WO2020119424A1 - 一种含有sumo抑制剂的组合物及应用 - Google Patents

一种含有sumo抑制剂的组合物及应用 Download PDF

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WO2020119424A1
WO2020119424A1 PCT/CN2019/120210 CN2019120210W WO2020119424A1 WO 2020119424 A1 WO2020119424 A1 WO 2020119424A1 CN 2019120210 W CN2019120210 W CN 2019120210W WO 2020119424 A1 WO2020119424 A1 WO 2020119424A1
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fxr
sumo
liver
activation
composition
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French (fr)
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郝海平
王洪
周济宇
王广基
崔双
潘晓洁
郭怡彤
黄宁宁
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中国药科大学
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Priority to EP19894708.7A priority Critical patent/EP3733207B1/en
Priority to US16/960,226 priority patent/US11541032B2/en
Priority to JP2020539275A priority patent/JP7121415B2/ja
Publication of WO2020119424A1 publication Critical patent/WO2020119424A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the invention relates to the technical field of medicine, in particular to a composition containing SUMO inhibitor and its application.
  • Liver diseases especially viral hepatitis, fatty liver disease, liver fibrosis and liver cancer, have a high incidence worldwide.
  • the incidence of non-alcoholic fatty liver in normal adults in Europe and America is 20 to 33%, and the incidence in obese people is 60 to 90%; in China, the incidence of ordinary adults is about 15% (FanJG, Farrell G. Epidemiology of non-alcoholic fatty liver disease in China. Journal of hepatology, 2009, 50(1): 204-10.).
  • Liver fibrosis (Fibrosis) is a pathological state, which refers to the transitional deposition of liver fibrous connective tissue.
  • Hepatic fibrosis is an intermediate link between chronic viral hepatitis, metabolic disorders and chronic alcoholic/non-alcoholic fatty liver and other chronic liver diseases that progress to cirrhosis.
  • Hepatic fibrosis is a common and important feature of chronic liver disease, and 25-40% Patients with chronic liver disease eventually develop cirrhosis or even liver cancer.
  • Studies in recent years have shown that liver fibrosis is a reversible disease, and cirrhosis is an irreversible disease. Therefore, inhibiting, preventing and reversing liver fibrosis is the central link in the treatment of various chronic liver diseases.
  • Hepatic Stellate Cells HSCs
  • HSCs are the most important cells that mediate the development of liver fibrosis.
  • HSCs are at rest; when stimulated by pathological factors, HSCs are activated into a fibroblast phenotype with proliferative ability. HSCs cells of this phenotype can secrete a large amount of fibrotic factors and collagen fibers, which eventually cause liver The development of fibrosis and cirrhosis. Therefore, HSCs are the determinants of the development of liver fibrosis, and inhibition of HSCs activation is an important strategy for the treatment and reversal of liver fibrosis.
  • the research and development of anti-liver fibrosis drugs is the current research and development hot spot of liver drugs, and has also made some research progress.
  • the currently reported therapeutic drugs related to the prevention and treatment of liver fibrosis mainly include the following categories: 1) Chinese herbal medicines and their extracts, such as turmeric, resveratrol, silibinin, allobromine and their salts (authorization announcement number CN10132721B ); 2) Chemical drugs and their preparations, such as pirfenidone and creatinine compositions (authorization bulletin number CN103550242B), Clestanone derivatives (authorization publication number CN104095857B); 3) biological agents, including recombinant proteins and gene drugs Etc., the former includes TGF ⁇ 1-inhibiting peptide (authorization announcement number CN1203091C), IL-4 (authorization announcement number CN101318013B), monoclonal antibody HAb18GC2 and its heavy and light chain variable genes and peptides (authorization announcement number CN
  • FXR Farnesoid X Receptor
  • NR1H4 Nuclear Receptor Subfamily 1, Group H, Member 4
  • FXR not only plays an important role in physiological processes such as bile acid, lipid and sugar metabolism, but also has a regulatory effect on a variety of pathological processes: FXR The FXR-FGF15/19 signaling pathway mediates the bile acid-promoting liver regeneration; FXR exerts an anti-inflammatory effect through the FXR/NF- ⁇ B negative feedback loop (Wang YD, Chen WD, Wang M, et al.
  • Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response. Hepatology,2008,48(5):1632-43.); FXR exerts an anti-autophagy effect by blocking the formation of CREB-CRTC2 complex and inhibiting the expression of downstream autophagy-related genes (Seok S, Fu T, Choi SE, et al. Transcriptional regulation of autophagy by an FXR-CREB axis.
  • FXR is closely related to the formation of liver tumors: FXR -/- mice are normally fed to All liver tumors occurred spontaneously at 15 months, and wild-type mice of the same age did not show the same changes (Yang F, Huang X, Yi T, et al. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor .Cancer Res 2007;67:863-867.). The above research shows that FXR is closely related to the occurrence and development of liver diseases.
  • FXR agonists are the main research strategy for liver protection and anti-fibrosis drugs, and there are a large number of related patent applications (Wang H, He Q, Wang G, et al. FXR modulators for enterohepatic and metabolic diseases. Expert Opin Ther Pat. 2018 Nov; 28 (11): 765-782), such as CN201210482982.3 (application number) provides the application of fibronectin compounds and pharmaceutically acceptable salts thereof in the preparation of drugs for the treatment of FXR-mediated diseases, CN201180067346.8 and CN201080043283.8 (application number) discloses the use of FXR activity regulators in pharmaceutical compositions, all of which have been authorized.
  • Obeticholic acid as a strong FXR agonist, its anti-NASH efficacy has just completed a clinical phase III study, and the results show that 21.0% of patients in the group taking 25mg OCA daily have fibrosis symptoms Improved (10.6% of patients in the placebo group improved), suggesting that OCA has a certain improvement in the treatment of liver fibrosis; but how to further improve its anti-liver fibrosis activity is an important scientific problem; in addition, as an antigenic bile liver Sclerosis (PBC) drugs have been approved by the US FDA in May 2016 (Markham A, Keam SJ. Obeticholic Acid: First Global Approval. Drugs. 2016 Aug; 76(12): 1221-6.), becoming FXR-based The first case of a drug successfully launched on the target site.
  • PBC antigenic bile liver Sclerosis
  • FXR agonists can inhibit the activation of HSC cells by stimulating the FXR signaling pathway in HSC cells (Fiorucci, Antonelli E, RizzoG,et.al.The nuclear receptor SHPmediatesinhibition ofhepatic stellate cellsby FXR and protectionsagainstliverfibrosis.Gastroenterology.2004Nov; 127(5):1497-512.FiorucciS1,RizzoE,Antonal Afarnesoidxreceptor-smallheterodimerpartnerregulatorycascademodulatestissuelometalloproteinaseinhibitor-1andmatrixmetalloproteaseexpressioninhepatictelestellatecellsandpromotesresolutionofofliverfibrosis.J.
  • the object of the present invention is to provide a composition containing a SUMO inhibitor, the composition comprising an FXR agonist and a SUMO inhibitor, which can significantly inhibit the high activation of hepatic stellate cells, reduce collagen fiber deposition, and thus significantly resist liver fibrosis .
  • the present invention provides a composition containing a SUMO inhibitor, the composition comprising an FXR agonist and a SUMO inhibitor.
  • the FXR agonist includes but is not limited to one or more of obeticholic acid, GW4064 and WAY-362450.
  • the SUMO inhibitor includes, but is not limited to, spectinomycin and/or ginkgo acid.
  • the invention also provides the application of the composition described in the above technical scheme in the preparation of anti-liver fibrosis medicine.
  • the present invention also provides the use of the composition described in the above technical solution in the preparation of a medicament against liver fibrosis symptoms.
  • the invention also provides the application of the composition described in the above technical scheme in the preparation of a drug for inhibiting the activation of hepatic stellate cells.
  • the invention also provides the application of the composition described in the above technical solution in the preparation of a drug for reducing collagen fiber deposition.
  • the dosage forms of the drugs independently include tablets, capsules, granules, pills, powders or injections.
  • the present invention provides a composition containing a SUMO inhibitor, the composition comprising an FXR agonist and a SUMO inhibitor.
  • FXR agonists do not have the effect of inhibiting the activation of hepatic stellate cells; the present invention can also activate FXR agonists and SUMO inhibitors in highly activated hepatic stellate cells
  • the FXR signaling pathway inhibits the activation of hepatic stellate cells, reduces the deposition of collagen fibers, and thus can resist liver fibrosis.
  • the important significance of the present invention is that for healthy humans, hepatic stellate cells are at rest and are reactive to FXR agonists; however, for patients with fibrosis, hepatic stellate cells have In the activated state, the cell's reactivity to the FXR agonist is reduced or lost, so the FXR agonist cannot inhibit the activation of the hepatic stellate cells in the activated state, so when the patient's liver appears fibrotic symptoms, take the FXR agonist Can not produce anti-liver fibrosis pharmacological activity; the present invention on the basis of explaining the mechanism of activated hepatic stellate cell loss of responsiveness to FXR agonist, innovatively proposes a combination method, that is, including SUMO inhibitor and FXR Application of agonist composition in the preparation of anti-hepatic fibrosis drugs: activated hepatic stellate cells still have good response to the complex, and the complex can significantly inhibit the activation of hepatic
  • Figure 1 is a graph showing the difference in FXR transcriptional activity between resting and activated HSCs, of which Figure 1-1 is: OCA analysis of mRNA levels of Fxr in resting and activated HSCs cells;
  • FIG 1-2 OCA analysis of Shp mRNA levels of HSCs cells in resting and activated states (TGF- ⁇ );
  • Figure 1-3 GW4064 analysis of mRNA levels of Fxr in resting and activated HSCs cells
  • FIG 1-4 GW4064 analysis of Shp mRNA levels of HSCs cells in resting and activated states (TGF- ⁇ );
  • Figure 1-5 is the analysis of Fxr mRNA level of WAY-362450 in resting and activated HSCs cells
  • Figure 1-6 WAY-362450 analysis of Shp mRNA levels of HSCs cells in resting and activated states (TGF- ⁇ ). *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 2 shows the analysis of the level of FXR protein SUMO after activation of HSCs. ***P ⁇ 0.001;
  • Figure 3 is the analysis of the SUMO inhibitory level of FXR protein in activated HSCs by SUMO inhibitors SP and GA, ***P ⁇ 0.001, ##P ⁇ 0.01, ###P ⁇ 0.001;
  • Figure 4 is the effect of FXR agonist combined with SUMO inhibitor on the FXR transcription activity of activated HSCs, of which Figure 4-1 is: the combination of OCA and SP on Fxr mRNA level analysis of activated HSCs cells;
  • Figure 4-2 is: OCA and SP combined to analyze the mRNA level of Shp in activated HSCs cells;
  • FIG. 4-3 GW4064 combined with SP to analyze the mRNA level of Fxr in activated HSCs cells
  • FIG. 4-4 GW4064 combined with SP to analyze the mRNA level of Shp in activated HSCs
  • Figure 4-5 is: WAY-362450 combined with SP to analyze the mRNA level of Fxr in activated HSCs cells;
  • Figure 4-6 is: WAY-362450 combined with SP to analyze the mRNA level of Shp in activated HSCs cells;
  • Figure 4-7 is: the combination of OCA and GA to analyze the mRNA level of Fxr in activated HSCs cells;
  • Figure 4-8 is: the combination of OCA and GA to analyze the mRNA level of Shp in activated HSCs cells, nsP>0.05, *P ⁇ 0.05, **P ⁇ 0.01;
  • Figure 5 shows the effect of the combination of FXR agonist and SUMO inhibitor on the activation of HSCs.
  • Figure 5-1 shows the analysis of Acta-2 mRNA expression level by the combination of OCA and SP;
  • Figure 5-2 GW4064 combined with SP analysis of Acta-2 mRNA expression level
  • Figure 5-3 Analysis of Acta-2 mRNA expression level in combination with WAY-362450 and SP;
  • Figure 5-4 Analysis of Acta-2 mRNA expression level by combination of OCA and GA, nsP>0.05, *P ⁇ 0.05;
  • Figure 6 is the effect of the combination of FXR agonist and SUMO inhibitor on liver FXR and its transcriptional activity under CCl 4 induced liver fibrosis, of which Figure 6-1 is: Fxr mRNA expression level analysis;
  • Figure 7 is the effect of FXR agonist combined with SUMO inhibitor on CCl 4 induced liver fibrosis, of which Figure 7-1 is: Acta-2 mRNA expression level analysis, **P ⁇ 0.01;
  • Figure 7-2 is: Sirius Red staining of liver.
  • the present invention provides a composition containing a SUMO inhibitor, the composition comprising an FXR agonist and a SUMO inhibitor.
  • the mass ratio of the FXR agonist and the SUMO inhibitor is not particularly limited, and any mass ratio can be used.
  • the FXR agonist preferably includes, but is not limited to, one or more of obeticholic acid, GW4064, and WAY-362450, and the plurality is specifically two or more.
  • the FXR agonist when there are two or more FXR agonists, the components are mixed in any quality.
  • the FXR agonist is not limited to the above three types, and its source is not particularly limited, and preferably includes various FXR agonists of natural origin, semi-synthetic, or chemical synthesis.
  • the SUMO inhibitor preferably includes, but is not limited to, spectinomycin and/or ginkgo acid.
  • the SUMO inhibitor when the SUMO inhibitor preferably includes azithromycin and ginkgolic acid, the kiwimycin and ginkgolic acid are mixed in any mass ratio.
  • the SUMO inhibitor is not limited to the above two, and its source is not particularly limited, and preferably includes various SUMO inhibitors of natural origin, semi-synthetic, or chemical synthesis.
  • highly activated hepatic stellate cells lose their responsiveness to FXR agonists, and FXR agonists do not have the effect of inhibiting the activation of hepatic stellate cells.
  • the SUMO inhibitor can significantly improve highly activated hepatic stellate cells The responsiveness of stromal cells to FXR agonists, and can significantly enhance the inhibitory effect of FXR agonists on the high activation of hepatic stellate cells; individuals with symptoms of liver fibrosis, whose hepatic stellate cells are already activated, use FXR alone
  • the agonist cannot produce a good anti-fibrosis effect, and the composition of the FXR agonist and the SUMO inhibitor provided by the present invention can significantly inhibit the activation of hepatic stellate cells, reduce collagen fiber deposition, and thus can resist liver fibrosis.
  • the invention also provides the application of the composition described in the above technical scheme in the preparation of anti-liver fibrosis medicine.
  • the present invention also provides the use of the composition described in the above technical solution in the preparation of a medicament against liver fibrosis symptoms.
  • the invention also provides the application of the composition described in the above technical scheme in the preparation of a drug for inhibiting the activation of hepatic stellate cells.
  • the invention also provides the application of the composition described in the above technical solution in the preparation of a drug for reducing collagen fiber deposition.
  • the dosage forms of the drugs independently preferably include tablets, capsules, granules, pills, powders or injections.
  • the preparation method of the above dosage form is not particularly limited, and it may be prepared by a conventional preparation method for preparing the corresponding dosage form and the corresponding auxiliary materials used.
  • the administration method of the composition preferably includes oral administration, intravenous injection, intravenous infusion, intramuscular injection, etc., and a combination of various administration methods.
  • the HSC-T6 cell line used in the present invention was purchased from the cell bank of Central South University;
  • Obeticholic acid (OCA), GW4064 and WAY-362450 used in the present invention were purchased from MCE company, TGF- ⁇ recombinant protein was purchased from R&D systems company, reverse transcription kit was purchased from Applied Biosystems company, Trizol RNAiso plus was purchased from TAKARA company The primers used were synthesized by Life Invitrogen, the SUMO-based ELISA detection kit was purchased from Epigentek, and the remaining reagents were all commercially available.
  • RNA pellet 1.0 ml of pre-chilled 75% ethanol, then centrifuge at 12000g for 5 min, discard the supernatant to obtain total RNA, and reconstitute with 10 ⁇ l DEPC water, dilute to 0.5 ⁇ g/ ⁇ l after quantification.
  • the PCR use conditions are as follows:
  • Stage 1 Pre-denatured at 95°C for 1min
  • Stage 2 PCR reaction 95 °C 15sec; such as 60 °C 30sec for 40 Cycles; 72 °C 30sec;
  • Stage 3 Melting curve analysis 65 ⁇ 95°C, 0.5°C/5s.
  • FXR agonists OCA, GW4064 and WAY-362450 can significantly induce the upregulation of the mRNA level of FXR target gene Shp ( Figure 1-2, Figure 1-4, Figure 1-6); At this time, the FXR agonists OCA, GW4064 and WAY-362450 could not significantly increase the mRNA level of the downstream target gene Shp of FXR ( Figure 1-2, Figure 1-4, Figure 1-6).
  • the SUMO inhibitors Spectinomycin (SP) and Ginkgolic acid (GA) were purchased from MCE.
  • 3.1 SUMO inhibitors can inhibit the level of SUMOylation of FXR protein in activated HSCs cells
  • 3.2 SUMO inhibitors can increase the responsiveness of activated HSCs to FXR agonists
  • Example 1 it was found that in activated HSCs cells, the regulation of FXR agonists OCA, GW4064 and WAY-362450 on the FXR signaling pathway was lost, which was manifested as a loss of induction of the Shp gene. In this part of the study, we plan to investigate the regulation of FXR agonists on the FXR signaling pathway in the presence of SUMO inhibitors.
  • 3.3 SUMO inhibitors can enhance the inhibitory effect of FXR agonists on HSCs cell activation
  • mice C57BL/6 were purchased from the Comparative Medicine Center of Yangzhou University;
  • CCl 4 was purchased from Shanghai Lingfeng Chemical Reagent Company, and mineral oil was purchased from Sigma-Aldrich Company.
  • mice After one week of adaptive feeding of the animals, the mice were randomly divided into 5 groups, 8 in each group, for a total of 40 mice. They are control group, model group, OCA single administration group, SP single administration group, OCA+SP combination administration group, in which the model group is given CCl 4 (20%, dissolved in mineral oil) by intraperitoneal injection, the control group The corresponding volume of solvent (mineral oil) was given twice a week for a total of 4 weeks. From the third week after modeling, OCA was intragastrically administered at a dose of 1.5 mg/kg once a day for two weeks. In addition, the OCA-administered group with SP was administered subcutaneously with SP from the first week at a dose of 200 mg/kg once a day. After the end of the dosing cycle, the mice were sacrificed and the liver was isolated and stored for future use.
  • liver tissue was fixed in 4% paraformaldehyde and sent to Wuhan Google Biotechnology Co., Ltd. (Wuhan, China) for double-blind analysis and detection.
  • the test item was Sirius Red staining analysis.
  • the SUMO inhibitor SP enhances the regulation of OCA on the FXR pathway in fibrotic mice
  • the SUMO inhibitor SP can enhance the anti-hepatic fibrosis effect of OCA
  • hepatic stellate cells in a highly activated state lose their responsiveness to FXR agonists, and FXR agonists do not have the effect of inhibiting the activation of hepatic stellate cells; the present invention combines FXR agonists and SUMO inhibitors After reconstitution, the hepatic stellate cells in the highly activated state restore responsiveness to the FXR agonist, and can inhibit the high activation of hepatic stellate cells, reduce the deposition of collagen fibers, and thus can resist liver fibrosis.

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Abstract

一种含有SUMO抑制剂的组合物，属于医药技术领域，所述组合物包括FXR激动剂和SUMO抑制剂。在肝星状细胞高活化状态下，FXR激动剂不具备抑制肝星状细胞活化的作用；将FXR激动剂和SUMO抑制剂复配后，在肝星状细胞高活化状态下也能够抑制其活化。伴有肝纤维化症状的个体，其肝星状细胞已处于活化态，单用FXR激动剂不能产生良好的抗纤维化作用；提供的FXR激动剂和SUMO抑制剂的复合物可以显著抑制肝星状细胞的活化，减少胶原纤维沉积，进而能够显著抗肝纤维化。

Description

一种含有SUMO抑制剂的组合物及应用
本申请要求于2018年12月14日提交中国专利局、申请号为201811534024.X、发明名称为“一种含有SUMO抑制剂的组合物及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医药技术领域，具体涉及一种含有SUMO抑制剂的组合物及应用。
背景技术
肝脏疾病尤其是病毒性肝炎、脂肪肝病、肝纤维化和肝癌等，在全球范围的发病率居高不下。如非酒精性脂肪肝在欧美普通成年人中的发病率为20～33％，肥胖人群中发病率达60～90％；在中国普通成年人患病率约为15％(Fan J G,Farrell G C.Epidemiology of non-alcoholic fatty liver disease in China.Journal of hepatology,2009,50(1):204-10.)。肝纤维化(Fibrosis)是一种病理状态，是指肝脏纤维结缔组织的过渡沉积。肝纤维化是慢性病毒性肝炎、代谢紊乱和慢性酒精性/非酒精性脂肪肝等慢性肝脏疾病进一步向肝硬化发展的中间环节，肝纤维化是慢性肝病的共同重要特征，且25～40％的慢性肝病患者最终发展为肝硬化甚至是肝癌。近几年的研究表明，肝纤维化是一个可以逆转的病变，而肝硬化是不可逆性病变。因此，抑制、阻止并逆转肝纤维化是治疗各种慢性肝病的中心环节。肝星状细胞(Hepatic Stellate Cells，HSCs)是介导肝纤维化发生发展的最主要细胞。正常肝脏中，HSCs处于静息态；当受到病理因素刺激时，HSCs活化为具有增殖能力的成纤维细胞表型，该表型的HSCs细胞可以分泌大量的纤维化因子和胶原纤维，最终引起肝纤维化和肝硬化的发生发展。因此，HSCs是肝纤维化发生发展的决定因素，抑制HSCs活化是治疗和逆转肝纤维化的重要策略。
抗肝纤维化药物的研发是当前肝药的研发热点，同时也取得了一定的研究进展。当前申报的防治肝纤维化相关的治疗药物主要有以下几类：1) 中草药及其提取物，如姜黄、白藜芦醇、水飞蓟宾、别隐品碱及其盐(授权公告号CN10132721B)；2)化学药物及其制剂，如吡非尼酮和肌酐组合物(授权公告号CN103550242B)、闭花木酮Cleistanone衍生物(授权公开号CN104095857B)；3)生物制剂，包括重组蛋白和基因药物等，前者包括TGFβ1-抑制肽(授权公告号CN1203091C)、IL-4(授权公告号CN101318013B)、单克隆抗体HAb18GC2及其重链和轻链可变去基因和多肽(授权公告号CN100586960C)；基因药物包括肝细胞核因子1α基因(授权公告号CN102552935B)、表达肝细胞生成素的基因药物(申请号200610145523.0)、人肝细胞生长因子基因(授权公告号CN1142272C)等。尽管治疗的药物很多，但是目前尚未发现对肝纤维化的特效药物。因此，寻找确切有效的抗肝纤维化药物是今后努力发展的方向。
法尼醇X受体(Farnesoid X Receptor，FXR)又称NR1H4(Nuclear Receptor Subfamily 1，Group H，Member 4)，是核受体超家族的一员。自1995年被克隆以来，该受体越来越多的功能被认识：FXR不仅在胆汁酸、脂质和糖代谢等生理过程中发挥重要作用，同时对多种病理进程具有调控作用：FXR通过FXR-FGF15/19信号通路介导了胆汁酸的促肝再生作用；FXR通过FXR/NF-κB负反馈环发挥抗炎作用(Wang Y D,Chen W D,Wang M,et al.Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response.Hepatology,2008,48(5):1632-43.)；FXR通过阻断CREB-CRTC2复合体形成并抑制下游自噬相关基因表达进而发挥抗自噬作用(Seok S,Fu T,Choi SE,et al.Transcriptional regulation of autophagy by an FXR-CREB axis.Nature 2014；516:108-U274.)；另外FXR与肝脏肿瘤的形成密切相关：FXR -/-小鼠正常饲养至15个月时全部自发性发生肝脏肿瘤，而同龄野生型小鼠未见同样变化(Yang F,Huang X,Yi T,et al.Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor.Cancer Res 2007；67:863-867.)。上述研究表明，FXR与肝脏疾病的发生发展密切相关。FXR激动剂是保肝抗纤维化药物的主要研究策略，有大量相关的专利申报(Wang H,He Q,Wang G,et al.FXR modulators for enterohepatic and metabolic diseases.Expert Opin  Ther Pat.2018 Nov；28(11):765-782)，如CN201210482982.3(申请号)提供了细格菌素类化合物及其药学上可接受的盐在制备治疗FXR介导疾病药物中的应用，CN201180067346.8和CN201080043283.8(申请号)公开了FXR活性调节剂在药物组合物中的应用，均已被授权。另外，奥贝胆酸(Obeticholic acid,OCA)作为一种强FXR激动剂，其抗NASH药效刚刚完成临床III期研究，结果显示每天服用25mg OCA的组别中有21.0％患者的纤维化症状得到改善(安慰组10.6％患者得到改善)，提示OCA对肝纤维化具有一定的改善治疗作用；但如何进一步提高其抗肝纤维化活性是一个重要的科学问题；此外作为抗原发性胆汁性肝硬化(PBC)药物已于2016年5月被美国FDA批准上市(Markham A,Keam SJ.Obeticholic Acid:First Global Approval.Drugs.2016 Aug；76(12):1221-6.)，成为以FXR为靶点成功上市的第一例药物。
前期部分动物实验结果表明，OCA等FXR激动剂对于肝纤维化具有显著的疗效，其作用机制在于FXR激动剂可以通过激动HSC细胞中FXR信号通路进而抑制HSC细胞的活化(Fiorucci S,Antonelli E,Rizzo G,et al.The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis.Gastroenterology.2004 Nov；127(5):1497-512.Fiorucci S1,Rizzo G,Antonelli E,et al.A farnesoid x receptor-small heterodimer partner regulatory cascade modulates tissue metalloproteinase inhibitor-1 and matrix metalloprotease expression in hepatic stellate cells and promotes resolution of liver fibrosis.J Pharmacol Exp Ther.2005 Aug；314(2):584-95.)；然而近期的研究发现，FXR激动剂对活化的HSC细胞的FXR信号通路没有作用，且对HSC细胞的活化没有改善作用(Kowdley KV,Luketic V,Chapman R,et al.A randomized trial of obeticholic acid monotherapy in patients with primary biliary cholangitis.Hepatology.2018 May；67(5):1890-1902.)；此外，近期的两项临床研究结果显示：与安慰剂相比较，OCA等FXR激动剂对PBC患者的肝纤维化指标并没有显著的改善作用(Nevens F,Andreone P,Mazzella G,et al.A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary Cholangitis. N Engl J Med.2016 Aug 18；375(7):631-43；Kowdley KV,Luketic V,Chapman R,et al.A randomized trial of obeticholic acid monotherapy in patients with primary biliary cholangitis.Hepatology.2018 May；67(5):1890-1902.)；对NASH患者的肝纤维化症状具有一定的改善作用，但其药效有限(改善率OCA 21.0％vs安慰剂10.6％)。
发明内容
本发明的目的在于提供一种含有SUMO抑制剂的组合物，所述组合物包括FXR激动剂和SUMO抑制剂，能够显著抑制肝星状细胞高活化，减少胶原纤维沉积，进而显著抗肝纤维化。
本发明提供了一种含有SUMO抑制剂的组合物，所述组合物包括FXR激动剂和SUMO抑制剂。
优选的，所述FXR激动剂包括但不仅限于奥贝胆酸、GW4064和WAY-362450中的一种或多种。
优选的，所述SUMO抑制剂包括但不仅限于奇霉素和/或银杏酸。
本发明还提供了上述技术方案所述的组合物在制备抗肝纤维化药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备对抗伴有肝纤维化症状的药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备抑制肝星状细胞活化药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备减少胶原纤维沉积药物中的应用。
优选的，所述药物的剂型独立的包括片剂、胶囊剂、颗粒剂、丸剂、散剂或注射剂。
本发明提供了一种含有SUMO抑制剂的组合物，所述组合物包括FXR激动剂和SUMO抑制剂。在肝星状细胞高活化状态下，FXR激动剂不具备抑制肝星状细胞活化的作用；本发明将FXR激动剂和SUMO抑制剂复配后，在高度活化的肝星状细胞中也能够激活FXR信号通路，抑制肝星状细胞的活化，减少胶原纤维沉积，进而能够抗肝纤维化。
此外，本发明的重要意义还在于：对于健康人体而言，肝星状细胞处于静息态，对FXR激动剂具有反应性；然而，对于伴有纤维化的患者 而言，肝星状细胞已处于活化态，此时该细胞对FXR激动剂的反应性降低或丧失，故而FXR激动剂不能抑制处于活化态的肝星状细胞的活化，因此当患者肝脏出现纤维化症状时，服用FXR激动剂不能产生抗肝纤维化的药理活性；本发明在阐释活化的肝星状细胞丧失对FXR激动剂反应性的机理的基础上，创新性的提出了一种组合方式，即包含SUMO抑制剂和FXR激动剂的组合物在制备抗肝纤维化药物中的应用：活化的肝星状细胞对该复合物仍具有良好的反应性，同时该复合物可以显著抑制处于活化态的肝星状细胞的活化，因此对于患有肝纤维化的患者而言，服用该组合物仍可以抑制肝星状细胞的活化，减少胶原纤维沉积，产生良好的抗肝纤维化的药理效应。因此，该组合物对于伴有纤维化症状的患者而言具有重要的实际价值和应用价值。
说明书附图
图1为静息态和活化态HSCs的FXR转录活性差异分析图，其中图1-1为：OCA对静息态(vehicle)和活化态(TGF-β)HSCs细胞Fxr的mRNA水平分析；
图1-2为：OCA对静息态(vehicle)和活化态(TGF-β)HSCs细胞Shp的mRNA水平分析；
图1-3为：GW4064对静息态(vehicle)和活化态(TGF-β)HSCs细胞Fxr的mRNA水平分析；
图1-4为：GW4064对静息态(vehicle)和活化态(TGF-β)HSCs细胞Shp的mRNA水平分析；
图1-5为WAY-362450对静息态(vehicle)和活化态(TGF-β)HSCs细胞Fxr的mRNA水平分析；
图1-6为：WAY-362450对静息态(vehicle)和活化态(TGF-β)HSCs细胞Shp的mRNA水平分析。*P<0.05，**P<0.01，***P<0.001；
图2为HSCs活化后FXR蛋白SUMO化水平分析。***P<0.001；
图3为SUMO抑制剂SP和GA对活化的HSCs中FXR蛋白SUMO化水平分析，***P<0.001，##P<0.01，###P<0.001；
图4为FXR激动剂与SUMO抑制剂联用对活化态HSCs的FXR转录活性的作用，其中图4-1为：OCA与SP联用对活化的HSCs细胞Fxr 的mRNA水平分析；
图4-2为：OCA与SP联用对活化的HSCs细胞Shp的mRNA水平分析；
图4-3为：GW4064与SP联用对活化的HSCs细胞Fxr的mRNA水平分析；
图4-4为：GW4064与SP联用对活化的HSCs细胞Shp的mRNA水平分析；
图4-5为：WAY-362450与SP联用对活化的HSCs细胞Fxr的mRNA水平分析；
图4-6为：WAY-362450与SP联用对活化的HSCs细胞Shp的mRNA水平分析；
图4-7为：OCA与GA联用对活化的HSCs细胞Fxr的mRNA水平分析；
图4-8为：OCA与GA联用对活化的HSCs细胞Shp的mRNA水平分析，ns P>0.05,*P<0.05，**P<0.01；
图5为FXR激动剂与SUMO抑制剂联用对HSCs活化的影响，其中图5-1为：OCA与SP联用对Acta-2 mRNA表达水平分析；
图5-2为：GW4064与SP联用对Acta-2 mRNA表达水平分析；
图5-3为：WAY-362450与SP联用对Acta-2 mRNA表达水平分析；
图5-4为：OCA与GA联用对Acta-2 mRNA表达水平分析，ns P>0.05,*P<0.05；
图6为FXR激动剂与SUMO抑制剂联用对CCl 4诱导的肝纤维化状态下肝脏FXR及其转录活性的作用，其中图6-1为：Fxr mRNA表达水平分析；
图6-2为：Shp mRNA表达水平分析，*P<0.05；
图7为FXR激动剂与SUMO抑制剂联用对CCl 4诱导的肝纤维化的作用，其中图7-1为：Acta-2 mRNA表达水平分析，**P<0.01；
图7-2为：肝脏Sirius Red染色。
备注：本发明的所有图中P<0.05时表示有统计学差异。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种含有SUMO抑制剂的组合物，所述组合物包括FXR激动剂和SUMO抑制剂。本发明对所述FXR激动剂和SUMO抑制剂的质量比没有特殊限定，任意质量比例皆可。
在本发明中，所述FXR激动剂优选包括但不仅限于奥贝胆酸、GW4064和WAY-362450中的一种或多种，所述多种具体为两种或两种以上。在本发明中，当所述FXR激动剂为两种或两种以上时，各组分任意质量混合。本发明对所述FXR激动剂不局限于上述3种，其来源没有特殊限定，优选包括天然来源、半合成或化学合成的各种FXR激动剂。
在本发明中，所述SUMO抑制剂优选包括但不仅限于奇霉素和/或银杏酸。在本发明中，当所述SUMO抑制剂优选包括奇霉素和银杏酸时，所述奇霉素和银杏酸以任意质量比混合。本发明对所述SUMO抑制剂不局限于上述2种，其来源没有特殊限定，优选包括天然来源、半合成或化学合成的各种SUMO抑制剂。
在本发明中，高度活化状态的肝星状细胞丧失对FXR激动剂的反应性，且FXR激动剂不具备抑制肝星状细胞活化的作用，所述SUMO抑制剂能够显著提高高度活化的肝星状细胞对FXR激动剂的反应性，且能显著增强FXR激动剂对肝星状细胞高活化的抑制作用；伴有肝纤维化症状的个体，其肝星状细胞已处于活化态，单用FXR激动剂不能产生良好的抗纤维化作用，而本发明提供的FXR激动剂和SUMO抑制剂的组合物可以显著抑制肝星状细胞的活化，减少胶原纤维沉积，进而能够抗肝纤维化。
本发明还提供了上述技术方案所述的组合物在制备抗肝纤维化药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备对抗伴有肝纤维化症状的药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备抑制肝星状细胞活化药物中的应用。
本发明还提供了上述技术方案所述的组合物在制备减少胶原纤维沉积药物中的应用。
在本发明中，所述药物的剂型独立的优选包括片剂、胶囊剂、颗粒剂、 丸剂、散剂或注射剂。本发明对上述剂型的制备方法没有特殊限定，采用常规制备相应剂型的制备方法和所用相应的辅料制备即可。
在本发明中，所述组合物的服用方法优选包括口服、静脉注射、静脉滴注、肌肉注射等，以及各种服用方式的组合。
下面结合实施例对本发明提供的一种含有SUMO抑制剂的组合物及应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
HSCs细胞活化后对FXR激动剂反应性降低原因分析
1 实验材料
本发明使用的HSC-T6细胞系购自中南大学细胞库；
本发明使用的奥贝胆酸(OCA)、GW4064和WAY-362450购自MCE公司，TGF-β重组蛋白购自R&D systems公司，逆转录试剂盒购自Applied Biosystems公司，Trizol RNAiso plus购自TAKARA公司，所用引物由Life Invitrogen公司合成，SUMO化ELISA检测试剂盒购自Epigentek公司，其余试剂均可由市售途径获得。
2 实验方法
2.1 HSC-T6细胞系培养和造模
HSC-T6细胞系培养至合适密度之后，更换含TGF-β(10mg/ml)或者OCA(5μM)或者GW40064(5μM)或者WAY-362450(5μM)的含药低血清培养基进行孵育12h后，收集细胞。
2.2 实时定量PCR
2.2.1 细胞样本总RNA提取
1)使用PBS清洗细胞后，加入800ul Trizol试剂，移液枪反复吹打后，转移至EP管中，室温静置5min。
2)加入160μl氯仿，剧烈振荡15sec，室温放置5min后12000g离心15min。样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。
3)小心转移上层水相300μl到新管中，并加入300μl异丙醇，颠倒混匀后，室温放置10min，而后12000g离心10min，弃去上清；
4)用预冷的75％乙醇1.0ml洗涤RNA沉淀，而后12000g离心5min，弃上清得到总RNA，并用10μl DEPC水复溶，定量后稀释至0.5μg/μl。
2.2.2 逆转录
按照说明书要求的体系配比将RNA溶液和试剂盒成分配制成总体积为20μl的体系并设定程序温度进行逆转录，具体配比要求如表1：
表1逆转录配比
Figure PCTCN2019120210-appb-000001
逆转录的使用条件如下：
Stage 1:逆转录37℃15min；
Stage 2:变性85℃5s。
2.2.3 PCR
PCR体系见表2：
表2 PCR体系
Figure PCTCN2019120210-appb-000002
Figure PCTCN2019120210-appb-000003
PCR使用条件如下：
Stage 1：预变性95℃1min；
Stage 2：PCR反应95℃15sec；如60℃30sec for 40 Cycles；72℃30sec；
Stage 3：融解曲线分析65～95℃，0.5℃/5s。
待测基因引物序列见表3：
表3引物序列
Figure PCTCN2019120210-appb-000004
Figure PCTCN2019120210-appb-000005
2.3 SUMO化ELISA检测试剂盒
按照ELISA检测试剂盒说明书要求提取细胞核蛋白之后，将一抗和细胞样本的核蛋白在板孔中共室温孵育60min，按照说明书要求接着补充加入SUMO特异检测抗体，进一步加入颜色增敏剂显色，之后迅速读取655nm的吸光度，进行相对定量分析。
3 实验结果
3.1 活化的HSCs对FXR激动剂反应性降低
在体外培养的HSCs细胞中，通过给予细胞TGF-β1刺激，促进HSCs细胞活化。根据实时RT-PCR的结果显示(图1)，在呈现高度活化状态的体外造模的HSC-T6细胞系中，存在FXR激动剂调控FXR下游靶基因能力减弱的现象。在静息态细胞中，FXR激动剂OCA、GW4064和WAY-362450可以显著诱导FXR靶基因Shp的mRNA水平的上调(图1-2,图1-4,图1-6)；而在高活化态下，FXR激动剂OCA、GW4064和WAY-362450均无法显著提高FXR下游靶基因Shp的mRNA水平(图1-2,图1-4,图1-6)。
3.2 活化的HSCs中FXR蛋白的SUMO化水平提高
根据针对SUMO化水平的ELISA试剂盒检测发现(图2)，在造模后高度活化的HSC细胞中的FXR蛋白SUMO化水平会随着培养时间的推进呈现逐渐提升。
实施例2
SUMO抑制剂在体外HSCs中对FXR通路的作用
1 实验材料
SUMO抑制剂奇霉素(Spectinomycin,SP)和银杏酸(Ginkgolic acid,GA)购自MCE公司。
其余实验材料同实施例1。
2 实验方法
2.1 HSC-T6细胞系培养和造模
具体方法同实施例1中2.1部分。
2.2 RT-PCR
具体方法同实施例1中2.2部分。
2.3 SUMO化ELISA检测试剂盒
具体方法同实施例1中2.3部分。
3 实验结果
3.1 SUMO抑制剂可以抑制活化的HSCs细胞中FXR蛋白的SUMO化水平
通过SUMO化ELISA试剂盒检测，发现SUMO化抑制剂SP和GA可以显著减少HSCs细胞内FXR蛋白的SUMO化水平(图3)。
3.2 SUMO抑制剂可以提高活化的HSCs细胞对FXR激动剂的反应性
在实施例1中，发现在活化的HSCs细胞中，FXR激动剂OCA、GW4064和WAY-362450对FXR信号通路的调控作用丧失，表现为对Shp基因的诱导作用丢失。在本部分的研究中，拟考察在SUMO抑制剂存在的条件下FXR激动剂对FXR信号通路的调控作用。根据RT-PCR的相对定量结果(图4)，发现在活化的HSCs细胞中，在给予SUMO抑制剂SP的条件下，FXR激动剂OCA、GW4064和WAY-36250对FXR下游靶基因Shp的诱导作用显著增强(图4-2,4-4和4-6)；此外在给予SUMO抑制剂GA的条件下，FXR激动剂OCA对Shp的诱导作用也显著增强(图4-8)。
3.3 SUMO抑制剂可以增强FXR激动剂对HSCs细胞活化的抑制作用
上述结果显示，SUMO抑制剂可以抑制活化的HSCs细胞中FXR的SUMO化水平，进而增强FXR激动剂OCA、GW4064和WAY-362450对FXR下游信号通路的调控作用。接下来，拟考察SUMO抑制剂对FXR激动剂抗HSCs活化的作用。同样根据RT-PCR的相对定量结果(图5)， 活化的HSCs细胞给予SP后，FXR激动剂OCA、GW4064和WAY36250给药组的肝星状细胞的纤维化相关基因Acta2的表达显著降低；活化的HSCs细胞给予GA后，FXR激动剂OCA可以显著抑制Acta2的表达。上述结果表明，SUMO抑制剂SP和GA可以降低FXR蛋白SUMO化水平，有利于FXR激动剂OCA、GW4064和WAY-362450调控减少纤维化相关基因的表达，从而提高抗纤维化的药效。
实施例3
SUMO抑制剂在小鼠肝纤维化状态下对FXR通路的作用
1 实验材料
实验小鼠(C57BL/6)购自扬州大学比较医学中心；
CCl 4购自上海凌峰化学试剂公司，矿物油购自Sigma-Aldrich公司。
其余实验材料同实施例1。
2 实验方法
2.1 SUMO抑制剂与OCA联用对CCl 4诱导的小鼠肝纤维化的作用。
动物适应性饲养一周后，将小鼠随机分为5组，每组8只，共计40只。分别为对照组、模型组、OCA单给药组、SP单给药组、OCA+SP联合给药组，其中模型组通过腹腔注射方式给予CCl 4(20％，溶解于矿物油)，对照组给予相应体积的溶媒(矿物油)，每周给2次，共持续4周。从造模后的第三周开始进行OCA腹腔灌胃给药，剂量为1.5mg/kg，每日一次为时两周。此外，联用SP的OCA给药组从第一周开始就进行SP的皮下注射给药，剂量为200mg/kg，每天一次。给药周期结束后处死小鼠，分离保存肝脏备用。
2.2 RT-PCR
具体同实施例1种2.2部分。
2.3 肝脏病理分析：
部分肝脏组织于4％多聚甲醛中固定后，送于武汉谷歌生物科技有限公司(武汉，中国)进行双盲分析检测，检测项目为Sirius Red染色分析。
3 实验结果
3.1 SUMO抑制剂SP增强OCA对纤维化小鼠FXR通路的调控作用
通过RT-PCR实验对肝脏组织中FXR下游基因的mRNA相对表达进行考察(图6)，发现OCA治疗性给药组小鼠的肝脏组织FXR下游基因的mRNA水平在和SP联用之后有了一定的回升。表明SUMO化抑制剂可以增强FXR激动剂OCA对纤维化小鼠的FXR通路的激动作用。
3.2 SUMO抑制剂SP可以增强OCA抗肝纤维化药效
根据RT-PCR实验对肝脏组织纤维化相关基因的表达考察以及肝脏病理分析的染色结果，发现联用SP的OCA给药组的OCA抗纤维化药效有了显著提升。RT-PCR结果显示，肝脏组织中主要纤维化基因Acta2的表达在联用组(SP+OCA)中也呈现较明显的下降，相对与单给药组的药效较为明显(图7-1)；此外，Sirius Red染色结果显示对照组肝脏只有较少的红色胶原纤维染色，而模型组以及OCA单给药组的切片中均因肝毒性而含有大量的红色胶原纤维，而采用OCA联用SP给药后，肝脏中红色胶原纤维增生减少(图7-2)。
由以上实施例可以得出，高活化状态的肝星状细胞丧失对FXR激动剂的反应性，且FXR激动剂不具备抑制肝星状细胞活化的作用；本发明将FXR激动剂和SUMO抑制剂复配后，高活化状态下的肝星状细胞对FXR激动剂恢复反应性，且能够抑制肝星状细胞的高活化作用，减少胶原纤维沉积，进而能够抗肝纤维化。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种含有SUMO抑制剂的组合物，其特征在于，所述组合物包括FXR激动剂和SUMO抑制剂。
  2. 根据权利要求1所述的组合物，其特征在于，所述FXR激动剂包括但不仅限于奥贝胆酸、GW4064和WAY-362450中的一种或多种。
  3. 根据权利要求1所述的组合物，其特征在于，所述SUMO抑制剂包括但不仅限于奇霉素和/或银杏酸。
  4. 权利要求1～3任一项所述的组合物在制备抗肝纤维化药物中的应用。
  5. 权利要求1～3任一项所述的组合物在制备对抗伴有肝纤维化症状的药物中的应用。
  6. 权利要求1～3任一项所述的组合物在制备抑制肝星状细胞活化药物中的应用。
  7. 权利要求1～3任一项所述的组合物在制备减少胶原纤维沉积药物中的应用。
  8. 根据权利要求4～7任一项所述的应用，其特征在于，所述药物的剂型独立的包括片剂、胶囊剂、颗粒剂、丸剂、散剂或注射剂。
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