WO2022178841A1 - Combination of adiponectin receptor agonist and elastin receptor inhibitor for prevention or treatment of non-alcoholic fatty liver disease - Google Patents

Combination of adiponectin receptor agonist and elastin receptor inhibitor for prevention or treatment of non-alcoholic fatty liver disease Download PDF

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WO2022178841A1
WO2022178841A1 PCT/CN2021/078192 CN2021078192W WO2022178841A1 WO 2022178841 A1 WO2022178841 A1 WO 2022178841A1 CN 2021078192 W CN2021078192 W CN 2021078192W WO 2022178841 A1 WO2022178841 A1 WO 2022178841A1
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seq
elastin
receptor agonist
adiponectin
pharmaceutical composition
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PCT/CN2021/078192
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French (fr)
Chinese (zh)
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刘琦
张斌智
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深圳市图微安创科技开发有限公司
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Priority to PCT/CN2021/078192 priority Critical patent/WO2022178841A1/en
Priority to CN202180094289.6A priority patent/CN116867796A/en
Publication of WO2022178841A1 publication Critical patent/WO2022178841A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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 belongs to the technical field of biochemistry, and particularly relates to the application of synergistic effects of both adiponectin receptor agonists and elastin receptor inhibitors in the treatment of non-alcoholic fatty liver disease and a combined pharmaceutical composition prepared by the two .
  • Non-alcoholic fatty liver disease is a clinical syndrome caused by excessive accumulation of triglycerides in the liver, which is similar to alcoholic hepatitis but without excessive drinking. It mainly includes simple steatosis, hepatitis , liver fibrosis and later liver cirrhosis and liver cancer (Ara JP. recent insigts into the pathogenosis of monalcoholic fatty liver disease. 2018. Cai J, Zhang XJ, Li H. The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis. Hepatology 2019. ). According to statistics, about 3-5% of people worldwide suffer from NAFLD, which has become another common cause of liver transplantation in the United States.
  • Polypeptide drugs generally refer to short peptides composed of 2-50 amino acids, many of which are endogenous peptides with low concentration but strong activity, which play a very important role in regulating the physiological functions of the body (Suzuki R, Brown GA, Christopher JA, Scully CCG, Congreve M. Recent Developments in Therapeutic Peptides for the Glucagon-like Peptide 1and 2Receptors. J Med Chem 2020;63:905-927).
  • Polypeptide drugs have the following advantages: first, most of them are endogenous, with clear structure and clear mechanism of action; second, the drug dose is small and the toxic and side effects are lower; third, compared with exogenous proteins, they are immunogenic It can be chemically synthesized, with high product purity and controllable quality (Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov Today 2015; 20: 122-128. Townsend SA, Newsome PN. Review article : new treatments in non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2017;46:494-507.). Therefore, for chronic diseases that require long-term medication, peptide drugs are undoubtedly the most ideal choice.
  • Adiponectin is an adipokine produced by adipocytes (Alzahrani B, Iseli T, Ramezani-Moghadam M, Ho V, Wankell M, Sun EJ, Qiao L, et al.
  • the role of AdipoR1 and AdipoR2 in liver fibrosis Biochim Biophys Acta Mol Basis Dis 2018;1864:700-708). It plays an important role in regulating the body's glucose and lipid metabolism, insulin resistance, inflammatory response, and oxidative stress by binding to receptors.
  • adiponectin is also closely related to atherosclerosis, diabetes, liver and kidney disease and other metabolic diseases (Otvos L, Jr., Knappe D, Hoffmann R, Kovalszky I, Olah J, Hewitson TD, Stawikowska R, et al. Development of second generation peptides modulating cellular adiponectin receptor responses. Front Chem 2014;2:93). Dual agonists of adiponectin receptor 1/2 were previously reported to reduce liver fibrosis by improving extracellular matrix metabolism and mitochondrial function.
  • the purpose of the present invention is to provide a combined pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, and the combined use of an adiponectin receptor agonist and an elastin receptor (ERC) inhibitor in the prevention or treatment of non-alcoholic fatty liver disease.
  • the application in liver disease, the adiponectin receptor agonist and the elastin receptor inhibitor have synergistic effect, and the combined drug has the effect of improving or treating non-alcoholic fatty liver disease, and has no toxic side effects or less toxic side effects, etc.
  • the present invention provides an application of a combination of an adiponectin receptor agonist and an elastin receptor inhibitor in the prevention or treatment of non-alcoholic fatty liver disease.
  • the amino acid sequence of the adiponectin receptor agonist is P-X2-L-Y-X5-F-X7; wherein at least one of X2, X5 and X7 is an unnatural amino acid, P is proline, L is leucine, Y is tyrosine and F is phenylalanine.
  • Amino acids other than 20 natural amino acids are collectively referred to as unnatural amino acids. Compared with natural amino acids, unnatural amino acids have the advantages of stable properties and long half-life. By introducing unnatural amino acids on the basis of existing proteins or peptides, site-specifically modified recombinant proteins or polypeptides can be obtained.
  • P-X2-L-Y-X5-F-X7 is a synthetic peptide based on the existing peptide Pep70 (structural sequence: PGLYYFD).
  • the peptide Pep70 can activate the downstream AMPK signaling pathway by binding to adiponectin receptor-1, and exert Regulation of glucose and lipid metabolism.
  • X2 in the amino acid sequence of the adiponectin receptor agonist is an unnatural amino acid.
  • X2 in the amino acid sequence of the adiponectin receptor agonist is norvaline.
  • X5 in the amino acid sequence of the adiponectin receptor agonist is tyrosine or serine.
  • X7 in the amino acid sequence of the adiponectin receptor agonist is alanine or aspartic acid.
  • the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen or acetyl.
  • the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen
  • X2 is norvaline
  • X5 is tyrosine
  • X7 is alanine
  • the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen
  • X2 is norvaline
  • X5 is tyrosine
  • X7 is aspartic acid.
  • the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen
  • X2 is norvaline
  • X5 is serine
  • X7 is aspartic acid
  • the proline in the amino acid sequence of the adiponectin receptor agonist is modified by an acetyl group
  • X2 is norvaline
  • X5 is serine
  • X7 is alanine
  • the adiponectin receptor agonist is selected from any of the following polypeptide compounds:
  • Compound A1 (SEQ ID NO.1):
  • Compound A2 (SEQ ID NO.2):
  • Compound A3 (SEQ ID NO.3):
  • Compound A4 (SEQ ID NO.4):
  • This elastin receptor inhibitor contains the parent peptide represented by the following amino acid sequence:
  • R 1 is: a lipophilic substituent or absent
  • Xa2 Val or Iva
  • Xa9 Asp or Glu
  • Xa10 Glu or Asp
  • R 1 when R 1 is a lipophilic substituent, the amino group of Val at position 1 in the amino acid sequence of the elastin receptor inhibitor is connected to the lipophilic substituent via a bridging group, and the bridging group comprises (PEG) m, Or include (PEG) m and ⁇ Glu, or include (PEG) m and Asp, and the connection mode is that the amino group of the first-position Val is modified by PEGylation of (PEG) m in the bridging group with the affinity.
  • the lipophilic substituent is CH 3 (CH 2 ) n C(O)- or HOOC(CH 2 ) n C(O)- and its acyl group is attached to the acyl group contained in the bridging group
  • the amino group forms an amide bond; wherein, m is an integer of 2-10; n is an integer of 14-20; the carboxyl terminal of the amino acid sequence has a bare carboxyl group, or is connected with an amino group to form a -CONH 2 group; the connection method is shown in the figure 10.
  • Xa2 Val or Iva
  • Xa9 Asp or Glu
  • Xa10 Glu or Asp
  • Xa14 Leu or Ala.
  • the amino acid sequence of the elastin receptor inhibitor is selected from the group consisting of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.9, SEQ ID NO.
  • the structure connected to the amino group of Val at position 1 in the amino acid sequence of the elastin receptor inhibitor is:
  • the elastin receptor inhibitor of the present invention is any of the following polypeptide compounds:
  • Compound B1 (SEQ ID NO.5):
  • Compound B3 (SEQ ID NO.7):
  • Compound B16 (SEQ ID NO. 20):
  • Compound B20 (SEQ ID NO. 24):
  • Compound B24 (SEQ ID NO. 28):
  • Nonalcoholic fatty liver disease includes nonalcoholic steatosis, nonalcoholic steatohepatitis, liver fibrosis, and cirrhosis associated with liver fibrosis.
  • the present invention does not particularly limit the order of administration of the adiponectin receptor agonist and the elastin receptor inhibitor, and the order of administration does not affect the therapeutic effect of the present invention; the present invention
  • the adiponectin receptor agonist and elastin receptor inhibitor can be administered simultaneously or sequentially; the order of sequential administration can either give the adiponectin receptor agonist first and then the elastin receptor inhibitor , or an elastin receptor inhibitor can be administered first followed by an adiponectin receptor agonist.
  • the present invention does not particularly limit the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor; preferably, the adiponectin receptor agonist and the elastin receptor inhibitor in the combined pharmaceutical composition of the present invention The concentration ratio of 10:1 to 3:1.
  • the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor in the combined pharmaceutical composition of the present invention is 6:1 to 4:1.
  • the present invention also provides a combined pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, the combined pharmaceutical composition comprising an adiponectin receptor agonist and an elastin receptor inhibitor.
  • the combined pharmaceutical composition of the present invention may be a single compound preparation, or may be a combination of two separate preparations, an adiponectin receptor agonist and an elastin receptor inhibitor, and the two separate preparations may be administered simultaneously or sequentially apply.
  • the combined pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the combined pharmaceutical composition of the present invention is suitable for various administration modes, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration and the like.
  • the pharmaceutical compositions of the present invention can be formulated into various suitable dosage forms.
  • Suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for application to the skin, aerosols, nasal sprays, and sterile injectable solutions.
  • the pharmaceutical composition of the present invention can be prepared as a solution or lyophilized powder for parenteral administration, and the powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier before use.
  • the liquid formulation is generally a buffer solution, etc. Osmotic solutions and aqueous solutions.
  • the present invention confirms that the adiponectin receptor agonist and the elastin receptor inhibitor have a synergistic effect, and the combination of the adiponectin receptor agonist and the elastin receptor inhibitor has a good effect. Amelioration or treatment of nonalcoholic fatty liver disease.
  • the present invention adopts the combined medication of adiponectin receptor agonist and elastin receptor inhibitor, and in the process of improving or treating non-alcoholic fatty liver disease, the adiponectin receptor agonist itself can significantly reduce the accumulation of extracellular matrix Furthermore, fibrosis can be improved, and the production of EDPs caused by the degradation of extracellular matrix by adiponectin receptor agonists can also be blocked by elastin receptor inhibitors. This synergistic effect greatly optimizes the drug effect.
  • the combined administration of the adiponectin receptor agonist and the elastin receptor inhibitor of the present invention has the effects of improving lipid metabolism, inflammation, accumulation of extracellular matrix and the like.
  • adiponectin receptor agonists and elastin receptor inhibitors not only exerts the advantages that adiponectin receptor agonists themselves can improve NAFLD, but also overcomes its drug side effects and achieves the purpose of low toxicity and long-term medication .
  • the present invention has been proved by a large number of experiments that the therapeutic effect of the combined use of the adiponectin receptor agonist and the elastin receptor inhibitor is far greater than that of the single use of the adiponectin receptor agonist or the elastin receptor inhibitor.
  • both adiponectin receptor agonists and elastin receptor inhibitors belong to polypeptide compounds, which have lower toxicity compared with some small molecules, have a larger safety window, and have the characteristics of no toxic side effects or less toxic side effects.
  • Figure 1 is a bar graph of the QPCR results of the extracellular matrix ⁇ -SMA in MCD-induced nonalcoholic steatohepatitis mice after administration.
  • Figure 2 H&E stained sections of MCD-induced nonalcoholic steatohepatitis mice after administration.
  • Figure 3 is a bar graph of the positive area of H&E staining in MCD-induced nonalcoholic steatohepatitis mice after administration.
  • Fig. 4 shows the CD68 immunostaining section of MCD-induced nonalcoholic steatohepatitis mice after administration.
  • Figure 5 is a bar graph of the positive area of CD68 immunostaining in MCD-induced nonalcoholic steatohepatitis mice after administration.
  • Figure 6 is a bar graph of the QPCR results of extracellular matrix ⁇ -SMA in mice with CCl 4 -induced liver fibrosis after administration.
  • Figure 7 H&E stained sections of mice with CCl 4 -induced liver fibrosis after administration.
  • Fig. 8 is a section diagram of Sirius red staining of mice with CCl 4 -induced liver fibrosis after administration.
  • Fig. 9 is a bar graph showing the positive area of Sirius red staining in mice with CCl 4 -induced liver fibrosis after administration.
  • Figure 10 is a schematic diagram showing the connection between the bridging group and the peptide chain in the elastin receptor inhibitor of the present invention.
  • the compound is synthesized from the carboxyl terminus to the amino terminus by the synthetic method of Fmoc solid-phase polypeptide, and is connected in sequence according to the above amino acid sequence sequence.
  • the indene detection resin becomes colorless, add piperidine to remove the protective agent, and carry out indene detection detection, if the protective group is removed, proceed to the next step in turn.
  • Tested drugs Adiponectin receptor agonists A1, A3 and elastin receptor inhibitors B1, B17, B22 were randomly selected and stored at -80°C.
  • mice Male C57BL/6J mice were provided by the Experimental Animal Center of Sun Yat-sen University, and the mice weighed 18-22 g. They were randomly divided into 3 groups of 78 mice, and each group was further randomly divided into 13 groups, each group is:
  • Control group (MCS) + normal saline, intraperitoneal injection, n 6;
  • Model group (MCD) + Compound A3 + Compound B1, intraperitoneal injection, n 6;
  • the administration concentration ratios of adiponectin receptor agonists (compounds A1, A3) and elastin receptor inhibitors (compounds B1, B17, B22) in the three groups were 3:1 (300 ⁇ g/kg: 100 ⁇ g/kg), 5:1 (500 ⁇ g/kg: 100 ⁇ g/kg) and 10:1 (1000 ⁇ g/kg: 100 ⁇ g/kg), adiponectin receptor agonists (compounds A1, A3) administered once daily, elastic Protein receptor inhibitors (compounds B1, B17, B22) were administered once every three days, and mice were sacrificed after 6 weeks of administration; MCD (methionine-choline deficient feed) and MCS feed were purchased from Medison, Jiangsu Biopharmaceutical Co., Ltd.
  • Hematoxylin-eosin (H&E) staining Take paraffin-embedded tissue sections and bake at 60°C for 1 h. Dewaxing and hydration: xylene for 20 minutes ⁇ xylene for 20 minutes ⁇ absolute alcohol for 15 minutes ⁇ absolute alcohol for 15 minutes ⁇ 95% alcohol for 10 minutes ⁇ 90% alcohol for 5 minutes ⁇ 80% alcohol for 5 minutes. Staining: hematoxylin for 7 minutes ⁇ rinse with tap water ⁇ differentiate with 1% hydrochloric acid and ethanol for 1 s ⁇ rinse with tap water ⁇ eosin staining for 15s-20s ⁇ rinse with tap water.
  • Immunohistochemistry drying, dewaxing, and soaking in double-distilled water for 5 minutes.
  • mice In the MCD-induced mouse steatosis model, it is characterized by massive accumulation of triglycerides around the central venous area, and massive inflammatory cell infiltration in the portal area and hepatic interlobular septa in the liver 6 weeks after induction. Three weeks after administration, the mice were anesthetized, and blood was collected from the retrobulbar venous plexus for serological assays. Liver samples were taken in which liver lobules were formalin-fixed and used for pathological analysis.
  • Figure 1 is a bar graph of the QPCR results of extracellular matrix ⁇ -SMA in mice with MCD-induced nonalcoholic steatohepatitis after administration. It can be seen from the results in Figure 1 that the combined use of any two drugs randomly selected in the present invention, compounds A1, A3 and compounds B1, B17 and B22, has a significant improvement effect on MCD-induced nonalcoholic steatohepatitis, and The improvement effect was better than that of single compound A1, A3 or single compound B1, B17, B22 administration.
  • adiponectin receptor agonist and elastin receptor inhibitor of the present invention has obvious improvement effect on nonalcoholic steatohepatitis, and the effect is far better than that of single use of adiponectin receptor agonist or elastin receptor inhibitor. Elastin receptor inhibitors.
  • Figure 2 is the H&E stained section diagram of MCD-induced non-alcoholic steatohepatitis mice after administration;
  • Figure 3 is a histogram of the positive area of H&E staining in MCD-induced non-alcoholic steatohepatitis mice after administration.
  • Figure 4 is a section diagram of CD68 immunostaining in MCD-induced non-alcoholic steatohepatitis mice after administration
  • Figure 5 is a bar graph of CD68 immunostaining positive area in MCD-induced non-alcoholic steatohepatitis mice after administration.
  • the combined use of an adiponectin receptor agonist and an elastin receptor inhibitor significantly reduced lipid accumulation. Since excessive accumulation of lipids leads to oxidative stress and inflammation, we further examined inflammation-related expressions. The results show that the combined use of adiponectin receptor agonists and elastin receptor inhibitors can significantly improve the infiltration of inflammatory cells and reduce the occurrence of inflammation.
  • Example 3 Improved therapeutic effect of combined action of adiponectin receptor agonist and elastin receptor inhibitor on CCl4 -induced liver fibrosis.
  • Tested drugs Adiponectin receptor agonists A1, A3 and elastin receptor inhibitors B1, B17, B22 were randomly selected and stored at -80°C.
  • mice Male C57BL/6J mice were provided by the Experimental Animal Center of Sun Yat-sen University, and the mice weighed 18-22 g. They were randomly divided into 3 groups of 78 mice, and each group was further randomly divided into 13 groups, each group is:
  • Control group (Oil) + normal saline, intraperitoneal injection, n 6;
  • the administration concentration ratios of adiponectin receptor agonists (compounds A1, A3) and elastin receptor inhibitors (compounds B1, B17, B22) in the three groups were 3:1 (300 ⁇ g/kg: 100 ⁇ g/kg), 5:1 (500 ⁇ g/kg: 100 ⁇ g/kg) and 10:1 (1000 ⁇ g/kg: 100 ⁇ g/kg), adiponectin receptor agonists (compounds A1, A3) administered once daily, elastic Protein receptor inhibitors (compounds B1, B17, B22) were administered once every three days, and the mice were sacrificed after 6 weeks of administration; CCl 4 and Oil were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
  • H&E staining drying slides, dewaxing; hematoxylin staining for 7 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid and ethanol for 1 s, rinsed with tap water, stained with eosin, rinsed with tap water, dehydrated and transparent, air-dried, and sealed with gum.
  • Sirius red staining bake the slides for 30 minutes, dewax, stand in double-distilled water for 5 minutes, stain with Sirius red for 60-80 minutes in a dark room, wash 5 times with 0.5% glacial acetic acid, and seal the slides in xylene.
  • liver fibrosis model In the CCl 4 -induced liver fibrosis model, it is characterized by infiltration of inflammatory cells around the central venous area, and deposition of a large number of collagen fibers in the portal area and hepatic lobular septa.
  • Figure 6 is a bar graph of the QPCR results of extracellular matrix ⁇ -SMA in mice with CCl 4 -induced liver fibrosis after administration. It can be seen from the results in Figure 6 that the combined use of any two drugs randomly selected in the present invention, compounds A1, A3 and compounds B1, B17, and B22, has a significant improvement effect on CCl4 -induced liver fibrosis, and both The improvement effect is better than that of compound A1, A3 alone or compound B1, B17, B22 alone.
  • adiponectin receptor agonist and elastin receptor inhibitor of the present invention has obvious improvement effect on liver fibrosis, and the effect is far better than that of single use of adiponectin receptor agonist or elastin receptor inhibitors.
  • Fig. 7 is a section view of H&E staining after administration of CCl 4 -induced liver fibrosis in mice.
  • inflammatory cell infiltration and cell necrosis occurred in the liver of mice 6 weeks after induction with CCl 4 , which were improved after administration.
  • the combined use of an adiponectin receptor agonist and an elastin receptor inhibitor can significantly reduce inflammatory cell infiltration around the central venous area, and the improvement effect of an adiponectin receptor agonist or an elastin receptor inhibitor alone Not significant.
  • Fig. 8 is a section diagram of Sirius red staining of mice with CCl 4 -induced hepatic fibrosis after administration
  • Fig. 9 is a bar graph of the positive area of Sirius red staining in mice with CCl 4 -induced hepatic fibrosis after administration.
  • Sirius red staining was performed, and the staining results were shown in Figure 8; the positive area in the Sirius red staining section was calculated and a histogram was made, and the results were shown in Figure 9.
  • Sirius red is a strong basic dye, which is easy to combine with basic groups in collagen molecules, so Sirius red can reflect the amount of collagen deposition. It can be seen from the results in Figures 8 to 9 that after drug treatment, collagen deposition was improved to varying degrees.
  • the combined use of adiponectin receptor agonists and elastin receptor inhibitors has less collagen deposition in the liver, indicating that the combined use of adiponectin receptor agonists and elastin receptor inhibitors improves the effect of liver fibrosis.
  • the effect is significantly better than that of adiponectin receptor agonists or elastin receptor inhibitors alone.
  • the combination of the adiponectin receptor agonist and the elastin receptor inhibitor of the present invention has achieved unexpected effects, and the adiponectin receptor agonist and the elastin receptor inhibitor act synergistically during the treatment process.
  • the effect of combined use of adiponectin receptor agonists and elastin receptor inhibitors is far superior to that of adiponectin receptor agonists or elastin receptor inhibitors alone.
  • the combined use of an adiponectin receptor agonist and an elastin receptor inhibitor in the present invention has a significant therapeutic effect on non-alcoholic fatty liver disease.

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Abstract

Disclosed is an application of a combination of an adiponectin receptor agonist and an elastin receptor inhibitor in the prevention or treatment of non-alcoholic fatty liver disease.

Description

脂联素受体激动剂和弹性蛋白受体抑制剂联合用药预防或治疗非酒精性脂肪肝病Combined use of adiponectin receptor agonists and elastin receptor inhibitors to prevent or treat nonalcoholic fatty liver disease 技术领域technical field
本发明属于生物化学技术领域，具体地涉及到脂联素受体激动剂和弹性蛋白受体抑制剂两者协同作用治疗非酒精性脂肪肝病中的应用及由两者制备的联合用药物组合物。The invention belongs to the technical field of biochemistry, and particularly relates to the application of synergistic effects of both adiponectin receptor agonists and elastin receptor inhibitors in the treatment of non-alcoholic fatty liver disease and a combined pharmaceutical composition prepared by the two .
背景技术Background technique
非酒精性脂肪性肝病(NAFLD)是一种由过量的甘油三酯积累于肝脏导致的一种病理变化与酒精性肝炎相似但无过量饮酒的临床综合症，其主要包括简单的脂肪变性、肝炎、肝纤维化以及后期的肝硬化和肝癌(Ara JP.recent insigts into the pathogenosis of monalcoholic fatty liver disease.2018。Cai J,Zhang XJ,Li H.The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis.Hepatology 2019.)。据统计，全球大约有3-5％的人患有NAFLD，已经成为了美国肝移植的又一大常见因素。另外，肝脏疾病导致的心血管疾病的发病率和死亡率较正常人群也不断的增加(Tilg H,Moschen AR,Roden M.NAFLD and diabetes mellitus.Nat Rev Gastroenterol Hepatol 2017；14:32-42。Meex RCR,Watt MJ.Hepatokines:linking nonalcoholic fatty liver disease and insulin resistance.Nat Rev Endocrinol 2017；13:509-520。Wree A,Broderick L,Canbay A,Hoffman HM,Feldstein AE.From NAFLD to NASH to cirrhosis-new insights into disease mechanisms.Nat Rev Gastroenterol Hepatol 2013；10:627-636.)。所以，日益增长的NAFLD发病率势必会造成医疗资源的进一步紧张和消耗。但是治疗NAFLD的药物开发并不是一帆风顺。2020年5月，Genfit公司宣布elafibranor在对2期或3期的纤维化的NASH患者的III期研究中未达到主要终点。就连研究最快、最有希望的Ocaliva上市要求也遭到了惨拒，所以目前该领域亟需新的有效治疗药物(Intercept Receives Complete Response Letter from FDA for Obeticholic Acid for the Treatment of Fibrosis Due to NASH.pdf)。双靶点药物、多靶点药物以及协同用药逐渐成为了人们研究的热点和重点。因为肝脏疾病的发病机理较为复杂，涉及到多个代谢相关因素。此外，NAFLD发病较为缓慢， 药物的治疗周期较长，单个药物靶点的药物很可能存在靶点的过度抑制情况，产生毒副作用，所以寻求更加安全、有效的治疗策略成为了一项艰巨的科学任务。Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome caused by excessive accumulation of triglycerides in the liver, which is similar to alcoholic hepatitis but without excessive drinking. It mainly includes simple steatosis, hepatitis , liver fibrosis and later liver cirrhosis and liver cancer (Ara JP. recent insigts into the pathogenosis of monalcoholic fatty liver disease. 2018. Cai J, Zhang XJ, Li H. The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis. Hepatology 2019. ). According to statistics, about 3-5% of people worldwide suffer from NAFLD, which has become another common cause of liver transplantation in the United States. In addition, the morbidity and mortality of cardiovascular disease caused by liver disease are also increasing compared with the normal population (Tilg H, Moschen AR, Roden M. NAFLD and diabetes mellitus. Nat Rev Gastroenterol Hepatol 2017;14:32-42. Meex RCR, Watt MJ. Hepatokines: linking nonalcoholic fatty liver disease and insulin resistance. Nat Rev Endocrinol 2017;13:509-520. Wree A, Broderick L, Canbay A, Hoffman HM, Feldstein AE. From NAFLD to NASH to cirrhosis-new insights into disease mechanisms. Nat Rev Gastroenterol Hepatol 2013;10:627-636.). Therefore, the increasing incidence of NAFLD is bound to cause further strain and consumption of medical resources. But drug development to treat NAFLD has not been smooth sailing. In May 2020, Genfit announced that elafibranor failed to meet the primary endpoint in a Phase III study of NASH patients with Phase 2 or 3 fibrosis. Even the fastest and most promising Ocaliva marketing request has been rejected, so there is an urgent need for new effective treatments in the field (Intercept Receives Complete Response Letter from FDA for Obeticholic Acid for the Treatment of Fibrosis Due to NASH. pdf). Dual-target drugs, multi-target drugs and synergistic drugs have gradually become the focus and focus of research. Because the pathogenesis of liver disease is complex, it involves multiple metabolic-related factors. In addition, the onset of NAFLD is relatively slow, and the treatment cycle of drugs is long. Drugs with a single drug target are likely to have excessive inhibition of the target, resulting in toxic side effects. Therefore, it has become a difficult science to seek safer and more effective treatment strategies. Task.
多肽药物一般是指2-50个氨基酸组成的短肽，很多都是内源性多肽，浓度低，但是活性强，在调节机体生理功能时发挥着非常重要的作用(Suzuki R,Brown GA,Christopher JA,Scully CCG,Congreve M.Recent Developments in Therapeutic Peptides for the Glucagon-like Peptide 1and 2Receptors.J Med Chem 2020；63:905-927)。多肽药物具有以下优点：第一，它们大多数是内源性的，结构清楚，作用机制明确；第二，用药剂量小、毒副作用更低；第三，它们与外源蛋白质相比，免疫原较低，而且可以化学合成，产品纯度高，质量可控(Fosgerau K,Hoffmann T.Peptide therapeutics:current status and future directions.Drug Discov Today 2015；20:122-128。Townsend SA,Newsome PN.Review article:new treatments in non-alcoholic fatty liver disease.Aliment Pharmacol Ther 2017；46:494-507.)。因此对于需要长期用药的慢性疾病，多肽药物无疑是最理想的选择。Polypeptide drugs generally refer to short peptides composed of 2-50 amino acids, many of which are endogenous peptides with low concentration but strong activity, which play a very important role in regulating the physiological functions of the body (Suzuki R, Brown GA, Christopher JA, Scully CCG, Congreve M. Recent Developments in Therapeutic Peptides for the Glucagon-like Peptide 1and 2Receptors. J Med Chem 2020;63:905-927). Polypeptide drugs have the following advantages: first, most of them are endogenous, with clear structure and clear mechanism of action; second, the drug dose is small and the toxic and side effects are lower; third, compared with exogenous proteins, they are immunogenic It can be chemically synthesized, with high product purity and controllable quality (Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov Today 2015; 20: 122-128. Townsend SA, Newsome PN. Review article : new treatments in non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2017;46:494-507.). Therefore, for chronic diseases that require long-term medication, peptide drugs are undoubtedly the most ideal choice.
脂联素是由脂肪细胞产生的一种脂肪细胞因子(Alzahrani B,Iseli T,Ramezani-Moghadam M,Ho V,Wankell M,Sun EJ,Qiao L,et al.The role of AdipoR1 and AdipoR2 in liver fibrosis.Biochim Biophys Acta Mol Basis Dis 2018；1864:700-708)。它通过与受体结合，对机体的糖脂代谢、胰岛素抵抗、炎症反应、氧化应激等发挥着重要的调节作用。此外，脂联素还与动脉粥样硬化、糖尿病、肝肾病等代谢疾病关系密切(Otvos L,Jr.,Knappe D,Hoffmann R,Kovalszky I,Olah J,Hewitson TD,Stawikowska R,et al.Development of second generation peptides modulating cellular adiponectin receptor responses.Front Chem 2014；2:93)。先前合成报道过脂联素受体1/2的双激动剂可以通过改善胞外基质的代谢和线粒体功能来减轻肝脏的纤维化。但是随着深入的研究发现胞外基质的代谢或降解具有一定的毒副作用，因为疾病状态下的胞外基质中含有大量的弹性蛋白，弹性蛋白的降解会产生一系列的具有不同的氨基酸序列的衍生多肽，它们可以通过激活下游通路进而调节一系列的生物活性。Adiponectin is an adipokine produced by adipocytes (Alzahrani B, Iseli T, Ramezani-Moghadam M, Ho V, Wankell M, Sun EJ, Qiao L, et al. The role of AdipoR1 and AdipoR2 in liver fibrosis . Biochim Biophys Acta Mol Basis Dis 2018;1864:700-708). It plays an important role in regulating the body's glucose and lipid metabolism, insulin resistance, inflammatory response, and oxidative stress by binding to receptors. In addition, adiponectin is also closely related to atherosclerosis, diabetes, liver and kidney disease and other metabolic diseases (Otvos L, Jr., Knappe D, Hoffmann R, Kovalszky I, Olah J, Hewitson TD, Stawikowska R, et al. Development of second generation peptides modulating cellular adiponectin receptor responses. Front Chem 2014;2:93). Dual agonists of adiponectin receptor 1/2 were previously reported to reduce liver fibrosis by improving extracellular matrix metabolism and mitochondrial function. However, with in-depth research, it is found that the metabolism or degradation of the extracellular matrix has certain toxic and side effects, because the extracellular matrix in the disease state contains a large amount of elastin, and the degradation of elastin will produce a series of different amino acid sequences. Derivative polypeptides that can modulate a range of biological activities by activating downstream pathways.
因此如何能在治疗NAFLD的同时而又不产生其他毒副作用是现阶段亟需解决的问题。Therefore, how to treat NAFLD without producing other toxic and side effects is an urgent problem to be solved at this stage.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种预防或治疗非酒精性脂肪肝病的联合用药物组合物以及脂联素受体激动剂和弹性蛋白受体(ERC)抑制剂联合用药在预防或治疗非酒精性脂肪肝病中的应用，该脂联素受体激动剂和弹性蛋白受体抑制剂之间具有协同作用，同时联合用药具有改善或治疗非酒精性脂肪肝病的效果，具有无毒副作用或毒副作用小等特点。The purpose of the present invention is to provide a combined pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, and the combined use of an adiponectin receptor agonist and an elastin receptor (ERC) inhibitor in the prevention or treatment of non-alcoholic fatty liver disease. The application in liver disease, the adiponectin receptor agonist and the elastin receptor inhibitor have synergistic effect, and the combined drug has the effect of improving or treating non-alcoholic fatty liver disease, and has no toxic side effects or less toxic side effects, etc. Features.
为达上述目的，本发明提供一种脂联素受体激动剂和弹性蛋白受体抑制剂联合用药在预防或治疗非酒精性脂肪肝病中的应用。In order to achieve the above object, the present invention provides an application of a combination of an adiponectin receptor agonist and an elastin receptor inhibitor in the prevention or treatment of non-alcoholic fatty liver disease.
该脂联素受体激动剂的氨基酸序列为P-X2-L-Y-X5-F-X7；其中X2、X5及X7中至少一个为非天然氨基酸，P为脯氨酸，L为亮氨酸，Y为酪氨酸，F为苯丙氨酸。氨基酸中20种天然氨基酸以外的氨基酸统称为非天然氨基酸，非天然氨基酸相比天然氨基酸具有性质稳定、半衰期长的优点。在现有蛋白或肽的基础上引入非天然氨基酸，能够得到位点特异性修饰的重组蛋白或多肽。P-X2-L-Y-X5-F-X7是以现有多肽Pep70(结构序列：PGLYYFD)为基础的合成肽，多肽Pep70能够通过结合脂联素受体-1，激活下游的AMPK信号通路，发挥调节糖脂代谢的作用。The amino acid sequence of the adiponectin receptor agonist is P-X2-L-Y-X5-F-X7; wherein at least one of X2, X5 and X7 is an unnatural amino acid, P is proline, L is leucine, Y is tyrosine and F is phenylalanine. Amino acids other than 20 natural amino acids are collectively referred to as unnatural amino acids. Compared with natural amino acids, unnatural amino acids have the advantages of stable properties and long half-life. By introducing unnatural amino acids on the basis of existing proteins or peptides, site-specifically modified recombinant proteins or polypeptides can be obtained. P-X2-L-Y-X5-F-X7 is a synthetic peptide based on the existing peptide Pep70 (structural sequence: PGLYYFD). The peptide Pep70 can activate the downstream AMPK signaling pathway by binding to adiponectin receptor-1, and exert Regulation of glucose and lipid metabolism.
优选的，脂联素受体激动剂氨基酸序列中的X2为非天然氨基酸。Preferably, X2 in the amino acid sequence of the adiponectin receptor agonist is an unnatural amino acid.
优选的，脂联素受体激动剂氨基酸序列中的X2是正缬氨酸。Preferably, X2 in the amino acid sequence of the adiponectin receptor agonist is norvaline.
优选的，脂联素受体激动剂氨基酸序列中的X5为酪氨酸或丝氨酸。Preferably, X5 in the amino acid sequence of the adiponectin receptor agonist is tyrosine or serine.
优选的，脂联素受体激动剂氨基酸序列中的X7为丙氨酸或天冬氨酸。Preferably, X7 in the amino acid sequence of the adiponectin receptor agonist is alanine or aspartic acid.
优选的，脂联素受体激动剂氨基酸序列中的脯氨酸由氢或乙酰基修饰。Preferably, the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen or acetyl.
优选的，脂联素受体激动剂氨基酸序列中的脯氨酸由氢修饰，X2是正缬氨酸，X5为酪氨酸，X7为丙氨酸。Preferably, the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen, X2 is norvaline, X5 is tyrosine, and X7 is alanine.
优选的，脂联素受体激动剂氨基酸序列中的脯氨酸由氢修饰，X2是正缬氨酸，X5为酪氨酸，X7为天冬氨酸。Preferably, the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen, X2 is norvaline, X5 is tyrosine, and X7 is aspartic acid.
优选的，脂联素受体激动剂氨基酸序列中的脯氨酸由氢修饰，X2是正缬氨酸，X5为丝氨酸，X7为天冬氨酸。Preferably, the proline in the amino acid sequence of the adiponectin receptor agonist is modified with hydrogen, X2 is norvaline, X5 is serine, and X7 is aspartic acid.
优选的，脂联素受体激动剂氨基酸序列中的脯氨酸由乙酰基修饰，X2是正缬氨酸，X5为丝氨酸，X7为丙氨酸。Preferably, the proline in the amino acid sequence of the adiponectin receptor agonist is modified by an acetyl group, X2 is norvaline, X5 is serine, and X7 is alanine.
最优选的，脂联素受体激动剂选自如下任一种多肽化合物：Most preferably, the adiponectin receptor agonist is selected from any of the following polypeptide compounds:
化合物A1(SEQ ID NO.1)：Compound A1 (SEQ ID NO.1):
P(Nva)LYYFAP(Nva)LYYFA
化合物A2(SEQ ID NO.2)：Compound A2 (SEQ ID NO.2):
P(Nva)LYYFDP(Nva)LYYFD
化合物A3(SEQ ID NO.3)：Compound A3 (SEQ ID NO.3):
P(NVa)LYSFDP(NVa)LYSFD
化合物A4(SEQ ID NO.4)：Compound A4 (SEQ ID NO.4):
P(NVa)LYSFA。P(NVa)LYSFA.
该弹性蛋白受体抑制剂含有以下氨基酸序列表示的母体肽：This elastin receptor inhibitor contains the parent peptide represented by the following amino acid sequence:
R 1-Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14， R 1 -Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14,
其中，in,
R 1为：亲脂性取代基或不存在； R 1 is: a lipophilic substituent or absent;
Xa2＝Val或Iva；Xa2=Val or Iva;
Xa9＝Asp或Glu；Xa9=Asp or Glu;
Xa10＝Glu或Asp；Xa10=Glu or Asp;
Xa14＝Leu或Ala；Xa14=Leu or Ala;
当R 1不存在，Xa2＝Val，且Xa14＝Leu时，母体肽为非直链肽。 When R1 is absent, Xa2 = Val, and Xa14=Leu, the parent peptide is a non-linear peptide.
其中，当R 1为亲脂性取代基时，弹性蛋白受体抑制剂的氨基酸序列中第1位Val的氨基经由桥接基团与亲脂性取代基连接，所述桥接基团包含(PEG) m、或者包含(PEG) m和γGlu、或者包含(PEG) m和Asp，所述连接方式为所述第1位Val的氨基通过所述桥接基团中(PEG) m的聚乙二醇化修饰与亲脂性取代基相连；并且，所述亲脂性取代基为CH 3(CH 2) nC(O)-或HOOC(CH 2) nC(O)-且其酰基与所述桥接基团中包含的氨基形成酰胺键；其中，m为2-10的整数；n为14-20的整数；所述氨基酸序列的羧基端裸露羧基，或连接有氨基形成-CONH 2基团；所述连接方式参见图10。 Wherein, when R 1 is a lipophilic substituent, the amino group of Val at position 1 in the amino acid sequence of the elastin receptor inhibitor is connected to the lipophilic substituent via a bridging group, and the bridging group comprises (PEG) m, Or include (PEG) m and γGlu, or include (PEG) m and Asp, and the connection mode is that the amino group of the first-position Val is modified by PEGylation of (PEG) m in the bridging group with the affinity. and the lipophilic substituent is CH 3 (CH 2 ) n C(O)- or HOOC(CH 2 ) n C(O)- and its acyl group is attached to the acyl group contained in the bridging group The amino group forms an amide bond; wherein, m is an integer of 2-10; n is an integer of 14-20; the carboxyl terminal of the amino acid sequence has a bare carboxyl group, or is connected with an amino group to form a -CONH 2 group; the connection method is shown in the figure 10.
其中，当R 1不存在时，弹性蛋白受体抑制剂的氨基酸序列中，第1位Val的氨基与第14位氨基酸的羧基通过形成酰胺键连接形成环肽化合物，其中所述的环肽化合物结构表示如下： Wherein, when R 1 is absent, in the amino acid sequence of the elastin receptor inhibitor, the amino group of the 1st Val and the carboxyl group of the 14th amino acid are connected by forming an amide bond to form a cyclic peptide compound, wherein the cyclic peptide compound The structure is represented as follows:
Figure PCTCN2021078192-appb-000001
Figure PCTCN2021078192-appb-000001
Xa2＝Val或Iva；Xa2=Val or Iva;
Xa9＝Asp或Glu；Xa9=Asp or Glu;
Xa10＝Glu或Asp；Xa10=Glu or Asp;
Xa14＝Leu或Ala。Xa14=Leu or Ala.
根据本发明的具体实施方式，弹性蛋白受体抑制剂的氨基酸序列选自SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29所示的氨基酸序列其中之一。According to a specific embodiment of the present invention, the amino acid sequence of the elastin receptor inhibitor is selected from the group consisting of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.9, SEQ ID NO. ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO .18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 One of the amino acid sequences shown in , SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29.
根据本发明的具体实施方式，弹性蛋白受体抑制剂的氨基酸序列的第1位Val的氨基所连接的结构为：According to a specific embodiment of the present invention, the structure connected to the amino group of Val at position 1 in the amino acid sequence of the elastin receptor inhibitor is:
Figure PCTCN2021078192-appb-000002
Figure PCTCN2021078192-appb-000002
Figure PCTCN2021078192-appb-000003
Figure PCTCN2021078192-appb-000003
优选的，本发明的弹性蛋白受体抑制剂为如下任一种多肽化合物：Preferably, the elastin receptor inhibitor of the present invention is any of the following polypeptide compounds:
化合物B1(SEQ ID NO.5)：Compound B1 (SEQ ID NO.5):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Leu-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Leu-NH 2
化合物B2(SEQ ID NO.6)：Compound B2 (SEQ ID NO. 6):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B3(SEQ ID NO.7)：Compound B3 (SEQ ID NO.7):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Leu-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Leu-NH 2
化合物B4(SEQ ID NO.8)：Compound B4 (SEQ ID NO. 8):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Leu-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Leu-NH 2
化合物B5(SEQ ID NO.9)：Compound B5 (SEQ ID NO. 9):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B6(SEQ ID NO.10)：Compound B6 (SEQ ID NO. 10):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala
化合物B7(SEQ ID NO.11)：Compound B7 (SEQ ID NO. 11):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B8(SEQ ID NO.12)：Compound B8 (SEQ ID NO. 12):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala
化合物B9(SEQ ID NO.13)：Compound B9 (SEQ ID NO. 13):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Ala-NH 2
化合物B10(SEQ ID NO.14)：Compound B10 (SEQ ID NO. 14):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Ala-NH 2
化合物B11(SEQ ID NO.15)：Compound B11 (SEQ ID NO. 15):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Leu-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Leu-NH 2
化合物B12(SEQ ID NO.16)：Compound B12 (SEQ ID NO. 16):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Leu-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Val-Val-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Asp-Ala-Ser-Pro-Leu-NH 2
化合物B13(SEQ ID NO.17)：Compound B13 (SEQ ID NO. 17):
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B14(SEQ ID NO.18)：Compound B14 (SEQ ID NO. 18):
(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B15(SEQ ID NO.19)：Compound B15 (SEQ ID NO. 19):
(PEG 2-PEG 2-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B16(SEQ ID NO.20)：Compound B16 (SEQ ID NO. 20):
(PEG 2-PEG 2-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2 (PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala-NH 2
化合物B17(SEQ ID NO.21)：Compound B17 (SEQ ID NO. 21):
Figure PCTCN2021078192-appb-000004
Figure PCTCN2021078192-appb-000004
化合物B18(SEQ ID NO.22)：Compound B18 (SEQ ID NO. 22):
Figure PCTCN2021078192-appb-000005
Figure PCTCN2021078192-appb-000005
化合物B19(SEQ ID NO.23)：Compound B19 (SEQ ID NO. 23):
Figure PCTCN2021078192-appb-000006
Figure PCTCN2021078192-appb-000006
化合物B20(SEQ ID NO.24)：Compound B20 (SEQ ID NO. 24):
Figure PCTCN2021078192-appb-000007
Figure PCTCN2021078192-appb-000007
化合物B21(SEQ ID NO.25)：Compound B21 (SEQ ID NO. 25):
Figure PCTCN2021078192-appb-000008
Figure PCTCN2021078192-appb-000008
化合物B22(SEQ ID NO.26)：Compound B22 (SEQ ID NO. 26):
Figure PCTCN2021078192-appb-000009
Figure PCTCN2021078192-appb-000009
化合物B23(SEQ ID NO.27)：Compound B23 (SEQ ID NO. 27):
Figure PCTCN2021078192-appb-000010
Figure PCTCN2021078192-appb-000010
化合物B24(SEQ ID NO.28)：Compound B24 (SEQ ID NO. 28):
Figure PCTCN2021078192-appb-000011
Figure PCTCN2021078192-appb-000011
化合物B25(SEQ ID NO.29)：Compound B25 (SEQ ID NO. 29):
Figure PCTCN2021078192-appb-000012
Figure PCTCN2021078192-appb-000012
非酒精性脂肪肝病包括非酒精性脂肪样变性、非酒精性脂肪肝炎、肝纤维化以及肝纤维化合并的肝硬化。Nonalcoholic fatty liver disease includes nonalcoholic steatosis, nonalcoholic steatohepatitis, liver fibrosis, and cirrhosis associated with liver fibrosis.
在预防或治疗非酒精性脂肪肝病时，本发明并不特别限制脂联素受体激动剂和弹性蛋白受体抑制剂的给药顺序，给药顺序并不影响本发明的治疗效果；本发明的脂联素受体激动剂和弹性蛋白受体抑制剂可以同时给药，也可以依次给药；依次给药的顺序既可以先给予脂联素受体激动剂再给予弹性蛋白受体抑制剂，也可以先给予弹性蛋白受体抑制剂再给予脂联素受体激动剂。In the prevention or treatment of non-alcoholic fatty liver disease, the present invention does not particularly limit the order of administration of the adiponectin receptor agonist and the elastin receptor inhibitor, and the order of administration does not affect the therapeutic effect of the present invention; the present invention The adiponectin receptor agonist and elastin receptor inhibitor can be administered simultaneously or sequentially; the order of sequential administration can either give the adiponectin receptor agonist first and then the elastin receptor inhibitor , or an elastin receptor inhibitor can be administered first followed by an adiponectin receptor agonist.
本发明并不特别限定脂联素受体激动剂和弹性蛋白受体抑制剂的浓度比；优选地，本发明的联合用药物组合物中脂联素受体激动剂和弹性蛋白受体抑制剂的浓度比为10：1至3：1。The present invention does not particularly limit the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor; preferably, the adiponectin receptor agonist and the elastin receptor inhibitor in the combined pharmaceutical composition of the present invention The concentration ratio of 10:1 to 3:1.
更优选地，本发明的联合用药物组合物中脂联素受体激动剂和弹性蛋白受体抑制剂的浓度比为6：1至4：1。More preferably, the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor in the combined pharmaceutical composition of the present invention is 6:1 to 4:1.
本发明还提供一种预防或治疗非酒精性脂肪肝病的联合用药物组合物，该联合用药物组合物包括脂联素受体激动剂和弹性蛋白受体抑制剂。The present invention also provides a combined pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, the combined pharmaceutical composition comprising an adiponectin receptor agonist and an elastin receptor inhibitor.
本发明的联合用药物组合物可以为单一的复方制剂，也可以为脂联素受体激动剂和弹性蛋白受体抑制剂两种单独制剂的组合，两种单独制剂可同时施用，也可依次施用。The combined pharmaceutical composition of the present invention may be a single compound preparation, or may be a combination of two separate preparations, an adiponectin receptor agonist and an elastin receptor inhibitor, and the two separate preparations may be administered simultaneously or sequentially apply.
本发明的联合用药物组合物还包含药学上可接受的载体或辅料。The combined pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or adjuvant.
本发明的联合用药物组合物适用于各种给药方式，例如口服给药、经皮给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。根据所采用的给药方式，可将本发明的药物组合物制成各种合适的剂型。The combined pharmaceutical composition of the present invention is suitable for various administration modes, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration and the like. Depending on the mode of administration employed, the pharmaceutical compositions of the present invention can be formulated into various suitable dosage forms.
适当剂型的实例为片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆、用于皮肤表面的油膏和药贴、气雾剂、鼻喷剂以及可用于注射的无菌溶液。Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for application to the skin, aerosols, nasal sprays, and sterile injectable solutions.
本发明的药物组合物可以制成溶液或者冻干粉以用于胃肠外给药，在使用前可加入适当溶剂或其他可药用的载体将粉末重新配制，液体配方一般是缓冲 液、等渗溶液和水溶液。The pharmaceutical composition of the present invention can be prepared as a solution or lyophilized powder for parenteral administration, and the powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier before use. The liquid formulation is generally a buffer solution, etc. Osmotic solutions and aqueous solutions.
本发明通过一系列的动物实验证实了脂联素受体激动剂和弹性蛋白受体抑制剂之间具有协同作用，同时脂联素受体激动剂和弹性蛋白受体抑制剂联合用药具有良好的改善或治疗非酒精性脂肪肝病的效果。Through a series of animal experiments, the present invention confirms that the adiponectin receptor agonist and the elastin receptor inhibitor have a synergistic effect, and the combination of the adiponectin receptor agonist and the elastin receptor inhibitor has a good effect. Amelioration or treatment of nonalcoholic fatty liver disease.
本发明采用脂联素受体激动剂和弹性蛋白受体抑制剂联合用药，在改善或治疗非酒精性脂肪肝病的过程中，脂联素受体激动剂本身可以显著的降低胞外基质的积累进而改善纤维化，同时脂联素受体激动剂降解胞外基质导致的EDPs的产生也能够被弹性蛋白受体抑制剂阻断，这样的协同作用极大的优化了药物效果。本发明脂联素受体激动剂和弹性蛋白受体抑制剂联合用药具有改善脂质代谢、炎症以及胞外基质的积累等效果。The present invention adopts the combined medication of adiponectin receptor agonist and elastin receptor inhibitor, and in the process of improving or treating non-alcoholic fatty liver disease, the adiponectin receptor agonist itself can significantly reduce the accumulation of extracellular matrix Furthermore, fibrosis can be improved, and the production of EDPs caused by the degradation of extracellular matrix by adiponectin receptor agonists can also be blocked by elastin receptor inhibitors. This synergistic effect greatly optimizes the drug effect. The combined administration of the adiponectin receptor agonist and the elastin receptor inhibitor of the present invention has the effects of improving lipid metabolism, inflammation, accumulation of extracellular matrix and the like.
脂联素受体激动剂和弹性蛋白受体抑制剂的联合使用不仅发挥了脂联素受体激动剂本身可以改善NAFLD的优势，并且还克服了其药物副作用，达到低毒，长久用药的目的。同时本发明经大量实验证实联合使用脂联素受体激动剂和弹性蛋白受体抑制剂的治疗效果远远大于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。The combined use of adiponectin receptor agonists and elastin receptor inhibitors not only exerts the advantages that adiponectin receptor agonists themselves can improve NAFLD, but also overcomes its drug side effects and achieves the purpose of low toxicity and long-term medication . At the same time, the present invention has been proved by a large number of experiments that the therapeutic effect of the combined use of the adiponectin receptor agonist and the elastin receptor inhibitor is far greater than that of the single use of the adiponectin receptor agonist or the elastin receptor inhibitor.
此外脂联素受体激动剂和弹性蛋白受体抑制剂都属于多肽化合物，与一些小分子相比具有较低的毒性，安全窗口更大，具有无毒副作用或毒副作用小等特点。In addition, both adiponectin receptor agonists and elastin receptor inhibitors belong to polypeptide compounds, which have lower toxicity compared with some small molecules, have a larger safety window, and have the characteristics of no toxic side effects or less toxic side effects.
附图说明Description of drawings
图1：为MCD诱导的非酒精性脂肪肝炎小鼠经给药后胞外基质α-SMA的QPCR结果柱状图。Figure 1 is a bar graph of the QPCR results of the extracellular matrix α-SMA in MCD-induced nonalcoholic steatohepatitis mice after administration.
图2：为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的H&E染色切片图。Figure 2: H&E stained sections of MCD-induced nonalcoholic steatohepatitis mice after administration.
图3：为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的H&E染色阳性面积柱状图。Figure 3 is a bar graph of the positive area of H&E staining in MCD-induced nonalcoholic steatohepatitis mice after administration.
图4：为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的CD68免疫染色切片图。Fig. 4 shows the CD68 immunostaining section of MCD-induced nonalcoholic steatohepatitis mice after administration.
图5：为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的CD68免疫染色阳性面积柱状图。Figure 5 is a bar graph of the positive area of CD68 immunostaining in MCD-induced nonalcoholic steatohepatitis mice after administration.
图6：为CCl 4诱导的肝纤维化小鼠经给药后胞外基质α-SMA的QPCR结果柱状图。 Figure 6 is a bar graph of the QPCR results of extracellular matrix α-SMA in mice with CCl 4 -induced liver fibrosis after administration.
图7：为CCl 4诱导的肝纤维化小鼠经给药后的H&E染色切片图。 Figure 7: H&E stained sections of mice with CCl 4 -induced liver fibrosis after administration.
图8：为CCl 4诱导的肝纤维化小鼠经给药后的天狼星红染色切片图。 Fig. 8 is a section diagram of Sirius red staining of mice with CCl 4 -induced liver fibrosis after administration.
图9：为CCl 4诱导的肝纤维化小鼠经给药后的天狼星红染色阳性面积柱状图。 Fig. 9 is a bar graph showing the positive area of Sirius red staining in mice with CCl 4 -induced liver fibrosis after administration.
图10：为本发明弹性蛋白受体抑制剂中桥接基团与肽链的连接示意图。Figure 10 is a schematic diagram showing the connection between the bridging group and the peptide chain in the elastin receptor inhibitor of the present invention.
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。除非另有说明，否则所用试剂或仪器均可以通过市购获得。The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. Unless otherwise stated, all reagents or instruments used are commercially available.
实例1、多肽化合物的合成Example 1. Synthesis of polypeptide compounds
1.材料：1.Material:
所有的氨基酸都购于NovaBiocH&Em公司。如果没有特别说明，其他所有试剂均为分析纯，购自Sigma公司。利用PH&Enomenex Luna C18制备柱(46mm x 250mm)用来纯化多肽。质谱分析采用Agilent质谱仪进行测定。All amino acids were purchased from NovaBioc H&Em. Unless otherwise specified, all other reagents were of analytical grade and were purchased from Sigma. A PH&Enomenex Luna C18 preparative column (46mm x 250mm) was used to purify the polypeptide. Mass spectrometry was measured using an Agilent mass spectrometer.
2.实验方法：2. Experimental method:
化合物采用Fmoc固相多肽的合成法，由羧基端向氨基端方向合成，按照上述氨基酸序列顺序依次连接。具体步骤如下：取Rink MBHA resin(1g，1.0eq，loading＝0.643mmol/g)置于多肽合成器中，加入5mL DCM，振摇30min活化树脂。加入20％的哌啶进行检测，树脂变蓝。将Fomc保护的氨基酸加入反应器中，通入氩气，反应2h。加入茚检试剂进行检测，茚检树脂变无色，加入哌啶脱掉保护剂，进行茚检检测，如果保护基脱掉，则依次进行下一步反应。其中茚检试剂的使用量为试剂1：试剂2：试剂3＝l：2：l(滴)。如此重复操作，直至合肽完成。最后利用冰乙醚洗涤沉淀切割后的多肽，然后进行高效液相色谱纯化。The compound is synthesized from the carboxyl terminus to the amino terminus by the synthetic method of Fmoc solid-phase polypeptide, and is connected in sequence according to the above amino acid sequence sequence. The specific steps are as follows: take Rink MBHA resin (1g, 1.0eq, loading=0.643mmol/g) and place it in a peptide synthesizer, add 5mL of DCM, and shake for 30min to activate the resin. 20% piperidine was added for detection and the resin turned blue. The Fomc-protected amino acid was added to the reactor, and argon was passed through to react for 2 h. Add the indene detection reagent for detection, the indene detection resin becomes colorless, add piperidine to remove the protective agent, and carry out indene detection detection, if the protective group is removed, proceed to the next step in turn. The usage amount of the indene detection reagent is reagent 1: reagent 2: reagent 3 = 1: 2: 1 (drops). This operation is repeated until the peptide synthesis is completed. Finally, the cleaved polypeptide was washed and precipitated with glacial ether, and then purified by high performance liquid chromatography.
实施列2、脂联素受体激动剂和弹性蛋白受体抑制剂联合作用对MCD诱 导的非酒精性脂肪肝炎的改善治疗作用。Column 2. Improved therapeutic effect of combined action of adiponectin receptor agonist and elastin receptor inhibitor on MCD-induced nonalcoholic steatohepatitis.
1.模型的建立：1. Model establishment:
受试药物：随机选取脂联素受体激动剂A1、A3和弹性蛋白受体抑制剂B1、B17、B22，保存条件-80℃。Tested drugs: Adiponectin receptor agonists A1, A3 and elastin receptor inhibitors B1, B17, B22 were randomly selected and stored at -80°C.
造模方法：由中山大学实验动物中心提供雄性C57BL/6J小鼠，小鼠体重18-22g，随机分成3个群组，每一群组78只小鼠，并将每一群组进一步随机分成13小组，各小组分别为：Modeling method: Male C57BL/6J mice were provided by the Experimental Animal Center of Sun Yat-sen University, and the mice weighed 18-22 g. They were randomly divided into 3 groups of 78 mice, and each group was further randomly divided into 13 groups, each group is:
1)对照组(MCS)+生理盐水，腹腔注射，n＝6；1) Control group (MCS) + normal saline, intraperitoneal injection, n=6;
2)模型组(MCD)+生理盐水，腹腔注射，n＝6；2) Model group (MCD) + normal saline, intraperitoneal injection, n=6;
3)模型组(MCD)+化合物A1，腹腔注射，n＝6；3) Model group (MCD) + compound A1, intraperitoneal injection, n=6;
4)模型组(MCD)+化合物A3，腹腔注射，n＝6；4) Model group (MCD) + compound A3, intraperitoneal injection, n=6;
5)模型组(MCD)+化合物B1，腹腔注射，n＝6；5) Model group (MCD) + compound B1, intraperitoneal injection, n=6;
6)模型组(MCD)+化合物B17，腹腔注射，n＝6；6) Model group (MCD) + compound B17, intraperitoneal injection, n=6;
7)模型组(MCD)+化合物B22，腹腔注射，n＝6；7) Model group (MCD) + compound B22, intraperitoneal injection, n=6;
8)模型组(MCD)+化合物A1+化合物B1，腹腔注射，n＝6；8) Model group (MCD) + Compound A1 + Compound B1, intraperitoneal injection, n=6;
9)模型组(MCD)+化合物A1+化合物B17，腹腔注射，n＝6；9) Model group (MCD) + Compound A1 + Compound B17, intraperitoneal injection, n=6;
10)模型组(MCD)+化合物A1+化合物B22，腹腔注射，n＝6；10) Model group (MCD) + Compound A1 + Compound B22, intraperitoneal injection, n=6;
11)模型组(MCD)+化合物A3+化合物B1，腹腔注射，n＝6；11) Model group (MCD) + Compound A3 + Compound B1, intraperitoneal injection, n=6;
12)模型组(MCD)+化合物A3+化合物B17，腹腔注射，n＝6；12) Model group (MCD) + Compound A3 + Compound B17, intraperitoneal injection, n=6;
13)模型组(MCD)+化合物A3+化合物B22，腹腔注射，n＝6。13) Model group (MCD)+Compound A3+Compound B22, intraperitoneal injection, n=6.
其中，3个群组中脂联素受体激动剂(化合物A1、A3)与弹性蛋白受体抑制剂(化合物B1、B17、B22)的给药浓度比分别为3:1(300μg/kg：100μg/kg)、5:1(500μg/kg：100μg/kg)和10:1(1000μg/kg：100μg/kg)，脂联素受体激动剂(化合物A1、A3)每天给药一次，弹性蛋白受体抑制剂(化合物B1、B17、B22)每三天给药一次，给药6周后处死小鼠；MCD(蛋氨酸-胆碱缺乏的饲料)和MCS饲料均购自于江苏美迪森生物医药有限公司。Among them, the administration concentration ratios of adiponectin receptor agonists (compounds A1, A3) and elastin receptor inhibitors (compounds B1, B17, B22) in the three groups were 3:1 (300 μg/kg: 100 μg/kg), 5:1 (500 μg/kg: 100 μg/kg) and 10:1 (1000 μg/kg: 100 μg/kg), adiponectin receptor agonists (compounds A1, A3) administered once daily, elastic Protein receptor inhibitors (compounds B1, B17, B22) were administered once every three days, and mice were sacrificed after 6 weeks of administration; MCD (methionine-choline deficient feed) and MCS feed were purchased from Medison, Jiangsu Biopharmaceutical Co., Ltd.
2.实验方法：2. Experimental method:
胞外基质蛋白α-SMA的QPCR实验：称取25mg组织，加入1mL Trizol，机械研磨，静置5分钟，加入1/5体积的氯仿，剧烈震荡15s，静置10分钟，12000rpm离心15分钟。吸取上清液，加入异丙醇沉淀RNA，静置10分钟， 12000rpm离心10分钟，弃上清。取沉淀物，加入75％的乙醇洗涤2次，7500rpm离心5分钟，弃上清。测蛋白浓度根据试剂盒的步骤对所提取的RNA进行反转和后续实验，试剂盒均购自于北京全式金生物技术公司。QPCR experiment of extracellular matrix protein α-SMA: Weigh 25 mg of tissue, add 1 mL of Trizol, grind mechanically, let stand for 5 minutes, add 1/5 volume of chloroform, shake vigorously for 15 s, let stand for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes. Aspirate the supernatant, add isopropanol to precipitate RNA, let stand for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant. The precipitate was taken, washed twice with 75% ethanol, centrifuged at 7500 rpm for 5 minutes, and the supernatant was discarded. To measure the protein concentration, the extracted RNA was reversed and the subsequent experiments were carried out according to the steps of the kit. The kits were purchased from Beijing Quanshijin Biotechnology Co., Ltd.
苏木素-伊红(H&E)染色：取石蜡包埋的组织切片，60℃烘1h。脱蜡水化：二甲苯20分钟→二甲苯20分钟→无水酒精15分钟→无水酒精15分钟→95％酒精10分钟→90％酒精5分钟→80％酒精5分钟。染色：苏木精7分钟→自来水冲洗干净→1％盐酸乙醇分化1s→自来水冲洗→伊红染色15s-20s→自来水冲洗。脱水透明：75％酒精1s→85％酒精1s→95％酒精1s→100％酒精1s→二甲苯1s→二甲苯1s。封片晾干30分钟，树胶封片。其中H&E染色液购自于上海生工生物工程有限公司。Hematoxylin-eosin (H&E) staining: Take paraffin-embedded tissue sections and bake at 60°C for 1 h. Dewaxing and hydration: xylene for 20 minutes → xylene for 20 minutes → absolute alcohol for 15 minutes → absolute alcohol for 15 minutes → 95% alcohol for 10 minutes → 90% alcohol for 5 minutes → 80% alcohol for 5 minutes. Staining: hematoxylin for 7 minutes → rinse with tap water → differentiate with 1% hydrochloric acid and ethanol for 1 s → rinse with tap water → eosin staining for 15s-20s → rinse with tap water. Dehydration and transparency: 75% alcohol for 1s→85% alcohol for 1s→95% alcohol for 1s→100% alcohol for 1s→xylene for 1s→xylene for 1s. The coverslips were air-dried for 30 minutes, and the coverslips were sealed with gum. The H&E staining solution was purchased from Shanghai Sangon Bioengineering Co., Ltd.
免疫组化：烘片、脱蜡，之后放入双蒸水中浸泡5分钟。抗原修复：将放病理切片的架子放于盛有柠檬酸的缓冲液(PH＝6.0)烧杯中，高温高压15分钟；取出烧杯，静置在室温，待温度下降至室温，然后取出片子，放入3％双氧水中10分钟，以阻断内源性过氧化物酶活性，之后用PBS洗涤3次，每次5分。利用1％BSA对组织进行封闭1h。去除1％BSA，按说明书推荐比例滴加抗体于组织上，4℃过夜；第二天取出病理切片，待恢复室温之后加入连有辣根过氧化物酶的二抗，37℃孵育60min，然后用DAB(二氨基联苯胺)(TH&Ermo Scientific，USA)显色；用苏木素染液复染，用自来水冲洗5min，1％盐酸乙醇分化，再用自来水洗5min，脱水，风干，封片，拍照。其中CD68抗体购自于CST公司。Immunohistochemistry: drying, dewaxing, and soaking in double-distilled water for 5 minutes. Antigen retrieval: put the rack for pathological slices in a beaker containing citric acid buffer (PH=6.0), high temperature and high pressure for 15 minutes; take out the beaker, let it stand at room temperature, wait for the temperature to drop to room temperature, then take out the slices, put Endogenous peroxidase activity was blocked by immersion in 3% hydrogen peroxide for 10 minutes, followed by three washes with PBS for 5 minutes each. Tissues were blocked with 1% BSA for 1 h. Remove 1% BSA, drop the antibody on the tissue according to the proportion recommended in the instructions, overnight at 4°C; take out the pathological section the next day, add the secondary antibody with horseradish peroxidase after returning to room temperature, incubate at 37°C for 60 min, and then DAB (diaminobenzidine) (TH&Ermo Scientific, USA) was used for color development; counterstained with hematoxylin staining solution, rinsed with tap water for 5 min, differentiated with 1% hydrochloric acid ethanol, washed with tap water for 5 min, dehydrated, air-dried, mounted, and photographed. The CD68 antibody was purchased from CST Company.
3.药效评价：3. Efficacy evaluation:
在MCD诱导的小鼠脂肪变性模型中，其表现特征为：中央静脉区域周围出现大量的甘油三酯积累，且诱导6周之后，肝脏中汇管区及肝小叶间隔有大量的炎症细胞浸润。给药三周之后，小鼠进行麻醉，并通过眼球后静脉丛取血进行血清学指标的检测。肝脏样本取其中肝脏小叶福尔马林固定并用于病理学分析。In the MCD-induced mouse steatosis model, it is characterized by massive accumulation of triglycerides around the central venous area, and massive inflammatory cell infiltration in the portal area and hepatic interlobular septa in the liver 6 weeks after induction. Three weeks after administration, the mice were anesthetized, and blood was collected from the retrobulbar venous plexus for serological assays. Liver samples were taken in which liver lobules were formalin-fixed and used for pathological analysis.
图1为MCD诱导的非酒精性脂肪肝炎小鼠经给药后胞外基质α-SMA的QPCR结果柱状图。从图1的结果可以看出，本发明随机选用的化合物A1、A3和化合物B1、B17、B22的任何两个药物的联合使用对于MCD诱导的非酒精性脂肪肝炎都具有明显的改善效果，且改善效果均优于单独化合物A1、 A3或单独化合物B1、B17、B22给药。由此可以证明本发明联合使用脂联素受体激动剂和弹性蛋白受体抑制剂对于非酒精性脂肪肝炎具有明显的改善效果，且效果远远优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。Figure 1 is a bar graph of the QPCR results of extracellular matrix α-SMA in mice with MCD-induced nonalcoholic steatohepatitis after administration. It can be seen from the results in Figure 1 that the combined use of any two drugs randomly selected in the present invention, compounds A1, A3 and compounds B1, B17 and B22, has a significant improvement effect on MCD-induced nonalcoholic steatohepatitis, and The improvement effect was better than that of single compound A1, A3 or single compound B1, B17, B22 administration. Therefore, it can be proved that the combined use of adiponectin receptor agonist and elastin receptor inhibitor of the present invention has obvious improvement effect on nonalcoholic steatohepatitis, and the effect is far better than that of single use of adiponectin receptor agonist or elastin receptor inhibitor. Elastin receptor inhibitors.
图2为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的H&E染色切片图；图3为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的H&E染色阳性面积柱状图。选取MCS组、MCD组、MCD+化合物A1组、MCD组+化合物B22组、MCD组+化合物A3组、MCD组+化合物B1组、MCD组+化合物A1+化合物B22组、MCD组+化合物A3+化合物B1组进行H&E染色，染色结果如图2所示；计算H&E染色切片图中的阳性面积并制作柱状图，结果如图3所示。从图2和图3的结果可以看出，在MCD诱导6周后，小鼠肝脏中出现大量的脂质积累，给予药物治疗后，可以明显的看出小鼠肝脏中脂质积累得到了不同程度的改善，且联合使用脂联素受体激动剂和弹性蛋白受体抑制剂的效果明显优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。Figure 2 is the H&E stained section diagram of MCD-induced non-alcoholic steatohepatitis mice after administration; Figure 3 is a histogram of the positive area of H&E staining in MCD-induced non-alcoholic steatohepatitis mice after administration. MCS group, MCD group, MCD+Compound A1 group, MCD group+Compound B22 group, MCD group+Compound A3 group, MCD group+Compound B1 group, MCD group+Compound A1+Compound B22 group, MCD group+Compound A3+Compound B1 group H&E staining was performed, and the staining results were shown in Figure 2; the positive area in the H&E staining section map was calculated and a histogram was made, and the results were shown in Figure 3. From the results in Figure 2 and Figure 3, it can be seen that after 6 weeks of MCD induction, a large amount of lipid accumulation occurs in the liver of the mice. After drug treatment, it can be clearly seen that the lipid accumulation in the liver of the mice is different. The degree of improvement, and the combined use of adiponectin receptor agonists and elastin receptor inhibitors is significantly better than the use of adiponectin receptor agonists or elastin receptor inhibitors alone.
图4为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的CD68免疫染色切片图；图5为MCD诱导的非酒精性脂肪肝炎小鼠经给药后的CD68免疫染色阳性面积柱状图。从图4至图5的结果可以看出，联合使用脂联素受体激动剂和弹性蛋白受体抑制剂明显的减少了脂质的积累。因为脂质的过度累计导致氧化应激以及炎症的发生，所以我们进一步检测了炎症相关表达。结果显示联合使用脂联素受体激动剂和弹性蛋白受体抑制剂能显著地改善炎症细胞的浸润，减少炎症的发生。Figure 4 is a section diagram of CD68 immunostaining in MCD-induced non-alcoholic steatohepatitis mice after administration; Figure 5 is a bar graph of CD68 immunostaining positive area in MCD-induced non-alcoholic steatohepatitis mice after administration. As can be seen from the results in Figures 4 to 5, the combined use of an adiponectin receptor agonist and an elastin receptor inhibitor significantly reduced lipid accumulation. Since excessive accumulation of lipids leads to oxidative stress and inflammation, we further examined inflammation-related expressions. The results show that the combined use of adiponectin receptor agonists and elastin receptor inhibitors can significantly improve the infiltration of inflammatory cells and reduce the occurrence of inflammation.
综合图1至图5胞外基质蛋白α-SMA的QPCR结果、H&E染色和CD68免疫染色结果可以明显看出，MCD造模6周后小鼠肝脏出现大量的空泡样变性并伴随着炎症细胞的浸润。给药治疗之后有不同程度的改善，联合使用脂联素受体激动剂和弹性蛋白受体抑制剂的效果远远优于单独使用联合使用脂联素受体激动剂或弹性蛋白受体抑制剂。Combining the QPCR results, H&E staining and CD68 immunostaining results of the extracellular matrix protein α-SMA from Figure 1 to Figure 5, it can be clearly seen that a large number of vacuolar degeneration and inflammatory cells appeared in the liver of mice 6 weeks after MCD modeling. infiltration. There are different degrees of improvement after administration and treatment. The effect of combined use of adiponectin receptor agonists and elastin receptor inhibitors is far better than that of combined use of adiponectin receptor agonists or elastin receptor inhibitors alone .
实施例3、脂联素受体激动剂和弹性蛋白受体抑制剂联合作用对CCl 4诱导的肝纤维化的改善治疗作用。 Example 3. Improved therapeutic effect of combined action of adiponectin receptor agonist and elastin receptor inhibitor on CCl4 -induced liver fibrosis.
1.模型的建立：1. Model establishment:
受试药物：随机选取脂联素受体激动剂A1、A3和弹性蛋白受体抑制剂 B1、B17、B22，保存条件-80℃。Tested drugs: Adiponectin receptor agonists A1, A3 and elastin receptor inhibitors B1, B17, B22 were randomly selected and stored at -80°C.
造模方法：由中山大学实验动物中心提供雄性C57BL/6J小鼠，小鼠体重18-22g，随机分成3个群组，每一群组78只小鼠，并将每一群组进一步随机分成13小组，各小组分别为：Modeling method: Male C57BL/6J mice were provided by the Experimental Animal Center of Sun Yat-sen University, and the mice weighed 18-22 g. They were randomly divided into 3 groups of 78 mice, and each group was further randomly divided into 13 groups, each group is:
1)对照组(Oil)+生理盐水，腹腔注射，n＝6；1) Control group (Oil) + normal saline, intraperitoneal injection, n=6;
2)模型组(CCl 4)+生理盐水，腹腔注射，n＝6； 2) Model group (CCl 4 ) + physiological saline, intraperitoneal injection, n=6;
3)模型组(CCl 4)+500μg/kg化合物A1，腹腔注射，n＝6； 3) Model group (CCl 4 )+500 μg/kg compound A1, intraperitoneal injection, n=6;
4)模型组(CCl 4)+500μg/kg化合物A3，腹腔注射，n＝6； 4) Model group (CCl 4 )+500 μg/kg compound A3, intraperitoneal injection, n=6;
5)模型组(CCl 4)+100μg/kg化合物B1，腹腔注射，n＝6； 5) Model group (CCl 4 )+100 μg/kg compound B1, intraperitoneal injection, n=6;
6)模型组(CCl 4)+100μg/kg化合物B17，腹腔注射，n＝6； 6) Model group (CCl 4 )+100 μg/kg compound B17, intraperitoneal injection, n=6;
7)模型组(CCl 4)+100μg/kg化合物B22，腹腔注射，n＝6； 7) Model group (CCl 4 )+100 μg/kg compound B22, intraperitoneal injection, n=6;
8)模型组(CCl 4)+500μg/kg化合物A1+100μg/kg化合物B1，腹腔注射，n＝6； 8) Model group (CCl 4 )+500 μg/kg Compound A1+100 μg/kg Compound B1, intraperitoneal injection, n=6;
9)模型组(CCl 4)+500μg/kg化合物A1+100μg/kg化合物B17，腹腔注射，n＝6； 9) Model group (CCl 4 )+500 μg/kg Compound A1+100 μg/kg Compound B17, intraperitoneal injection, n=6;
10)模型组(CCl 4)+500μg/kg化合物A1+100μg/kg化合物B22，腹腔注射，n＝6； 10) Model group (CCl 4 )+500 μg/kg Compound A1+100 μg/kg Compound B22, intraperitoneal injection, n=6;
11)模型组(CCl 4)+500μg/kg化合物A3+100μg/kg化合物B1，腹腔注射，n＝6； 11) Model group (CCl 4 )+500 μg/kg Compound A3+100 μg/kg Compound B1, intraperitoneal injection, n=6;
12)模型组(CCl 4)+500μg/kg化合物A3+100μg/kg化合物B17，腹腔注射，n＝6； 12) Model group (CCl 4 )+500 μg/kg Compound A3+100 μg/kg Compound B17, intraperitoneal injection, n=6;
13)模型组(CCl 4)+500μg/kg化合物A3+100μg/kg化合物B22，腹腔注射，n＝6。 13) Model group (CCl 4 )+500 μg/kg Compound A3+100 μg/kg Compound B22, intraperitoneal injection, n=6.
其中，3个群组中脂联素受体激动剂(化合物A1、A3)与弹性蛋白受体抑制剂(化合物B1、B17、B22)的给药浓度比分别为3:1(300μg/kg：100μg/kg)、5:1(500μg/kg：100μg/kg)和10:1(1000μg/kg：100μg/kg)，脂联素受体激动剂(化合物A1、A3)每天给药一次，弹性蛋白受体抑制剂(化合物B1、B17、B22)每三天给药一次，给药6周后处死小鼠；CCl 4和Oil均购自于上海阿拉丁生化科技股份有限公司。 Among them, the administration concentration ratios of adiponectin receptor agonists (compounds A1, A3) and elastin receptor inhibitors (compounds B1, B17, B22) in the three groups were 3:1 (300 μg/kg: 100 μg/kg), 5:1 (500 μg/kg: 100 μg/kg) and 10:1 (1000 μg/kg: 100 μg/kg), adiponectin receptor agonists (compounds A1, A3) administered once daily, elastic Protein receptor inhibitors (compounds B1, B17, B22) were administered once every three days, and the mice were sacrificed after 6 weeks of administration; CCl 4 and Oil were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
2.实验方法：2. Experimental method:
胞外基质蛋白α-SMA的QPCR实验：称取25mg组织，加入1mL Trizol，机械研磨，静置5分钟，加入1/5体积的氯仿，剧烈震荡15s，静置10分钟，12000rpm离心15分钟。吸取上清液，加入异丙醇沉淀RNA，静置10分钟，12000rpm离心10分钟，弃上清。取沉淀物，加入75％的乙醇洗涤2次，7500rpm离心5分钟，弃上清。测蛋白浓度后续根据试剂盒的步骤对所提取的RNA进行反转和后续实验，试剂盒均购自于北京全式金生物技术公司。QPCR experiment of extracellular matrix protein α-SMA: Weigh 25 mg of tissue, add 1 mL of Trizol, grind mechanically, let stand for 5 minutes, add 1/5 volume of chloroform, shake vigorously for 15 s, let stand for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes. Aspirate the supernatant, add isopropanol to precipitate RNA, let stand for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant. The precipitate was taken, washed twice with 75% ethanol, centrifuged at 7500 rpm for 5 minutes, and the supernatant was discarded. After measuring the protein concentration, the extracted RNA was reversed and the subsequent experiments were carried out according to the steps of the kit. The kits were purchased from Beijing Quanshijin Biotechnology Co., Ltd.
H&E染色：烘片、脱蜡；苏木素染色7分钟、自来水冲洗干净、1％盐酸乙醇分化1s、自来水冲洗、伊红染色、自来水冲洗、脱水透明、晾干、树胶封片。H&E staining: drying slides, dewaxing; hematoxylin staining for 7 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid and ethanol for 1 s, rinsed with tap water, stained with eosin, rinsed with tap water, dehydrated and transparent, air-dried, and sealed with gum.
天狼星红染色：烘片30分钟、脱蜡、双蒸水中静置5分钟、暗室里天狼星红染色60-80分钟、0.5％冰醋酸洗5下、最后在二甲苯中，未干封片。Sirius red staining: bake the slides for 30 minutes, dewax, stand in double-distilled water for 5 minutes, stain with Sirius red for 60-80 minutes in a dark room, wash 5 times with 0.5% glacial acetic acid, and seal the slides in xylene.
3.药效评价：3. Efficacy evaluation:
在CCl 4诱导的肝脏纤维化模型中，其表现特征为：中央静脉区域周围有炎性细胞浸润，以及汇管区及肝小叶间隔有大量胶原纤维沉积等。 In the CCl 4 -induced liver fibrosis model, it is characterized by infiltration of inflammatory cells around the central venous area, and deposition of a large number of collagen fibers in the portal area and hepatic lobular septa.
图6为CCl 4诱导的肝纤维化小鼠经给药后胞外基质α-SMA的QPCR结果柱状图。从图6的结果可以看出，本发明随机选用的化合物A1、A3和化合物B1、B17、B22的任何两个药物的联合使用对于CCl 4诱导的肝纤维化都具有明显的改善效果，且均改善效果均优于单独使用化合物A1、A3或单独使用化合物B1、B17、B22。由此可以证明本发明联合使用脂联素受体激动剂和弹性蛋白受体抑制剂对于肝纤维化具有明显的改善效果，且效果远远优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。 Figure 6 is a bar graph of the QPCR results of extracellular matrix α-SMA in mice with CCl 4 -induced liver fibrosis after administration. It can be seen from the results in Figure 6 that the combined use of any two drugs randomly selected in the present invention, compounds A1, A3 and compounds B1, B17, and B22, has a significant improvement effect on CCl4 -induced liver fibrosis, and both The improvement effect is better than that of compound A1, A3 alone or compound B1, B17, B22 alone. Therefore, it can be proved that the combined use of adiponectin receptor agonist and elastin receptor inhibitor of the present invention has obvious improvement effect on liver fibrosis, and the effect is far better than that of single use of adiponectin receptor agonist or elastin receptor inhibitors.
图7为CCl 4诱导的肝纤维化小鼠经给药后的H&E染色切片图。从图7的结果可以看出，由CCl 4诱导6周之后，小鼠肝脏出现炎性细胞浸润、细胞坏死的情况，在给药之后得到了改善。联合使用脂联素受体激动剂和弹性蛋白受体抑制剂能明显地减少中央静脉区周围的炎性细胞浸润，且单独使用脂联素受体激动剂或弹性蛋白受体抑制剂的改善效果并不显著。 Fig. 7 is a section view of H&E staining after administration of CCl 4 -induced liver fibrosis in mice. As can be seen from the results in FIG. 7 , inflammatory cell infiltration and cell necrosis occurred in the liver of mice 6 weeks after induction with CCl 4 , which were improved after administration. The combined use of an adiponectin receptor agonist and an elastin receptor inhibitor can significantly reduce inflammatory cell infiltration around the central venous area, and the improvement effect of an adiponectin receptor agonist or an elastin receptor inhibitor alone Not significant.
图8为CCl 4诱导的肝纤维化小鼠经给药后的天狼星红染色切片图；图9为CCl 4诱导的肝纤维化小鼠经给药后的天狼星红染色阳性面积柱状图。选取MCS组、MCD组、MCD+化合物A1组、MCD组+化合物B22组、MCD组+化合物A3组、MCD组+化合物B1组、MCD组+化合物A1+化合物B22组、 MCD组+化合物A3+化合物B1组进行天狼星红染色，染色结果如图8所示；计算天狼星红染色切片图中的阳性面积并制作柱状图，结果如图9所示。天狼星红是强碱性染料，易与胶原分子中的碱性基团结合，所以天狼星红可以反应胶原的沉积量。从图8至图9的结果可以看出，给予药物治疗之后，胶原沉积出现不同程度的改善。其中联合使用脂联素受体激动剂和弹性蛋白受体抑制剂小鼠肝脏中胶原的沉积量较少，说明联合使用脂联素受体激动剂和弹性蛋白受体抑制剂改善肝纤维化的效果明显优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。 Fig. 8 is a section diagram of Sirius red staining of mice with CCl 4 -induced hepatic fibrosis after administration; Fig. 9 is a bar graph of the positive area of Sirius red staining in mice with CCl 4 -induced hepatic fibrosis after administration. MCS group, MCD group, MCD+Compound A1 group, MCD group+Compound B22 group, MCD group+Compound A3 group, MCD group+Compound B1 group, MCD group+Compound A1+Compound B22 group, MCD group+Compound A3+Compound B1 group Sirius red staining was performed, and the staining results were shown in Figure 8; the positive area in the Sirius red staining section was calculated and a histogram was made, and the results were shown in Figure 9. Sirius red is a strong basic dye, which is easy to combine with basic groups in collagen molecules, so Sirius red can reflect the amount of collagen deposition. It can be seen from the results in Figures 8 to 9 that after drug treatment, collagen deposition was improved to varying degrees. Among them, the combined use of adiponectin receptor agonists and elastin receptor inhibitors has less collagen deposition in the liver, indicating that the combined use of adiponectin receptor agonists and elastin receptor inhibitors improves the effect of liver fibrosis. The effect is significantly better than that of adiponectin receptor agonists or elastin receptor inhibitors alone.
综合图7至图9的结果，在给予化合物治疗肝脏纤维化小鼠后，H&E染色和天狼星红染色结果可以看出，由CCl 4诱导肝纤维化之后出现的炎性细胞浸润，肝脏纤维化的情况在联合使用脂联素受体激动剂和弹性蛋白受体抑制剂之后得到了显著的改善，说明本发明联合使用脂联素受体激动剂和弹性蛋白受体抑制剂可以很好的改善肝脏纤维化。同时联合使用脂联素受体激动剂和弹性蛋白受体抑制剂的效果明显的远远优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。 Combining the results of Figures 7 to 9, it can be seen from the results of H&E staining and Sirius red staining that after administration of compounds to treat liver fibrosis mice, the inflammatory cell infiltration after the induction of liver fibrosis by CCl 4 , and the infiltration of liver fibrosis. The situation was significantly improved after the combined use of adiponectin receptor agonists and elastin receptor inhibitors, indicating that the combined use of adiponectin receptor agonists and elastin receptor inhibitors in the present invention can very well improve the liver fibrosis. The combined use of adiponectin receptor agonists and elastin receptor inhibitors at the same time is significantly more effective than single use of adiponectin receptor agonists or elastin receptor inhibitors.
综上所述，本发明脂联素受体激动剂和弹性蛋白受体抑制剂联合用药取得了意想不到的效果，在治疗过程中脂联素受体激动剂和弹性蛋白受体抑制剂起协同作用，联合使用脂联素受体激动剂和弹性蛋白受体抑制剂的效果远远优于单独使用脂联素受体激动剂或弹性蛋白受体抑制剂。本发明联合使用脂联素受体激动剂和弹性蛋白受体抑制剂对于非酒精性脂肪肝病具有显著的治疗效果。To sum up, the combination of the adiponectin receptor agonist and the elastin receptor inhibitor of the present invention has achieved unexpected effects, and the adiponectin receptor agonist and the elastin receptor inhibitor act synergistically during the treatment process. The effect of combined use of adiponectin receptor agonists and elastin receptor inhibitors is far superior to that of adiponectin receptor agonists or elastin receptor inhibitors alone. The combined use of an adiponectin receptor agonist and an elastin receptor inhibitor in the present invention has a significant therapeutic effect on non-alcoholic fatty liver disease.
当然，本发明还可有其它多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形，但这些相应的改变和变形都应属于本发明所附的权利要求书所界定的保护范围。Of course, the present invention can also have other various embodiments, without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding Changes and deformations should all belong to the protection scope defined by the appended claims of the present invention.

Claims (18)

  1. 脂联素受体激动剂和弹性蛋白受体抑制剂联合用药在预防或治疗非酒精性脂肪肝病中的应用。Application of adiponectin receptor agonist and elastin receptor inhibitor combination in the prevention or treatment of nonalcoholic fatty liver disease.
  2. [根据细则26改正08.04.2021]　
    根据权利要求1所述的应用，其特征在于，所述脂联素受体激动剂的氨基酸序列为P-X2-L-Y-X5-F-X7；其中X2、X5及X7中至少一个为非天然氨基酸，P为脯氨酸，L为亮氨酸，Y为酪氨酸，F为苯丙氨酸。     [Corrected 08.04.2021 according to Rule 26]
    The use according to claim 1, wherein the amino acid sequence of the adiponectin receptor agonist is P-X2-LY-X5-F-X7; wherein at least one of X2, X5 and X7 is non-natural Amino acid, P is proline, L is leucine, Y is tyrosine, F is phenylalanine.
  3. 根据权利要求2所述的应用，其特征在于，所述脂联素受体激动剂的氨基酸序列选自SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4中的一种。The application according to claim 2, wherein the amino acid sequence of the adiponectin receptor agonist is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 one of the.
  4. 根据权利要求1至3任一项所述的应用，其特征在于，所述弹性蛋白受体抑制剂含有以下氨基酸序列表示的母体肽：The use according to any one of claims 1 to 3, wherein the elastin receptor inhibitor contains a parent peptide represented by the following amino acid sequence:
    R 1-Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14， R 1 -Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14,
    其中，in,
    R 1为：亲脂性取代基或不存在； R 1 is: a lipophilic substituent or absent;
    Xa2＝Val或Iva；Xa2=Val or Iva;
    Xa9＝Asp或Glu；Xa9=Asp or Glu;
    Xa10＝Glu或Asp；Xa10=Glu or Asp;
    Xa14＝Leu或Ala；Xa14=Leu or Ala;
    当R 1不存在，Xa2＝Val，且Xa14＝Leu时，母体肽为非直链肽。 When R1 is absent, Xa2 = Val, and Xa14=Leu, the parent peptide is a non-linear peptide.
  5. 根据权利要求4所述的应用，其特征在于，所述弹性蛋白受体抑制剂的氨基酸序列选自SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28和SEQ ID NO.29中的一种。The application according to claim 4, wherein the amino acid sequence of the elastin receptor inhibitor is selected from the group consisting of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO. 25. One of SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29.
  6. 根据权利要求1所述的应用，其特征在于，所述脂联素受体激动剂和所述弹性蛋白受体抑制剂同时给药。The use according to claim 1, wherein the adiponectin receptor agonist and the elastin receptor inhibitor are administered simultaneously.
  7. 根据权利要求1所述的应用，其特征在于，所述脂联素受体激动剂和所述弹性蛋白受体抑制剂依次给药。The use according to claim 1, wherein the adiponectin receptor agonist and the elastin receptor inhibitor are administered sequentially.
  8. 根据权利要求7所述的应用，其特征在于，所述依次给药的顺序为先给予所述脂联素受体激动剂再给予所述弹性蛋白受体抑制剂。The use according to claim 7, characterized in that, the sequence of sequential administration is to administer the adiponectin receptor agonist first and then administer the elastin receptor inhibitor.
  9. 根据权利要求7所述的应用，其特征在于，所述依次给药的顺序为先给予所述弹性蛋白受体抑制剂再给予所述脂联素受体激动剂。The use according to claim 7, characterized in that, the sequence of sequential administration is to administer the elastin receptor inhibitor first and then administer the adiponectin receptor agonist.
  10. 根据权利要求1所述的应用，其特征在于，所述脂联素受体激动剂和弹性蛋白受体抑制剂的浓度比为10：1至3：1。The use according to claim 1, wherein the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor is 10:1 to 3:1.
  11. 根据权利要求10所述的应用，其特征在于，所述脂联素受体激动剂和弹性蛋白受体抑制剂的浓度比为6：1至4：1。The use according to claim 10, wherein the concentration ratio of the adiponectin receptor agonist and the elastin receptor inhibitor is 6:1 to 4:1.
  12. 根据权利要求1所述的应用，其特征在于，所述非酒精性脂肪肝病包括非酒精性脂肪样变性、非酒精性脂肪肝炎、肝纤维化以及肝纤维化合并的肝硬化。The use according to claim 1, wherein the non-alcoholic fatty liver disease comprises non-alcoholic steatosis, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis combined with liver fibrosis.
  13. 一种联合用药物组合物，适用于权利要求1-12任一项所述的预防或治疗非酒精性脂肪肝病中的应用，其特征在于，所述联合用药物组合物包括脂联素受体激动剂和弹性蛋白受体抑制剂。A combined pharmaceutical composition, suitable for use in the prevention or treatment of non-alcoholic fatty liver disease according to any one of claims 1-12, wherein the combined pharmaceutical composition comprises an adiponectin receptor Agonists and elastin receptor inhibitors.
  14. 根据权利要求13所述的联合用药物组合物，其特征在于，所述联合用药物组合物为单一的复方制剂。The combined pharmaceutical composition according to claim 13, wherein the combined pharmaceutical composition is a single compound preparation.
  15. 根据权利要求13所述的联合用药物组合物，其特征在于，所述联合用药物组合物为脂联素受体激动剂和弹性蛋白受体抑制剂两种单独制剂的组合。The combined pharmaceutical composition according to claim 13, wherein the combined pharmaceutical composition is a combination of two separate preparations, an adiponectin receptor agonist and an elastin receptor inhibitor.
  16. 根据权利要求13所述的联合用药物组合物，其特征在于，所述联合用药物组合物还包含药学上可接受的载体或辅料。The combined pharmaceutical composition according to claim 13, characterized in that, the combined pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.
  17. 根据权利要求13所述的联合用药物组合物，其特征在于，所述联合用药物组合物为药学上可接受的剂型。The combined pharmaceutical composition according to claim 13, wherein the combined pharmaceutical composition is in a pharmaceutically acceptable dosage form.
  18. 根据权利要求17所述的联合用药物组合物，其特征在于，所述剂型为片剂、胶囊、糖衣片剂、粒剂、口服溶液、糖浆、用于皮肤表面的油膏和药贴、气雾剂、鼻喷剂以及用于注射的无菌溶液中的至少一种。The combination pharmaceutical composition according to claim 17, wherein the dosage form is tablet, capsule, sugar-coated tablet, granule, oral solution, syrup, ointment and patch for skin surface, gas At least one of aerosols, nasal sprays, and sterile solutions for injection.
PCT/CN2021/078192 2021-02-26 2021-02-26 Combination of adiponectin receptor agonist and elastin receptor inhibitor for prevention or treatment of non-alcoholic fatty liver disease WO2022178841A1 (en)

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