one embodiment, the step of said mensuration TGF-'beta ' activity utilizes two luciferase reporter gene detection systems on cellular level, to carry out.

Description of drawings

Fig. 1: (A) Sorafenib, Erlotinib (Erlotinib), gefitinib (Gefitinib), Da Bufeilong (Darbufelone), celecoxib small-molecule drugs such as (Celecoxib) are to the influence of the TGF-signal beta of AML12 cell; (B) Sorafenib of 0,1,2,5 and 10 μ M is to the influence of the TGF-signal beta of AML12 cell; (C) in the AML12 cell, the Sorafenib of various dose is to the inhibitory action of the beta induced Smad2 protein phosphorylation of TGF-; (D) Sorafenib is to the inhibitory action of the mRNA level of the beta induced Smad7 of TGF-.*, p＜0.01 promptly has significant difference on the statistics.

Fig. 2: (A) Sorafenib of various dose (0,1,5 and 10 μ M) is to epithelium--the inhibitory action of mesenchyme conversion (EMT) of the TGF-β 1 inductive Mouse Liver parenchyma AML12 generation of 5ng/ml; (B) Sorafenib of various dose (0,1,5 and 10 μ M) is induced the influence of level of epithelial cell marker and the mesenchymal cell mark of AML12 cellular expression to the TGF-β 1 of 5ng/ml; (C and D) Sorafenib is to the inhibiting time dependence of AML12 cell EMT process.

Fig. 3: the Sorafenib of various dose (0,1,5 and 10 μ M) is to the apoptotic inhibitory action of TGF-β 1 inductive AML12 of 5ng/ml.*, p＜0.01 promptly has significant difference on the statistics.

Fig. 4: (A) influence of STAT3 phosphorylation level in 1 pair of AML12 cell of TGF-β of variable concentrations (0,0.5,1,2,5 and 10ng/ml); (B) after the TGF-β 1 of 5ng/ml handles AML12 cell different time (0,1,2,4,8 and 16h), the influence of STAT3 phosphorylation level in the pair cell; (C) in the AML12 cell, the inhibitory action that the Sorafenib of various dose (0,1,5 and 10 μ M) raises to TGF-β 1 inductive STAT3 phosphorylation level; (D) the AML12 cell is after 5 μ M Sorafenibs are handled, the proteic location of STAT3 in the cell.*, p＜0.01 promptly has significant difference on the statistics.

Fig. 5: (A) in the Mouse Liver primary cell, 5 μ M Sorafenibs are to the influence of TGF-signal beta; (B) detect in the Mouse Liver primary cell 10 μ M Sorafenibs to the influence of the level of TGF-β 1 inductive epithelial cell mark and mesenchymal cell mark through the Western blotting; (C) detect in the Mouse Liver primary cell 5 μ M Sorafenibs to the influence of the level of TGF-β 1 inductive epithelial cell mark ZO-1 and mesenchymal cell mark fibronectin through immunofluorescence.

Fig. 6: (A) can obviously reduce CCl through tissue section strain method proof Sorafenib ₄Induce the hepatic tissue collagen deposition in the mouse model of hepatic fibrosis; (B) verify further that through the variation of hydroxyproline content Sorafenib can obviously reduce the murine liver tissue collagen deposition in the mouse model that CCl4 induces hepatic fibrosis; *, p＜0.01 promptly has significant difference on the statistics; (C) Sorafenib obviously reduces the α-SMA albumen in the liver organization of mouse model that CCl4 induces hepatic fibrosis.

The specific embodiment

1. definition

Except as otherwise noted, term used herein has the implication of those skilled in the art's common sense.

" pharmaceutically acceptable salt " refers to the pharmaceutically acceptable salt of certain chemical compound, and this salt can comprise, for example sodium, potassium, calcium, magnesium, ammonium, tetra-allkylammonium etc. derived from various organic and inorganic counter ion counterionsl gegenions well known in the art; When molecule contains basic functionality, comprise the salt of organic or mineral acid, example hydrochloric acid salt, hydrobromate, tartrate, mesylate, acetate, maleate, oxalates etc.

" pharmacy effective dose " and " treatment effective dose " refers to be enough to treat particular condition or disease or its one or more symptoms and/or prevent said disease or the consumption of the chemical compound that disease takes place.

" pharmaceutically acceptable excipient or carrier " refers to can be used for the excipient or the carrier of medical applications; It is generally inertia, includes but not limited to excipient or the carrier put down in writing among " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences) of this paper and E.W.Martin.

" TGF-β " is transforming growth factor-beta; It is one type of important cell growth factor; A plurality of biology of incident (Rahimi and Leof such as the synthetic and early embryonic development of the growth of wide participation cell, differentiation, migration, apoptosis, angiogenesis, immunoreation, extracellular matrix; 2007), can also promote the transfer of EMT and cancerous cell.

" EMT " refers to epithelium--mesenchyme transforms, and promptly epithelial cell loses the epithelial cell characteristic and obtained a kind of phenomenon (Zavadil and Bottinger, 2005) of mesenchymal cell characteristic.EMT is the important phenomenon in early embryo development and the organ generative process, participates in former bowel movement, the neural lophocyte travel motion from neurocele, and the processes such as formation of cardiovascular valve.The injury repairing of adult tissue simultaneously, the infiltration of the fibrosis of kidney, liver, lung, especially malignant tumor and transfer, (Thiery etc., 2009) all are closely related with EMT.

Unless otherwise indicated herein, term " treatment " refers to therapeutic and preventative means, and purpose is prevention or slow down (weakening) bad physiological change or imbalance, like the development of fibrotic disease or spread.For the object of the invention; Useful or required clinical effectiveness includes but not limited to: can detect or undetectable remission, disease degree alleviate, morbid state is stablized (promptly not worsening), and PD postpones or slows down; Morbid state improves or alleviates, and alleviation (partly or entirely)." treatment " also can refer to compare with the expectation survival period of not receiving treatment, and survival period prolongs.The object that need treat comprises the object of suffering from disease or imbalance and tends to the object of ill or imbalance or need prevent disease or the object of imbalance.

" disease that TGF-is beta mediated " refers to the disease that TGF-β possibly cause under situation such as gene mutation, abnormal expression, phosphorylation level change, activity change, celluar localization change; Include but not limited to: the fibrosis lesion of multiple internal organs, like pulmonary fibrosis, renal fibrosis, hepatic fibrosis etc.; Multiple cardiovascular disease, as hereditary hemorrhagic telangiectasia (hereditary hemorrhagic telangiectasia, HTT), primary pulmonary hypertension (primary pulmonary hypertension, PPT); And Marfan's syndrome (Marfan Syndrome, MFS) or the like.

" disease of TGF-β and STAT3 mediation " refers to the disease that TGF-β and STAT3 possibly cause under situation such as unconventionality expression, phosphorylation level change, activity change, celluar localization change; Refer to that more specifically TGF-β influences characteristics such as the expression of its downstream albumen STAT3, phosphorylation level, activity, celluar localization under situation such as abnormal expression, phosphorylation level change, activity change, celluar localization change; Thereby possibly cause the disease that takes place; Include but not limited to: the fibrosis lesion of multiple internal organs, like pulmonary fibrosis, renal fibrosis, hepatic fibrosis etc.; Leukemia; Tumor; Multiple cardiovascular disease is like glycoprotein (GP) 13O dependency myocardial hypertrophy.

" disease of EMT mediation " refers to the disease that epithelium-the mesenchyme conversion process causes when unusual, includes but not limited to: the fibrosis lesion of multiple internal organs, and like pulmonary fibrosis, renal fibrosis, hepatic fibrosis etc.

2. pharmaceutical composition and medication

In other embodiments, the Sorafenib that comprises effective dose and the pharmaceutical composition of pharmaceutically acceptable carrier or excipient have been described.These compositionss are applicable to veterinary or human administration.

Pharmaceutical composition of the present invention can be any composition forms that gives the patient.For example, said composition can be solid, liquid or gas (aerosol) form.General route of administration includes but not limited to: oral, local, gastrointestinal tract is outer, in the Sublingual, rectum, vagina, eye, tumor and intranasal.The gastrointestinal tract external administration comprises subcutaneous injection, intravenous, intramuscular, intrathoracic injection or infusion.In one aspect, parenteral administration said composition.Aspect another, intravenous gives said pharmaceutical composition.Aspect another, the said pharmaceutical composition of orally give.

But the compounding pharmaceutical compositions makes its effective ingredient after giving patient's said composition, and promptly Sorafenib can be by biological utilisation.Pharmaceutical composition can be taked the form of single or multiple dose unit, and for example, tablet can be a single dosage unit, and the container of aerosol can hold one or more a plurality of dosage units.

The material that is used to prepare this pharmaceutical composition should be nontoxic under its consumption.Those of ordinary skills will understand that the optimal dose of active component in this pharmaceutical composition depends on various factors.Correlative factor includes but not limited to: the concrete form of type of animal (like the people), Sorafenib, administering mode and concrete composition.

Pharmaceutically acceptable carrier can be graininess, so said composition can be, for example tablet or powder type.When carrier was liquid, said composition can be, for example oral syrup or injection.In addition, carrier can be gaseous state or microgranule, to be provided for the aerosol combination of (for example) inhalation.

When oral administration, preferred solid of said composition or liquid form, semisolid, semiliquid, suspension and gel form are also included within the solid or liquid form that this paper considers.

As the solid composite of oral administration, can said composition be processed powder, granule, compressed tablets, pill, capsule, chewing gum, wafer etc.This solid composite generally contains one or more inert diluents.In addition, can there be one or more following materials: binding agent such as carboxymethyl cellulose, ethyl cellulose, microcrystalline Cellulose or gelatin; Excipient such as starch, lactose or dextrin, disintegrating agent such as alginic acid, sodium alginate, original gel (Primogel), corn starch etc.; Lubricant such as magnesium stearate; Fluidizer such as silica sol; Sweeting agent such as sucrose or glucide, flavoring agent such as Herba Menthae, cresotic acid or the agent of orange flavor; And coloring agent.

When compositions is a capsule, during like the gelatine capsule form, except that the material of the above-mentioned type, it also can contain liquid carrier such as Polyethylene Glycol, cyclodextrin or fatty oil.

Said composition can be a liquid form, like elixir, syrup, solution, emulsion or suspending agent.Liquid can be used for oral administration or injected delivery.When oral administration, compositions can contain one or more in sweeting agent, antiseptic, dyestuff/coloring agent and the flavour reinforcers.In the compositions of drug administration by injection, also can comprise in surfactant, antiseptic, wetting agent, dispersant, suspending agent, buffer, stabilizing agent and the isotonic agent one or more.

The fluid composition that no matter is solution, suspension or other similar type also can comprise one or more following materials: sterile diluent such as water for injection; Saline solution; Preferred normal saline, Ringer's solution, isotonic sodium chloride can be used as fixed oil such as synthetic monoglyceride or diglyceride, Polyethylene Glycol, glycerol, cyclodextrin, propylene glycol or other solvent of solvent or suspension media; Antibacterial such as benzyl alcohol or methyl parahydroxybenzoate; Antioxidant such as ascorbic acid or sodium sulfite; Chelating agen such as ethylenediaminetetraacetic acid; Buffer such as acetate, citrate or phosphate buffer, and the material of adjustment of tonicity such as sodium chloride or dextrose.Can the outer compositions of gastrointestinal tract be packaged in ampoule, disposable syringe or the multiple dose vials of being processed by glass, plastics or other material.Normal saline is exemplary co-adjuvant.Injectable composition is preferably aseptic.

The effective dose of reactive compound depends on the character of disease or disease in treatment disease specific or disease, and the available standards clinical technology is measured effective dose.In addition, can randomly carry out external or in vivo test help to identify the optimal dose scope.The accurate dosage that is used for compositions also depends on the seriousness of route of administration and disease or imbalance, should decide according to doctor's judgement and each patient's situation.

Said composition contains the reactive compound of treating effective dose, to obtain appropriate dose.This treatment effective dose generally is at least about 0.01% of said composition weight.When oral administration, this treatment effective dose is about the 0.1%-80% of said composition weight.In one aspect, Orally administered composition can contain the reactive compound that accounts for said composition weight 4%-50%.Aspect another, the preparation present composition is so that gastrointestinal tract other unit dosage contains the reactive compound of the 0.01 weight %-2 weight % that has an appointment.

This pharmaceutical composition can contain the reactive compound of the about 0.01-1000mg of every kg body weight.In one aspect, this pharmaceutical composition can comprise the reactive compound of every approximately kg body weight 0.1-100mg.In one aspect, this pharmaceutical composition can comprise the reactive compound of every approximately kg body weight 1-100mg.On the other hand, dosage is about the reactive compound of 0.1-25mg/kg body weight.On the other hand, dosage is about the reactive compound of 1-10mg/kg body weight.

In therapeutic scheme; The dosage that gives this pharmaceutical composition every day can be about 0.01-1000mg reactive compound/kg body weight; Preferred 0.1-100mg reactive compound/kg body weight, more preferably 1-10mg reactive compound/kg body weight, most preferably 2-5mg reactive compound/kg body weight.

Reactive compound or pharmaceutical composition can for example through infusing or injecting administration, absorb through epithelium or mucous layer (like oral mucosa, rectum and intestinal mucosa etc.) through any approach easily.Can whole body or topical.Known various delivery system, as be wrapped in liposome, microgranule, microcapsule, the capsule etc., they can be used for giving reactive compound or pharmaceutical composition.In some embodiments, reactive compound or the pharmaceutical composition with more than one gives the patient.

For example also can prepare with short aerosol, or carry out pulmonary administration through fluorocarbon or the perfusion of synthetic lung surfactant through using inhaler or aerosol apparatus.

In another embodiment, reactive compound or compositions can be sent by controlled release system, such as but not limited to: use pump or various polymeric material.In another embodiment; Can with controlled release system place reactive compound or compositions target (like brain) near; Thereby only need the whole body dosage a part (referring to for example Goodson, " medical application of controlled release " (Medical Applications ofControlled Release), the same; The 2nd volume, 115-138 page or leaf (1984)).Other controlled release system of discussing in the available Langer summary (Science 249:1527-1533 (1990)).

Pharmaceutically acceptable carrier can be a liquid, and Ru Shui and oil comprise oil, animal, plant or synthetic liquid of originating, like Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods.Pharmaceutically acceptable carrier can be saline, Radix Acaciae senegalis, gelatin, gelatinized corn starch, Pulvis Talci, keratin, colloidal silica, carbamide etc.In addition, can use adjuvant, stabilizing agent, thickening agent, lubricant and coloring agent.In one embodiment, when giving the patient, reactive compound or compositions and pharmaceutically acceptable carrier are aseptic.When intravenous gave reactive compound, water was exemplary carrier.The aqueous solution of saline solution and dextrose and glycerol also can be used as liquid-carrier, especially injection solution.Suitable pharmaceutical carrier also comprises excipient such as starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Pulvis Talci, sodium chloride, defatted milk powder, glycerol, propylene, glycol, water, ethanol etc.If desired, the present composition also can contain a small amount of wetting agent or emulsifying agent, or the pH buffer agent.

The present composition can be taked solution, suspension, and emulsion, tablet, pill, micro tablet, capsule contains the capsule form of liquid, powder, slow releasing agent, suppository, Emulsion, aerosol, spray, suspending agent or the form of other suitable use.Other example of other suitable pharmaceutical carrier is seen E.W.Martin's " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences).

In one embodiment, according to conventional methods reactive compound is made into suitable intravenous and gives animal, especially people's pharmaceutical composition.The carrier that is used for intravenous administration generally is a sterile isotonic aqueous buffer solution.When needing, said composition also can comprise solubilizing agent.The compositions that is used for intravenous administration can randomly comprise local anesthetic such as lignocaine, to alleviate the pain of injection site.Usually, in the sealed container of lined out activity material consumption such as ampoule or medicine bag with unit dosage forms separately or mixed form, for example, as freeze-dried powder or do not have aqueous concentrate and provide.When giving reactive compound through infusion, available, for example contain the pharmaceutical grade sterilized water or brinish infusion bottle makes up a prescription.When giving reactive compound, can provide Injectable sterile water or brinish ampoule are housed, so that before administration, mix these compositions through injection.

Said composition can be used for topical, and this moment, carrier can be solution, emulsion, ointment or gel-type vehicle form.If be used for transdermal administration, said composition can be transdermal patch or iontophoresis device form.Topical formulations can comprise concentration and be about 0.05%-50%w/v (weight in the per unit volume compositions), is the reactive compound of 0.1%-10%w/v on the other hand.This can pass through, such as but not limited to: the intra-operative local infusion; Local application is like postoperative and wound dressing coupling; Injection; The conduit mode; The suppository mode; Or implantation mode (implant is porous, imporosity or gelatin materials, comprise film such as silicon mould (sialastic) film or fiber) realizes.

Said composition can be used for rectally, and its form is for example in rectum, to melt the also suppository of release of active compounds.

Said composition can comprise the various materials of the physical form that changes solid or liquid dosage unit.For example, said composition can be included in the material that active component forms the coating shell on every side.The material that forms the coating shell generally is inert, for example can be selected from: sugar, Lac and other enteric coating material.Perhaps, can active component be packaged in the gelatine capsule.

Said composition can be made up of the gaseous state dosage unit, for example, can be aerosol form.The term aerosol agent is used to refer to various systems, from the colloidal nature system to by packing the system that forms.Can send through liquefied gas or Compressed Gas or through the pumping system of dispersed activity composition compatibly.

No matter be solid, liquid or gas form, pharmaceutical composition of the present invention all can comprise the other medicines that are used to treat fibrotic disease.

3. the purposes of Sorafenib

The inventor suppresses the EMT process of hepatic parenchymal cells through experiment showed, Sorafenib through the proteic phosphorylation of STAT3 that suppresses TGF-β and its downstream, thereby reverses hepatic fibrosis.Below prove this purposes of Sorafenib through embodiment.It will be understood by those skilled in the art that the following embodiment that provides is merely illustration purpose, the protection domain that does not limit the present invention in any way.

Embodiment

The inhibitor of embodiment 1 screening TGF-signal beta

The applicant chooses present at tens kinds of micromolecular compounds of clinical use, can suppress the chemical compound of TGF-signal beta through the method screening of luciferase reporter gene activity analysis.

Experiment material:

Mice AML12 hepatic parenchymal cells system: available from American type culture collection (American Type Culture Collection, ATCC).

TGF-β 1 albumen: available from U.S. R&D system house (R&D Systems, USA).

Luciferase detection system: the two luciferase reporter gene detection systems (Dual Luciferase Reporter Gene Assay System) that adopt U.S. Pu Luomaige company (Promega) to produce.

Reporter gene (CAGA) ₁₂-Lux: present by professor Chen Yeguang of Tsing-Hua University.

Plasmid pRL-SV40: available from U.S. Pu Luomaige company (Promega).

Lipofection reagent (Lipofectamine Plus/Reagent) test kit: available from U.S. hero life technology company limited (Invitrogen).

The experimental technique summary:

1.AML12 cell transfecting

Use the plasmid of lipofection reagent (Lipofectamine Plus/Reagent) test kit cultured cells transfection various combination in 24 porocyte culture plates.With the AML12 cell is example, and every hole contains 5 * 10 approximately ⁴Individual cell, the plasmid transfection amount is about 200ng.Every hole comprises the TGF-β reporter gene (CAGA) of 200ng ₁₂The confidential reference items reporter gene pRL-SV40 of-Lux, 5ng.Each sample repeats 3 times, changes serum-free DMEM culture medium behind the transfection 24h into, and to add final concentration be that 5ng/ml TGF-β 1 is the micromolecular compound to be measured of 5 μ M with final concentration, cultivates 8h for 37 degrees centigrade.

2. the active detection of reporter gene expression

24 orifice plate cells use the PBS buffer to wash 2 times.Add 100 μ l1 * passive (Passive) lysis buffer, cell lysis 15min at room temperature rocks frequently cell is split away off from culture plate.Collect behind the lysate with centrifuge with the centrifugal 30s of 12000rpm, get supernatant and partly carry out luciferase activity mensuration.Sampling amount is generally 10 μ l, measures the activity (being the activity of Lampyridea luciferase) of target reporting gene earlier, measures the activity of confidential reference items reporter gene Renila (being the sea pansy luciferase) then.The consumption of substrate is 17 μ l during two kinds of enzyme activity determinations.

3. date processing

The relative activity of target reporting gene=target reporting gene activity measured value/confidential reference items reporter gene activity measured value, i.e. Lampyridea luciferase/sea pansy luciferase.Every group of experiment repeated the back recorded data for three times carry out statistical analysis, calculating mean value and standard error, and utilize Xue Shengshi t check (Student ' s t-test) that every group of data are carried out significance analysis.*, p＜0.05, significant difference; *, p＜0.01, utmost point significant difference.

Through detecting the influence of tens kinds of small-molecule drugs such as Sorafenib, Erlotinib, gefitinib, Da Bufeilong, celecoxib to the TGF-signal beta, the result finds that the cancer therapy drug Sorafenib can obviously suppress TGF-signal beta (Figure 1A).

Embodiment 2 Sorafenibs are the inhibitory action to the TGF-signal beta

After screening Sorafenib, the applicant has studied the dose-effect relationship of Sorafenib to TGF-signal beta depression effect.

Experiment material and method: with embodiment 1.

The result finds that under the stimulation of 5ng/ml TGF-β 1, Sorafenib has stronger inhibitory action to the TGF-signal beta, and this inhibitory action is dose dependent (Figure 1B).

In addition, the inventor also measures the influence of Sorafenib to the mRNA level of the target gene Smad7 of the phosphorylation level of TGF-β 1 downstream molecules Smad2 and TGF-β 1 path respectively through Western blotting and real-time fluorescence quantitative PCR.

Experiment material:

P-Smad2/3 and Smad2 antibody: available from U.S. cell signalling technology company (CellSignaling Technology).

Beta-actin antibody: available from U.S. Sigma aldrich company (Sigma-Aldrich).

RNA reverse transcription test kit: available from Canada rich enzyme Tai Si company (Fermentas).

Real-time fluorescence quantitative PCR detection kit: available from Canada rich enzyme Tai Si company (Fermentas).

Primer: give birth to worker bio-engineering corporation by Shanghai and synthesize.

Experimental technique:

1.Western blotting

Sorafenib with 5ng/ml TGF-β 1 and variable concentrations is handled the AML12 cell.Behind the 48h, cell adds 1ml cell pyrolysis liquid (50mMTris-Cl pH=7.5,150mM NaCl, 2mM EDTA pH 8.0,0.5%Nonidet P-40 and protease inhibitor) with PBS buffer washing secondary in the 60mm plate, place cracking 10min on ice.Cell pyrolysis liquid is transferred in the EP pipe, and the centrifugal 10min of 12000rpm gets supernatant, and 100 ℃ are boiled 10min.Sample is transferred to protein sample on the pvdf membrane behind the SDS-PAGE electrophoresis, and with p-Smad2/3 and Smad2 antibody test destination protein.With the colour developing of ECL chromogenic substrate, the exposure of X-ray sheet tabletting is preserved experimental result in the dark place after development and photographic fixing.

2. real-time fluorescence quantitative PCR

The AML12 cell extracts cell total rna behind 5ng/ml TGF-

β

1 and 5 μ M Sorafenibs processing 24h.The method that cell total rna extracts is seen " molecular cloning handbook (second edition) ".Use the RNA reverse transcription test kit of rich enzyme Tai Si company (MBI), to specifications operation.Carry out the real-time fluorescence quantitative PCR detection of genes of interest as template with the reverse transcription product cDNA of 1/20 volume.Internal control gene commonly used is a beta-actin in the real-time fluorescence quantitative PCR reaction, and the primer sequence of beta-actin is a forward: 5 '-TGAGCGCAAGTACTCTGTGTGGAT-3 ' (SEQ ID NO:1); Oppositely: 5 '-TAGAAGCATTTGCGGTGCACGATG-3 ' (SEQ ID NO:2).The Smad7 primer sequence is forward: 5 '-TCCTGCTGTGCAAAGTGTTC-3 ' (SEQ ID NO:3); Oppositely: 5 '-AGTAAGGAGGAGGGGGAGAC-3 ' (SEQ ID NO:4).

The result proves, can cause the proteic phosphorylation of downstream molecules Smad2 after TGF-β 1 handles cell, and Sorafenib can obviously suppress the proteic phosphorylation level of downstream molecules Smad2 (Fig. 1 C).In addition, Sorafenib can also suppress the target gene Smad7mRNA level (Fig. 1 D) of TGF-signal beta.These prove that further the small-molecule drug Sorafenib is the inhibitor of TGF-signal beta.

Embodiment 3. Sorafenibs suppress the beta induced cell EMT process of TGF-

In the present embodiment, the inventor utilizes TGF-β 1 to induce the AML12 cell that the influence of the cell model checking Sorafenib pair cell EMT process of EMT takes place.

Experiment material:

E-cadherin (E-cadherin) antibody: available from U.S. cell signalling technology company;

Fibronectin (fibronectin) antibody and beta-actin antibody: available from U.S. Sigma aldrich company;

ZO-1 antibody: available from U.S. hero company (Invitrogen);

Vimentin (vimentin) antibody: available from U.S. Santa Cruz biotech company (Santa Cruz Biotechnology).

Experimental technique: the Western blotting is seen embodiment 2.

We have set up TGF-β 1 and have induced AML 12 cells that the cell model of EMT takes place.Under the stimulation of 5ng/mlTGF-β 1, the Mouse Liver parenchyma is that tangible metamorphosis has taken place AML12: the forfeiture of AML12 cell its epithelial cell form and characteristic obtain mesenchymal cell form and characteristic simultaneously.In addition, in cell culture medium, add the Sorafenib of variable concentrations (1,5,10 and 20 μ M) after, handle cell with the TGF-β of 5ng/ml 1.The result shows that after 48 hours, 10 μ M Sorafenibs can significantly suppress the EMT process of TGF-β 1 inductive hepatic parenchymal cells.This inhibition effect has concentration and relies on effect, promptly along with the increase of Sorafenib concentration, suppresses the effect also more obvious (Fig. 2 A) of EMT.

In the EMT of hepatic parenchymal cells process, except cellular morphology generation significant change, significant variation has also taken place in the expression of a plurality of marker gene.For example, TGF-β 1 stimulates the AML12 cell after 48 hours, and epithelial cell mark E-cadherin and the proteic expression of ZO-1 obviously reduce, and the expression of mesenchymal cell mark fibronectin and Vimentin obviously increases (Fig. 2 B).Stimulate under the situation of AML12 cell at TGF-β 1, add certain density Sorafenib again, the E-cadherin of AML12 cell and the proteic expression of ZO-1 will increase, and the corresponding minimizing of the expression of fibronectin and Vimentin (Fig. 2 B).

In addition, Sorafenib also has time dependence to the inhibitory action of AML12 cell EMT process.Handle cell respectively after 24 hours and 48 hours with Sorafenib, no matter still be the expression of marker gene from cellular morphology on, we can both find that Sorafenib can effectively suppress the inductive cell EMT of TGF-signal beta process (Fig. 2 C and 2D).

Embodiment 4. Sorafenibs suppress TGF-β 1 inductive apoptosis

The inventor utilizes dna ladder method, flow cytometry method to detect Sorafenib to TGF-β 1 inductive apoptotic inhibitory action.

Experiment material:

Annexin V (Annexin V) apoptosis test regent box: visit dimension Shengong department (Biovision) available from the U.S..

Experimental technique:

1. flow cytometry analysis apoptosis

Handled the AML12 cell 48 hours with the TGF-β 1 of 5ng/ml and the Sorafenib of variable concentrations, centrifugal collecting cell uses the U.S. to visit the annexin V apoptosis test regent box of dimension Shengong department afterwards, and pair cell carries out dying operation to specifications.After operation was accomplished, sample sent Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences's molecular biology platform to analyze and statistics.

2. the dna segment detection (dna ladder) of apoptotic cell

In cell culture medium, add the TGF-β 1 of 5ng/ml and the Sorafenib of variable concentrations, add behind the mixing and cultivate the AML12 cell in 6 orifice plates.After 48 hours, the centrifugal apoptotic cell of collecting suspension adds the cell pyrolysis liquid (containing 10mM Tris-Cl (pH 8.0), 25mM EDTA (pH 8.0), 0.25% triton (Triton) X-100 and 5mg/ml RNA enzyme A) of 500 μ l together with adherent cell.Be put in static 30min on ice, the centrifugal 15min of 12000g.Add 56 ℃ of incubated overnight of E.C. 3.4.21.64 (2.5mg/ml) in the supernatant.Next day, add the cold dehydrated alcohol deposit D NA of 1/10 volume 3M sodium acetate and 2.5 times of volumes.Behind the centrifugal 15min of 12000g sedimentary DNA is dissolved in the water, 1.2% agarose gel electrophoresis analysis, EB dyeing is also taken a picture.

The result: the TGF-signal beta takes place also can induce the generation apoptosis the EMT except inducing the AML12 cell.Add up apoptotic quantity through flow cytometer, we find that TGF-β 1 can impel about 60% cell generation apoptosis, and after in cell culture, adding Sorafenib, apoptotic quantity obviously reduces.Under the effect of 10 μ M Sorafenibs, 5% the cell generation apoptosis of only having an appointment, this and blank group basic identical (Fig. 3 A).

During cell generation apoptosis, occur tangible karyopycnosis in the cell, and present fragmented.Through agarose gel electrophoresis, can be observed the dna fragmentation of 180-200bp integral multiple, present stair-stepping dna ladder.After the processing of 1 μ M Sorafenib, the dna fragmentation number reduces; After 5 μ M and 10 μ M Sorafenibs were handled, dna fragmentation disappeared (Fig. 3 B), and the quantity that this explanation Sorafenib is handled back cell generation apoptosis obviously reduces.

So far; We are through the apoptotic method of multiple detection; Find that the small-molecule drug Sorafenib suppresses the caused apoptosis of TGF-β, and present dose-dependent effect within the specific limits: promptly the concentration of Sorafenib is high more, and it is obvious more to suppress apoptotic effect.

Embodiment 5. Sorafenibs suppress the proteic phosphorylation level of STAT3

The inventor utilizes the method for Western blotting and immunofluorescence to verify that Sorafenib is to the inhibitory action of the proteic phosphorylation level of STAT3 with to STAT3 protein subcellular location influence respectively.

Experiment material:

P-STAT3 (Tyr ⁷⁰⁵) and STAT3 antibody: available from U.S. cell signalling technology company;

Beta-actin antibody: available from U.S. Sigma aldrich company.

Experimental technique:

1.Western blotting is seen embodiment 2.

2. cellular immunofluorescence

The AML12 cell is layered on the coverslip, and length is taken out coverslip after certain density, wash secondary with PBS.After selecting 4% formalin or 4% paraformaldehyde fixed cell 20min for use, wash secondary with PBS.With the penetrating processing cell of 1 ‰ triton x-100s 5min, secondary is washed with PBS in penetrating back.10%BSA seals 60min.Add the anti-diluent (1: 1000) contain STAT3 antibody, incubated at room 2h or 4 ℃ spend the night.PBST rinsing 3 times, each 5min.Afterwards, add the fluorescence two anti-diluents (1: 1000) that FITC is coupled, incubated at room 1h notes lucifuge.PBST rinsing 3 times, each 5min.Add the DAPI diluent at last and hatch 1min, and use the distilled water rinsing.After the mounting, the proteic Subcellular Localization of fluorescence microscope STAT3.

The result: take place in EMT and the apoptosis process at TGF-β 1 inducing mouse AML12 cell, we find that the phosphorylation level of transcription factor STAT3 also increases thereupon.Even under the TGF-β 1 of low concentration (for example 1ng/ml) stimulated, in the short time (for example 2 hours), the phosphorylation level of STAT3 obviously increased (Fig. 4 A and 4B).After adding Sorafenib in the cell, the phosphorylation level of STAT3 has been suppressed (Fig. 4 C).In addition, we find that also Sorafenib also disturbs the Subcellular Localization of transcription factor STAT3.Under normal circumstances, be positioned at nucleus, work the function that makes gene transcription regulation as the STAT3 of transcription factor.Cell is after Sorafenib is handled, and change has taken place the proteic Subcellular Localization of STAT3: most STAT3 albumen are positioned at Cytoplasm.Consider that STAT3 is the key factor that EMT takes place cell; In conjunction with discovery (Fig. 2 and Fig. 3) before; We think just because of Sorafenib impel transcription factor STAT3 from nucleus to intracytoplasmic location, thereby stoped the functional transcription of STAT3, and then suppressed the EMT process of cell.

Embodiment 6. Sorafenibs suppress the EMT process of mice primary hepatocyte

The inventor verifies the influence of Sorafenib to mice primary hepatocyte EMT process through the method for luciferase reporter gene activity analysis.

Experiment material:

C57BL/6 mice: available from zoopery center, Chinese Academy of Sciences Shanghai;

IV Collagen Type VI enzyme and α-SMA antibody: available from U.S. Sigma aldrich company;

All the other experiment materials are seen embodiment 2.

Experimental technique:

1. embodiment 1 is seen in the active detection of reporter gene expression.

2.Western blotting is seen embodiment 2.

3. cellular immunofluorescence is seen embodiment 5.

4. the separation of mice primary hepatocyte and cultivation

Get the C57BL/6 mice, adopt pentobarbital sodium (100mg/kg) anesthesia; Open mouse peritoneal, remove small intestinal to the left, expose postcava and portal vein, import into from postcava, until near hepatic vein with No. 21 trocars; Employing contains the buffer perfusion liver of 1mM EGTA and 0.1% glucose, cuts off portal vein simultaneously rapidly infusion liquid is flowed out from portal vein, and rate of flooding is 4ml/min; Change behind the perfusion 20ml and contain 0.5mg/ml IV Collagen Type VI enzyme and 5mM CaCl ₂Infusion liquid continue perfusion 10min, carefully take out liver at once, place the 10mM culture dish that contains buffer, cut off liver peplos with shears, rock gently hepatocyte discharged; Adopt the cellular filter of 70 μ m to filter; The DMEM washed cell of use 10ml 2 times is collected hepatocyte after 800rpm is centrifugal.Hepatocyte after the separation is dyeed with 4% trypan blue at once; Examine under a microscope, nuclear is the blue dead cell that is, the cell of refusing to dye is a living cells; With cell inoculation in the 12 hole culture dishs that collagen encapsulates; Employing contains 10% hyclone, and the DMEM culture fluid of 100u/ml penicillin and streptomycin is at 5%CO ₂, 37 ℃ of cultivations.

The result finds that in the mice primary hepatocyte, Sorafenib can significantly suppress TGF-signal beta (Fig. 5 A) equally.Through the method for western trace and immunofluorescence, we find that also Sorafenib can also suppress significant expression of gene variation (Fig. 5 B and 5C) in the beta induced EMT process of TGF-.This just explains that Sorafenib not only can suppress the TGF-signal beta and the EMT process of AML12 cell line, and the mice primary hepatocyte is also had same effect.

Embodiment 7. Sorafenibs are to CCl ₄The therapeutic effect of inductive hepatic fibrosis in mice

The inventor passes through CCl ₄The animal model of inductive hepatic fibrosis in mice is verified the curative effect of Sorafenib to hepatic fibrosis.

Experiment material: C57BL/6 mice: available from zoopery center, Chinese Academy of Sciences Shanghai.

Modeling method:

In order further to study the probability of Sorafenib treatment hepatic fibrosis, the applicant has set up CCl ₄The animal model of inductive hepatic fibrosis in mice.Promptly choose suitable mice as model group, weekly the CCl of secondary lumbar injection 50% ₄/ olive oil solution.After two weeks, the mice of model group is being accepted lumbar injection CCl ₄The basis on, accept the pharmaceutical carrier (DMSO is as placebo) of doses every day through gavage.After 8 weeks, choose the mouse liver BIAO and BEN, carry out H&E dyeing and Sirius red colouring after the section, detect liver collagen density.

Experimental technique:

The mice of experimental group is being accepted lumbar injection CCl ₄The basis on, accept the Sorafenib of doses (4mg/kg) every day through gavage.Under this dosage, the medicine Sorafenib does not influence physical signs (table 1) such as survival and the body weight of mice.

Table 1 small-molecule drug Sorafenib does not influence physical signs such as survival and the body weight of mice

Divide into groups	Quantity (n)	Survival rate (%)	Body weight (g)
				Normal group (contrast)	12	100.0	25.74±2.15
Model group (placebo)	15	86.67	20.63±2.15
				Sorafenib group (administration)	15	93.33	22.22±0.89

After 8 weeks, after the whole execution of every group mice, cut the hepatic tissue of mice, FFPE carries out HE dyeing and Sirius red colouring after section.Through check, to compare with the mice of normal group (contrast), a large amount of collagen depositions appear in the hepatic tissue of model group mice, and the fibrosis lesion process of hepatic tissue is obvious.With respect to model group, the collagen deposition of experimental mice hepatic tissue obviously reduces (Fig. 6 A).Through the statistical analysis to area of collagen behind Sirius red colouring of every group of 20 hepatic tissue sections, we think that Sorafenib is very significant (p＜0.01, Fig. 6 A) to the effect that reduces the hepatic tissue collagen deposition.

In addition; One of principal character sexual element of body collagen protein is a hydroxyproline; Therefore it accounts for 13% of collagen, amino acid total amount, measures the hydroxyproline content of liver organization, can clear and definite collagen aggregate level; Evaluation fibrosis lesion degree, it is the direct indicator of reflection hepatic fibrosis degree.We are also through measuring the influence of hydroxyproline assessment Sorafenib to the fibrosis lesion degree.

Experimental technique:

Get hepatic tissue 2g, put into Potter-Elvehjem Tissue Grinders homogenate fast after shredding, regulate homogenate pH value to 2.5 with acetic acid, adding pepsin (60mg/ml) digests 72h, adds Tris liquid stopped reaction, and the bag filter dialysed overnight of packing into obtains interior, bag outer two samples of bag.Add 6N hydrochloric acid more respectively, the rearmounted 110 ℃ of following hydrolysis 18h of sealing.At last, sample is used the paradime thylaminobenzaldehyde determination of color, detects absorption value at the 560nm place.

The result:

We have measured the hydroxyproline content in three groups of murine liver tissue, find that the hydroxyproline content in the murine liver tissue of model group is higher, and the hydroxyproline content in the murine liver tissue obviously reduce (Fig. 6 B) behind the medicine feed Sorafenib.

In addition, the proteic expression of α-SMA is the important symbol in the fibrosis lesion process of hepatic tissue.We are through the influence of SABC method checking Sorafenib to the α-expression of SMA albumen in murine liver tissue.

Experiment material:

α-SMA antibody and enhanced sensitivity diaminobenzidine (DAB) colour developing liquid: available from U.S. Sigma aldrich company.Experimental technique: after 8 weeks of modeling, after the whole execution of three groups mices, cut the hepatic tissue of mice.4% paraformaldehyde fixing organization, dehydration back FFPE is by the thickness section of 5 μ M.H&E dyeing or immunohistochemical analysis are carried out in the section of hepatic tissue.With the PBST sealing 60min that contains 10% serum.Afterwards, add an anti-diluent (1: 1000) that contains α-SMA antibody, incubated at room 2h.PBST rinsing 3 times, each 5min.Add the two anti-diluents (1: 1000) that HRP is coupled, incubated at room 1h.PBST rinsing 3 times, each 5min.At last, detect expression and the distribution of α-SMA with enhanced sensitivity diaminobenzidine (DAB) colour developing liquid.

The result finds, through CCl ₄Induce that the α-SMA albumen in the liver organization obviously reduces (Fig. 6 C) behind the mouse feeding Sorafenib of hepatic fibrosis.

At last, we also carry out pathology statistics through the fibrosis to murine liver tissue, find that the collagen deposition of liver organization obviously reduces behind the mouse feeding medicine Sorafenib, fibrosis obviously alleviates (table 2).These data explain that all Sorafenib has reparation and therapeutic effect preferably to hepatic fibrosis in mice.

The pathology statistics of table 2 murine liver tissue fibrosis

List of references:

Chen?YL，Liu?B，Zhou?ZN，Hu?RY，Fei?C，Xie?ZH，Ding?XY.J.Biol.Chem.，2009，284(35)：23481-23490.

Friedman SL.J.Hepatol., 2003,38 (supplementary issue 1): S38-S53.

Gilliland DG and Griffin JD.Curr.Opin.Hematol., 2002,9:274-281.

Gines?P，Cardenas?A，Arroyo?V，Rodes?J.N.Engl.J.Med.，2004，350：1646-1654.

Gressner AM, Weiskirchen R, Breitkopf K etc., Front.Biosci, 2002,7:d793-d807.

Heinrich MC, Blanke CD, Druker BJ and Corless CL.J.Clin.Oncol., 2002,20:1692-1703.

Henderson NC and Forbes SJ.Toxicology, 2008,254:130-135.

Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF etc., N.Engl.J.Med., 2008,359:378-390.

Rahimi RA and Leof EB.J.Cell Biochem., 2007,102:593-608.

Sebolt-Leopold?JS，English?JM.Nature，2006，441(7092)：457-462.

Shimizu A, O ' brien KP, Sjoblom T etc., Cancer Res., 1999,59:3719-3723.

Thiery?JP，Acloque?H，Huang?RY，Nieto?MA.Cell，2009，139：871-890.

Zavadil J and Bottinger EP.Oncogene, 2005,24:5764-5774.

Zeisberg?M，Yang?C，Martino?M，Duncan?MB，Rieder?F，Tanjore?H，Kalluri?R.J.Biol.Chem.，2007，282：23337-23347.

Claims

1. Sorafenib or its pharmaceutically acceptable salt application in the medicine of the beta mediated disease of preparation treatment TGF-.

2. application as claimed in claim 1 is characterized in that, said disease is the disease of TGF-β and STAT3 mediation.

3. application as claimed in claim 2 is characterized in that, said disease is the disease of EMT mediation.

4. like each described application among the claim 1-3, it is characterized in that said disease is a fibrotic disease.

5. application as claimed in claim 4 is characterized in that, said fibrotic disease is hepatic fibrosis or pulmonary fibrosis.

6. one kind with Sorafenib or its salt method at vitro inhibition TGF-β.

7. method as claimed in claim 6 is characterized in that, said method can also be at vitro inhibition STAT3.

8. method as claimed in claim 7 is characterized in that, said method can also be in vitro inhibition EMT process.

9. method of screening the medicine of treatment hepatic fibrosis or pulmonary fibrosis; Said method comprises waiting to sieve the step that medicine contacts with TGF-β and measures the TGF-'beta ' activity, if contact back TGF-'beta ' activity reduces this is waited to sieve the medicine of drug identification for treatment hepatic fibrosis or pulmonary fibrosis.

10. method as claimed in claim 9 is characterized in that, the step of said mensuration TGF-'beta ' activity utilizes two luciferase reporter gene detection systems on cellular level, to carry out.