WO2021249143A1 - 靶向于纤连蛋白衍生肽的抑制剂多肽化合物及其用途 - Google Patents

靶向于纤连蛋白衍生肽的抑制剂多肽化合物及其用途 Download PDF

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WO2021249143A1
WO2021249143A1 PCT/CN2021/094680 CN2021094680W WO2021249143A1 WO 2021249143 A1 WO2021249143 A1 WO 2021249143A1 CN 2021094680 W CN2021094680 W CN 2021094680W WO 2021249143 A1 WO2021249143 A1 WO 2021249143A1
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ala
seq
peg
pro
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蒋先兴
宋娜资
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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 AU2021289870A priority Critical patent/AU2021289870B2/en
Priority to EP21821478.1A priority patent/EP4163289A1/en
Priority to KR1020237000148A priority patent/KR20230019941A/ko
Priority to JP2023516640A priority patent/JP2023527483A/ja
Publication of WO2021249143A1 publication Critical patent/WO2021249143A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the technical field of biochemistry. Specifically, the present invention relates to an inhibitor polypeptide compound targeted to fibronectin-derived peptides (EDPs).
  • the polypeptide compound can be used to treat or prevent organ fibrosis and organ diseases. Use in medicines for fibrotic disorders; especially liver fibrosis and fibrotic disorders accompanied by liver diseases and idiopathic pulmonary fibrosis and fibrotic disorders accompanied by lung diseases.
  • Organ fibrosis is a characteristic manifestation of end-stage organ failure of many chronic diseases, which seriously endangers the health of the people and is an important cause of death of patients.
  • the main mechanism of fibrosis is that the body initiates a protective repair process in response to traumatic stimuli, but repeated abnormal healing and repair lead to disturbances in the internal environment of the tissue, excessive accumulation of extracellular matrix (ECM), and destruction of tissue structure. This led to the occurrence of fibrosis (DCRockey, PDBell, JAHill, The New England journal of medicine 2015, 372, 1138-1149.).
  • liver fibrosis is the continuous occurrence of liver damage in the liver.
  • the ECM is continuously synthesized and degraded, resulting in a pathological process caused by imbalanced matrix deposition.
  • Liver fibrosis is an important pathological feature of chronic liver disease, and it is also the main intermediate relief for the further development of liver cirrhosis.
  • the repair reaction after injury will cause many changes in the ECM microenvironment.
  • liver fibrosis and cirrhosis There are many reasons that affect liver fibrosis and cirrhosis. For example, in Western countries, excessive drinking, hepatitis virus infection and fatty liver disease are the most common inducing factors. In addition, chronic immune-mediated damage can also lead to liver fibrosis and cirrhosis, such as primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH) ( K. Bottcher, M. Pinzani, Advanced drug delivery reviews 2017, 121, 3-8.).
  • PSC primary sclerosing cholangitis
  • PBC primary biliary cholangitis
  • AIH autoimmune hepatitis
  • liver fibrosis is not controlled and treated, it will develop into cirrhosis, which will eventually lead to liver damage and liver necrosis. Once cirrhosis develops, the patient is at a very high risk of hepatocellular carcinoma (AHAli, KDLindor, Expert opinion on pharmacotherapy 2016, 17, 1809-1815.), which brings great pain and even threats to the patient. Life, therefore, must rely on liver transplantation for treatment. Therefore, it can be seen that liver fibrosis is a serious threat to the health of people in our country and the world, and effective drugs are urgently needed for treatment!
  • OCA obeticholic acid
  • FDA U.S. Food and Drug Administration
  • OCA has prominent adverse reactions such as itching and high-density lipoprotein cholesterol reduction during treatment.
  • OCA is expensive, and pharmacoeconomic analysis believes that OCA is not optimistic in terms of cost-effectiveness ratio, and it needs to be further optimized and improved. Based on the above reasons, it is particularly important to develop drugs with fewer side effects, more targeted and more economical treatments for fibrosis.
  • Idiopathic pulmonary fibrosis is a chronic, progressive, and fibrotic interstitial pneumonia with unknown etiology and pathogenesis. It is characterized by abnormal deposition of ECM leading to extensive lung remodeling (L.Richeldi, HRCollard, MG Jones, The Lancet 2017, 389, 1941-1952.). It is more common in the elderly, with a median survival rate of 2-3 years. Pathogenesis and risk factors, including genetic factors, environmental exposure, smoking, chronic viral infections and certain comorbidities. Histopathological examination usually shows extensive alveolar scar formation, that is, fibrous scars containing myofibroblasts replace normal alveoli. IPF cannot be cured.
  • the purpose of treatment is to delay disease progression, improve life, and prolong survival, including anti-fibrotic treatment with nintedanib and pirfenidone, non-drug treatment and pulmonary fibrosis (DJLederer, FJ Martinez, The New England journal of medicine 2018,378,1811-1823.).
  • anti-fibrotic treatment with nintedanib and pirfenidone
  • non-drug treatment and pulmonary fibrosis
  • COPD chronic obstructive pulmonary disease
  • small airway obstruction chronic obstructive bronchiolitis
  • emphysema Airway examination confirmed poor reversibility of airway obstruction, resulting in air retention in the lungs and shortness of breath for physical activity.
  • the most common risk factor for COPD is smoking. Although the mechanism of COPD is still poorly understood, the disease is associated with chronic inflammation that is often resistant to corticosteroids.
  • COPD involves accelerated lung aging and abnormal repair mechanisms that may be driven by oxidative stress (K.F. Rabe, H.
  • Fibronectin is the most stable protein in ECM. It is the main component of elastic fibers. It exists in a variety of elastic soft tissues, such as arterial walls, ligaments, lungs, bladder, and skin (ROBERT M.SENIOR, J . Clin. Invest. 1982, 70, 614-618.). Elastin is synthesized from the fetal period, reaches its peak at birth, and gradually decreases or even disappears after puberty. The half-life of elastic fiber is as long as 70 years, and the re-synthesis rate is low. Under normal pathological conditions, elastic fibers are the main component of tissue connections and are used to maintain tissue elasticity. But in the pathological state of the disease, elastic fibers will be abnormally synthesized and degraded.
  • Degradation of elastic fibers will produce a series of derived peptides (EDPs), which have different amino acid sequences, which can regulate a series of cell signals by activating downstream receptors, such as Ras-Raf-1-MEK1 /2-ERK1/2; Gi-p110 ⁇ -Raf-1-MEK1/2-ERK1/2; cAMP-PKA-B-Raf-MEK1/2-ERK1/2; NO-cGMP-PKG-Raf-1-MEK1 /2-ERK1/2 or Gi-p110 ⁇ -Akt-caspase9-Bad-Foxo3A (L.Duca, C.
  • EDPs derived peptides
  • EDPs are closely related to the development of many diseases. Studies have found that excessive production of EDPs can lead to metabolic disorders in the liver, the accumulation of inflammation, and promote the development of liver diseases. Therefore, inhibiting the activity of EDPs must have a certain improvement effect on liver fibrosis (C. Ntayi, ALLabrousse, R. Debret, P. Birembaut, G. Bellon, F. Antonicelli, W. Hornebeck, P.
  • the patent of the present invention relates to a new type of inhibitor polypeptide compound targeting fibronectin-derived peptides (EDPs), which can bind to EDPs circulating in the body to block its biological effects.
  • EDPs fibronectin-derived peptides
  • the present invention designs a biologically active polypeptide inhibitor that binds to EDPs based on the conformation and binding site of EDPs, which is bound to provide new ideas and scientific research directions in the development of drugs for treating organ fibrosis.
  • the purpose of the present invention is to provide a novel inhibitor polypeptide compound targeting fibronectin-derived peptides.
  • the present invention has proved that the inhibitor polypeptide compound has no adverse reactions and can be used for the treatment of organ fibrosis and organ disease-associated fibrosis; preferably, the organ fibrosis and organ disease-associated fibrosis Symptoms are: liver fibrosis and fibrotic disorders accompanied by liver disease and idiopathic pulmonary fibrosis and fibrotic disorders accompanied by lung disease.
  • Another object of the present invention is to provide the above-mentioned novel inhibitor polypeptide compound for the treatment or prevention of organ fibrosis and organ disease accompanied by fibrotic conditions, especially the novel inhibitor polypeptide compound can be used as a new generation of liver treatment Fibrosis and fibrosis accompanied by liver disease and idiopathic pulmonary fibrosis and pulmonary disease accompanied by fibrotic disease drugs.
  • the present invention provides an inhibitor polypeptide compound targeted to a fibronectin-derived peptide, which contains a parent peptide represented by the following amino acid sequence:
  • R 1 is: lipophilic substituent or absent
  • Xa2 Val or Iva
  • Xa9 Asp or Glu
  • Xa10 Glu or Asp
  • R 1 is a lipophilic substituent
  • the amino group of Val at position 1 in the amino acid sequence of the parent peptide is connected to the lipophilic substituent via a bridging group, and the bridging group includes (PEG) m , or Containing (PEG) m and ⁇ Glu, or (PEG) m and Asp
  • the connection mode is that the amino group of Val at position 1 is modified by PEGylation and lipophilicity of (PEG) m in the bridging group
  • the substituents are connected; and the lipophilic substituent is CH 3 (CH 2 ) n C(O)- or HOOC(CH 2 ) n C(O)- and its acyl group is connected to the amino group contained in the bridging group
  • An amide bond is formed; wherein m is an integer of 2-10; n is an integer of 14-20; the carboxyl end of the amino acid sequence has a naked carboxyl group, or an amino group is connected to form a
  • Xa2 Val or Iva
  • Xa9 Asp or Glu
  • Xa10 Glu or Asp
  • Xa14 Leu or Ala.
  • the amino acid sequence of the parent peptide is selected from SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, 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 One of the amino acid sequences shown in ID NO.24 and SEQ ID NO.25.
  • the structure connected to the amino group of Val at position 1 in the amino acid sequence of the parent peptide is:
  • the present invention provides any of the following polypeptide compounds:
  • Another aspect of the present invention is to provide a pharmaceutical composition containing the novel inhibitor polypeptide compound of the present invention.
  • the novel inhibitor polypeptide compound is used as an active ingredient and a pharmaceutically acceptable carrier and/or auxiliary materials are added to prepare a pharmaceutical composition. .
  • Another aspect of the present invention is to provide the pharmaceutical use of the novel inhibitor polypeptide compound of the present invention.
  • Cell and animal experiments have shown that the novel inhibitor polypeptide compound of the present invention has no adverse reactions and can be used for the treatment of organ fibrosis and organ disease-associated fibrotic conditions; preferably, the organ fibrosis and organ-disease-associated fiber
  • the metastatic disease is: liver fibrosis and fibrotic disease accompanying liver disease and idiopathic pulmonary fibrosis and fibrotic disease accompanying lung disease.
  • the parent peptide in the novel inhibitor polypeptide compound mentioned in the present invention is a homologous polypeptide.
  • the homologous polypeptide in the present invention means that the polypeptide originally has the amino acid sequence of the natural parent peptide, but one or more of the amino acid residues have been replaced by different amino acid residues, and these amino acid residues are conserved among each other.
  • the obtained polypeptide can be used to implement the present invention.
  • cell and animal experiments show that compared with the natural parent peptide when the mass number is the same, the parent peptide in the inhibitor polypeptide compound of the present invention has better biological and pharmacological activities, and its therapeutic effect is better than that of the natural parent peptide.
  • the pharmaceutical composition of the present invention is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, transnasal administration, and the like.
  • the pharmaceutical composition of the polypeptide compound of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the polypeptide compound of the present invention and at least one pharmaceutically acceptable drug.
  • suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the surface of the skin, aerosols, nasal sprays, and sterile solutions that can be used for injection.
  • the pharmaceutical composition containing the polypeptide compound of the present invention can be made into a solution or freeze-dried powder for parenteral administration.
  • the powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier before use.
  • the liquid formulation is generally buffered. Liquid, isotonic solution and aqueous solution.
  • the dosage of the pharmaceutical composition of the present invention can be varied within a relatively large range, and those skilled in the art can easily depend on some objective factors, such as the type of disease, the severity of the disease, the weight of the patient, the dosage form, and the route of administration. To be determined.
  • Boc is tert-butoxycarbonyl
  • Fmoc is fluorene methoxycarbonyl
  • t-Bu is tert-butyl
  • resin is resin
  • TFA is trifluoroacetic acid
  • EDT 1,2-ethanedithiol
  • FBS is fetal bovine serum
  • BSA bovine serum albumin
  • HPLC high performance liquid phase
  • mPEG monomethoxy polyethylene glycol
  • Ser is serine
  • D-Ser is D-serine
  • Gln is glutamine
  • Gly glycine
  • Glu glutamine Acid
  • Ala is alanine
  • Asp is aspartic acid
  • Leu is leucine
  • Pro is proline
  • Val is valine
  • Iva is isovaline.
  • Figure 1 is a statistical histogram of the activity evaluation of compound 1-25 in the LX2 cell model in Example 2 (**: indicates a confidence level >99%, the difference between the two is very significant (P ⁇ 0.01); ***: Indicates that the confidence level is >99.9%, the difference between the two is very significant (P ⁇ 0.001)).
  • Figure 2 shows the HE stained pathological section of mouse liver in Example 3.
  • Figure 3 shows the pathological section and statistical histogram of Collgen I immunohistochemical staining of mouse liver in Example 3 (**: indicates a confidence level >99%, the difference between the two is very significant (P ⁇ 0.01); ** *: Indicates that the confidence level is >99.9%, the difference between the two is extremely significant (P ⁇ 0.001)).
  • Figure 4 shows the pathological section and statistical histogram of Sirius Red stained mouse liver in Example 3 (*: indicates the confidence level>95%, the difference between the two groups is significant (P ⁇ 0.05); **: indicates the confidence level> 99%, the difference between the two is very significant (P ⁇ 0.01); ***: means the confidence level is >99.9%, the difference between the two is very significant (P ⁇ 0.001)).
  • Figure 5 shows the HE staining, Masson staining, ⁇ -SMA and Collagen1 immunohistochemical pathological slices and statistical histograms of the mouse lungs in Example 4 (**: indicates a confidence level of >99%, the difference between the two is very significant (P ⁇ 0.01); ***: means confidence level>99.9%, the difference between the two is very significant (P ⁇ 0.001); ****: means confidence level>99.99%, the difference between the two is very significant Meaning (p ⁇ 0.0001)).
  • Figure 6 is a statistical histogram of the expression of TGF ⁇ and MMP9 cytokines in the serum of each group of mice detected by ELISA in Example 4 (**: indicates the confidence level>99%, the difference between the two is very significant (P ⁇ 0.01) ;***: means confidence level>99.9%, the difference between the two is significant (P ⁇ 0.001); ****: means confidence level>99.99%, the difference between the two is significant (p ⁇ 0.0001 )).
  • Figure 7 is a schematic diagram of the connection between the bridging group and the peptide chain in the compound of the present invention.
  • the peptide sequence is: (PEG 2 -PEG 2- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala-NH 2 .
  • the column was eluted with a gradient of 40-60% acetonitrile-0.1% trifluoroacetic acid/H 2 O at 19 mL/min for 60.0 minutes, the components containing C5 were collected, concentrated to remove acetonitrile, and then trans-salt lyophilized. 145 mg of pure product with a purity of 98.83% by HPLC was obtained.
  • the separated products were analyzed by liquid mass spectrometry and the separated products were analyzed by liquid mass spectrometry. It was found that the m/2z value of the protonated molecular ion peak was 1050.4 and the theoretical value was 2099.4.
  • the sequence structure is:
  • the resin was dispersed in the DMF solution, 0.15 equivalents of tetratriphenylphosphine palladium and 10 equivalents of morphine were added, and reacted for 12 hours under a nitrogen atmosphere, followed by washing with DCM (10mL*3), DMF (10mL*3) and draining.
  • Step 5 Cleavage of peptides and removal of protecting groups
  • LX2 cells are human liver activated stellate cells, they can be directly used as an in vitro liver fibrotic cell evaluation model
  • LX2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), the peptide compound 1- 25. Perform a preliminary screening for active anti-fibrosis activity to detect the expression of ⁇ -SMA protein, a marker of myofibroblast activation.
  • the polypeptide compound 1-25 was incubated with two concentrations (1.0 ⁇ M and 10.0 ⁇ M) for 48 hours.
  • the control group was given the same volume of Dulbecco's Modified Eagle Medium (DMEM) (purchased from Gibco, USA), and GAPDH (purchased from Beijing Quanshijin Biological Co., Ltd.) as an internal control.
  • DMEM Dulbecco's Modified Eagle Medium
  • GAPDH purchased from Beijing Quanshijin Biological Co., Ltd.
  • ⁇ -actin ⁇ -SMA is a hallmark feature that distinguishes myofibroblasts from myofibroblasts.
  • Tested drug peptide compound analogue. Storage conditions: -20°C.
  • CCl 4 20% CCl 4 was given to the mice, and the mice in the control group were given corn oil injections of the same volume and frequency according to the administration volume of 5.0 ⁇ L/g of the body weight of the mice.
  • Dosing frequency CCl 4 was given to mice once for three days for 6 weeks; when the model administration group was given CCl 4 to mice for 3 weeks, the peptide compounds 1, 5, 7, 11, 12, and 5 were given from the 4th week. 13, 17, 18, 21 and 22 for 3 weeks.
  • CCl 4 and Oil were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
  • liver fibrosis model its manifestations are: inflammatory cell infiltration around the central vein area, hepatocyte edema and degeneration, and a large amount of collagen fiber deposition in the portal area and the liver lobule interval.
  • mice were processed and samples were taken, blood was taken from the vein behind the eyeball for serological index detection, and liver tissue was taken for pathological analysis.
  • Hematoxylin-Eosin (H&E) staining Take paraffin-embedded tissue sections and bake at 60°C for 1 hour. Dewaxing and hydration: 20 minutes for xylene ⁇ 20 minutes for xylene ⁇ 15 minutes for anhydrous alcohol ⁇ 15 minutes for anhydrous alcohol ⁇ 10 minutes for 95% alcohol ⁇ 5 minutes for 90% alcohol ⁇ 5 minutes for 80% alcohol. Staining: hematoxylin for 7 minutes ⁇ rinse with tap water ⁇ 1% hydrochloric acid and ethanol for 1 s ⁇ rinse with tap water ⁇ stain with eosin for 15 s-20s ⁇ rinse with tap water. Dehydration transparent: 75% alcohol 1s ⁇ 85% alcohol 1s ⁇ 95% alcohol 1s ⁇ 100% alcohol 1s ⁇ xylene 1s ⁇ xylene 1s. Mount the slides to dry for 30 minutes, then mount the slides with gum.
  • Sirius red staining baking sheet, dewaxing; let stand for 5.0 minutes in double distilled water; staining with Sirius red in a dark room for 60-80 minutes; rinse with 0.5% glacial acetic acid for 5 seconds; dehydrated and transparent, mount the slides, and take pictures.
  • Immunohistochemistry baking sheet, dewaxing, and soaking in double distilled water for 5 minutes.
  • Figure 2 shows: model administration group (CCl 4 ) + 100.0 ⁇ g/kg series of compounds 1, 5, 7, 11, 12, 13, 17, 18, 21 and 22, after the above-mentioned compound administration treatment ,
  • the aggregation of inflammatory cells in the liver of mice in each treatment model group was significantly improved; the liver fibrosis of mice in each model treatment group was treated with 1,5,7,11,12,13,17,18,21 and Significant improvement has been obtained after 22.
  • compounds 1, 5, 7, 11, 12, 13, 17, 18, 21 and 22 can inhibit the accumulation of ECM very well, and treat and improve liver fibrosis.
  • Figure 3 shows: the model administration group (CCl 4 ) + 100.0 ⁇ g/kg series of compounds 1, 5, 7, 11, 12, 13, 17, 18, 21 and 22, after the above-mentioned compound administration treatment, each The expression of Collagen I in the liver of mice in the treatment model group was significantly improved.
  • Figure 4 shows: model administration group (CCl 4 ) + 100.0 ⁇ g/kg series of compounds 1, 5, 7, 11, 12, 13, 17, 18, 21, and 22. After the above-mentioned compound is administered and treated, each The collagen deposition in the liver of mice in the treatment model group showed significant improvement in treatment.
  • Tested drug peptide compound analogue. Storage conditions: -20°C.
  • mice in the model treatment group were treated with administration (dose frequency: daily administration/150 ⁇ g/kg, intraperitoneal injection), and samples were taken 14 days after administration , Record for pathological observation.
  • H&E staining Take paraffin-embedded tissue sections and bake at 60°C for 1 hour. Dewaxing and hydration: 20 minutes for xylene ⁇ 20 minutes for xylene ⁇ 15 minutes for anhydrous alcohol ⁇ 15 minutes for anhydrous alcohol ⁇ 10 minutes for 95% alcohol ⁇ 5 minutes for 90% alcohol ⁇ 5 minutes for 80% alcohol. Staining: hematoxylin for 7 minutes ⁇ rinse with tap water ⁇ 1% hydrochloric acid and ethanol for 1 s ⁇ rinse with tap water ⁇ stain with eosin for 15 s-20s ⁇ rinse with tap water. Dehydration transparent: 75% alcohol 1s ⁇ 85% alcohol 1s ⁇ 95% alcohol 1s ⁇ 100% alcohol 1s ⁇ xylene 1s ⁇ xylene 1s. Mount the slides to dry for 30 minutes, then mount the slides with gum.
  • Masson staining baking sheet, dewaxing; mordant dyeing; lapis lazuli staining solution dripping for 2-3min, washing; Mayer hematoxylin staining solution dripping and washing; acidic ethanol differentiation solution for several seconds, washing with tap water; Ponceau red magenta staining solution Drip dyeing, rinse with distilled water; treat with phosphomolybdic acid solution; drip into aniline blue dye solution for 5 minutes; treat with weak acid solution for 2 minutes; dehydrate transparently, dry, and seal with gum.
  • Immunohistochemistry baking sheet, dewaxing, and soaking in double distilled water for 5 minutes.
  • H&E staining The lung tissue of the mice in the normal control group and the sham-operated saline control group is clear, the alveolar wall is not thickened, the alveolar epithelial structure is intact, and no inflammatory cell infiltration is seen; Masson staining: normal In the control group and the sham-operated saline control group, only a small amount of collagen fibers were stained blue.
  • the mice in each group of the model treatment group were treated with 150.0 ⁇ g/kg series of compounds 1, 5, 7, 11, 12, 13, 17, 18, 21 and 22. Pulmonary fibrosis in the model group mice has been significantly improved by treatment.
  • H&E staining and Masson staining showed that the alveolar septum in the bleomycin group was significantly widened, and a large number of fibroblasts and matrix filling were seen; administration 1, 5, 7, 11, 12, 13, 17, 18 After treatment with, 21 and 22 polypeptide compounds, it can be seen that the degree of pulmonary fibrosis in each treatment group was significantly less than that of the bleomycin control group.
  • MMPs Matrix metalloproteinases
  • MMP-9 are proteases that degrade the matrix and degrade all the components of the extracellular matrix. They are involved in the pathophysiological process of pulmonary fibrosis, play an important role in the abnormal remodeling of extracellular matrix and the destruction of basement membrane, and promote the recruitment of inflammatory cells and fibroblasts.
  • the expression of MMP-9 increases in animal models and patients of pulmonary fibrosis. Higher than normal levels of MMP-9 may destroy normal tissue structures and increase the migration of inflammatory cells to the diseased site.
  • the polypeptide compound of the present invention inhibits the accumulation of ECM and the expression of ⁇ -SMA and Collagen 1 in lung tissue, and can be used to treat liver fibrosis and fibrosis accompanied by liver disease and idiopathic pulmonary fibrosis and Fibrotic conditions associated with lung disease.

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Abstract

本发明涉及一种靶向于纤连蛋白衍生肽的抑制剂多肽化合物及其用途，该多肽化合物含有以下氨基酸序列表示的母体肽：R 1-Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14，其具有更好的生物学活性，安全性和稳定性较高以及合成产率高、成本低等特点。该多肽化合物可用于治疗肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症。

Description

靶向于纤连蛋白衍生肽的抑制剂多肽化合物及其用途 技术领域
本发明属于生物化学技术领域，具体而言，本发明涉及一种靶向于纤连蛋白衍生肽(EDPs)的抑制剂多肽化合物，该多肽化合物可用于治疗或预防器官纤维化及器官疾病伴随的纤维化病症的药物中的用途；特别是肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症。
背景技术
器官纤维化是许多慢性疾病终末期器官衰竭的特征性表现，严重危害了人民群众的身体健康，是造成患者死亡的重要原因。目前认为纤维化主要机制是机体应对损伤性刺激启动了保护性的修复过程，但是反复异常的愈合修复导致了组织内环境的紊乱，细胞外基质(ECM)的过度积累，以及组织结构的破坏，从而造成了纤维化的发生(D.C.Rockey,P.D.Bell,J.A.Hill,The New England journal of medicine2015,372,1138-1149.)。
很多慢性疾病末期器官都会出现纤维化的特征，例如：肝脏纤维化是肝脏持续的发生肝损伤，组织发生修复反应时ECM不断地合成、降解所导致基质沉积不平衡而引起的病理学过程，是涉及复杂的细胞及分子机制的动态过程。肝纤维化是慢性肝病重要的病理特征，也是进一步向肝硬化发展的主要中间缓解。损伤后的修复反应会引起很多ECM微环境的改变，在这个过程中，静止的肝星状细胞会被激活变成活化的肝星状细胞，导致肝星状细胞形态及功能的改变，具体表现为ECM的异常合成与降解(L.Petitclerc,G.Sebastiani,G.Gilbert,G.Cloutier,A.Tang,Journal of magnetic resonance imaging:JMRI2017,45,1276-1295.)。而ECM的合成与降解之间的不平衡就会导致ECM的大量积累进而形成纤维化，而纤维化的发生会进一步的加重肝星状细胞的激活，使得纤维化不断地加重。影响肝纤维化和肝硬化的原因有很多，比如在西方国家，过量的饮酒，肝炎病毒的感染和脂肪肝疾病的发生是最为常见的诱导因素。此外， 慢性免疫介导的损伤也可以导致肝纤维化和肝硬化的发生，如原发性硬化性胆管炎(PSC)、原发性胆道胆管炎(PBC)和自身免疫性肝炎(AIH)(K.Bottcher,M.Pinzani,Advanced drug delivery reviews2017,121,3-8.)。
肝脏纤维化如果不加以控制和治疗，就会发展为肝硬化，最终将会导致肝功能受损和肝坏死的发生。而一旦发展为肝硬化，患者则处于极高危的患肝细胞癌的风险中(A.H.Ali,K.D.Lindor,Expert opinion on pharmacotherapy2016,17,1809-1815.)，给患者带来巨大的痛苦甚至威胁其生命，从而必须依靠肝脏移植来治疗。所以可见肝纤维化严重威胁着我国以及世界人民的健康，亟待有效的药物来治疗！
然而迄今为止，经美国食品药品监督管理局(FDA)批准的用于治疗纤维化的药物只有奥贝胆酸(OCA)。但是OCA在治疗过程中存在较为突出的瘙痒和高密度脂蛋白胆固醇降低等不良反应，部分患者甚至因为严重的瘙痒而被迫停药，且有部分患者还出现严重的心血管事件。目前OCA价格昂贵，药物经济学分析认为OCA在费用-疗效比方面并不乐观，尚有待进一步优化提高。综合以上各种原因，研发更少副作用、更强针对性及更经济的治疗纤维化的药物显得尤为重要。
而另一个受人们普遍关注的就是肺纤维化的发生。特发性肺纤维化(IPF)是一种病因及发病机制不明的慢性、进行性、纤维化性的间质性肺炎，其特征是ECM的异常沉积导致广泛的肺重构(L.Richeldi,H.R.Collard,M.G.Jones,The Lancet2017,389,1941-1952.)。老年人多见，中位生存率2-3年。发病机制及危险因素，包括遗传因素、环境暴露、吸烟、慢性病毒感染和某些合并症。其组织病理学检查通常显示广泛的肺泡瘢痕形成，即用含有成肌纤维细胞的纤维性瘢痕代替正常肺泡。IPF不可能治愈，治疗目的是延缓疾病进展、改善生活治疗、延长生存期，包括以尼达尼布、吡非尼酮等抗纤维化药物治疗、非药物治疗和肺纤维化(D.J.Lederer,F.J.Martinez,The New England journal of medicine2018,378,1811-1823.)。尽管我们对IPF病理的认识有了新进展，但仍然没有治疗IPF的方法，目前可用的抗纤维化治疗方法只延缓了该病的进展，不能完全停止该病的进展。
此外在引起肺纤维化方面，慢性阻塞性肺疾病(COPD)是一种具有高全球发病率和死亡率的常见疾病，特征包括小气道阻塞(慢性阻塞性细支气管炎)和肺气肿，通过气道检查证实了气道阻塞的可逆性较差，导致肺内空气滞留和 对体力活动的呼吸短促。COPD最常见的危险因素是吸烟。尽管对COPD的机制仍知之甚少，但该疾病与通常对皮质类固醇耐药的慢性炎症有关。此外，COPD涉及加速的肺部衰老和可能由氧化应激驱动的异常修复机制(K.F.Rabe,H.Watz,The Lancet2017,389,1931-1940.)。除了鼓励戒烟之外，有效的长效支气管扩张剂、家庭氧疗可以控制稳定疾病，但这些药物不能减缓潜在的疾病进程，因此不会降低疾病的进展或死亡率。需要更多的研究来更好地了解疾病的机制，并开发出减少疾病活动和进展的新疗法。
纤连蛋白(Elastin)是ECM中最稳定的一种蛋白，是弹性纤维的主要成分，存在于多种弹性软组织中，如动脉壁、韧带、肺、膀胱和皮肤等(ROBERT M.SENIOR,J.Clin.Invest.1982,70,614-618.)。弹性蛋白从胎儿时期进行合成，出生时达到巅峰，青春期后逐渐的减少甚至合成消失。弹性纤维的半衰期长达70年之久，重新合成率较低。在正常的病理状态下，弹性纤维是组织连接的主要成份，用于维持组织的弹性。但是在疾病的病理状态下，弹性纤维会被异常的合成以及降解。弹性纤维降解会产生一系列的衍生肽(EDPs)，这种由弹性纤维降解的衍生肽具有不同的氨基酸序列，通过激活下游受体进而调节一系列的细胞信号，比如Ras-Raf-1-MEK1/2-ERK1/2；Gi-p110γ-Raf-1-MEK1/2-ERK1/2；cAMP-PKA-B-Raf-MEK1/2-ERK1/2；NO-cGMP-PKG-Raf-1-MEK1/2-ERK1/2或者Gi-p110γ-Akt-caspase9-Bad-Foxo3A(L.Duca,C.Blanchevoye,B.Cantarelli,C.Ghoneim,S.Dedieu,F.Delacoux,W.Hornebeck,A.Hinek,L.Martiny,L.Debelle,The Journal of biological chemistry2007,282,12484-12491.)。EDPs与很多疾病的发展都具有非常密切的联系。研究发现，EDPs的过量产生可导致肝脏的代谢紊乱，炎症的积累，促进肝脏疾病的发展。所以抑制EDPs的活性肯定对肝纤维化具有一定改善作用(C.Ntayi,A.L.Labrousse,R.Debret,P.Birembaut,G.Bellon,F.Antonicelli,W.Hornebeck,P.Bernard,The Journal of investigative dermatology2004,122,256-265.)。虽然IPF的发病机制仍不清楚，但ECM重塑异常被认为有助于胶原肺部疤痕组织的不断沉积，有研究推测ECM的异常重构在疾病的发展和(或)进展中起着重要的作用。
综上所述，目前临床研究的药物在治疗纤维化疾病方面仍存在较大的不足和安全风险，抗纤维化领域更加安全、有效的新靶点药物研发仍是一项艰巨的科学任务，而相应的药物分子的设计合成更是迫在眉睫！
本发明专利涉及一种新型的靶向于纤连蛋白衍生肽(EDPs)的抑制剂多肽 化合物，可以与体内循环的EDPs结合进而阻断其生物效应。通过体外细胞模型活性筛选和体内动物模型活性筛选，对系列活性多肽进行药效学评价。目前还没有类似关于本领域化合物与纤维化相关性的研究文献报道。所以本发明基于EDPs的构象以及结合位点设计一种具有生物活性的结合EDPs的多肽抑制剂，势必在治疗器官纤维化药物研发方面提供了新的思路及科学研究方向。
发明内容
本发明的目的在于提供一种靶向于纤连蛋白衍生肽的新型的抑制剂多肽化合物。本发明经过大量的实验研究证明该抑制剂多肽化合物，没有不良反应发生，可用于器官纤维化及器官疾病伴随的纤维化病症的治疗；优选地，所述器官纤维化及器官疾病伴随的纤维化病症为：肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症。
本发明的另一个目的在于提供上述新型的抑制剂多肽化合物在治疗或预防所述器官纤维化及器官疾病伴随的纤维化病症的应用，尤其是该新型的抑制剂多肽化合物可作为新一代治疗肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症药物。
本发明的技术方案如下：
本发明提供一种靶向于纤连蛋白衍生肽的抑制剂多肽化合物，含有以下氨基酸序列表示的母体肽:
R 1-Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14，
其中，
R 1为：亲脂性取代基或不存在；
Xa2＝Val或Iva；
Xa9＝Asp或Glu；
Xa10＝Glu或Asp；
Xa14＝Leu或Ala；
当R1不存在，Xa2＝Val，且Xa14＝Leu时，母体肽为非直链肽。
其中，当R 1为亲脂性取代基时，所述的母体肽的氨基酸序列中第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；所述的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7。
其中，当R 1不存在时，所述的母体肽的氨基酸序列中，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:
Figure PCTCN2021094680-appb-000001
Xa2＝Val或Iva；
Xa9＝Asp或Glu；
Xa10＝Glu或Asp；
Xa14＝Leu或Ala。
根据本发明的具体实施方式，所述母体肽的氨基酸序列选自SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、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和SEQ ID NO.25所示的氨基酸序列其中之一。
根据本发明的具体实施方式，所述母体肽中氨基酸序列的第1位Val的氨基所连接的结构为：
Figure PCTCN2021094680-appb-000002
Figure PCTCN2021094680-appb-000003
根据本发明的具体实施方式，本发明提供如下任一种多肽化合物：
化合物1(SEQ ID NO.1)：
(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
化合物2(SEQ ID NO.2)：
(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
化合物3(SEQ ID NO.3)：
(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
化合物4(SEQ ID NO.4)：
(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
化合物5(SEQ ID NO.5)：
(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
化合物6(SEQ ID NO.6)：
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala
化合物7(SEQ ID NO.7)：
(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
化合物8(SEQ ID NO.8)：
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala
化合物9(SEQ ID NO.9)：
(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
化合物10(SEQ ID NO.10)：
(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
化合物11(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-Leu-NH 2
化合物12(SEQ ID NO.12)：
(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
化合物13(SEQ ID NO.13)：
(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
化合物14(SEQ ID NO.14)：
(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
化合物15(SEQ ID NO.15)：
(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
化合物16(SEQ ID NO.16)：
(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
化合物17(SEQ ID NO.17)：
Figure PCTCN2021094680-appb-000004
化合物18(SEQ ID NO.18)：
Figure PCTCN2021094680-appb-000005
化合物19(SEQ ID NO.19)：
Figure PCTCN2021094680-appb-000006
化合物20(SEQ ID NO.20)：
Figure PCTCN2021094680-appb-000007
化合物21(SEQ ID NO.21)：
Figure PCTCN2021094680-appb-000008
化合物22(SEQ ID NO.22)：
Figure PCTCN2021094680-appb-000009
化合物23(SEQ ID NO.23)：
Figure PCTCN2021094680-appb-000010
化合物24(SEQ ID NO.24)：
Figure PCTCN2021094680-appb-000011
化合物25(SEQ ID NO.25)：
Figure PCTCN2021094680-appb-000012
本发明的另一方面是提供含有本发明的新型的抑制剂多肽化合物的药物组合物，以所述新型的抑制剂多肽化合物作为活性成分添加药学上可接受载体和/或辅料制成药物组合物。
本发明的再一方面是提供本发明的新型的抑制剂多肽化合物的药物用途。细胞和动物实验显示，本发明的新型的抑制剂多肽化合物没有不良反应发生，可用于器官纤维化及器官疾病伴随的纤维化病症的治疗；优选地，所述器官纤维化及器官疾病伴随的纤维化病症为：肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症。
本发明中提到的新型的抑制剂多肽化合物中的母体肽为同源性多肽。本发明中的同源性多肽是指，多肽本来具有天然母体肽的氨基酸序列，但其中一个或多个氨基酸残基己被不同的氨基酸残基取代，这些氨基酸残基彼此之间是保守的，所得到的多肽可用于实施本发明。并且细胞和动物实验显示，在质量数相同时与天然母体肽相比，本发明的抑制剂多肽化合物中的母体肽，具有更好的生物学、药学活性，其治疗效果优于天然母体肽。
本领域技术人员可以理解，本发明的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，可将本发明的多肽化合物的药物组合物制成各种合适的剂型，其中包含至少一种有效剂量的本发明的多肽化合物和至少一种药学上可接受的药用载体。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。
含有本发明多肽化合物的药物组合物可以制成溶液或者冻干粉末以用于胃肠外给药，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置，液体配方一般是缓冲液、等渗溶液和水溶液。
本发明的药物组合物的用量可以在一个较大范围内变动，本领域技术人员可以根据一些客观的因素，如根据疾病的种类、病情严重程度、病人体重、剂型以及给药途径等因素很容易的加以确定。
本发明的优点：
1)在质量数相同时，与天然母体肽相比，具有更好的生物学活性；
2)在质量数相同时，与天然母体肽相比，具有更好的治疗肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症的药效作用；
3)在质量数相同时，与天然母体肽相比，在药物的药代实验中显示出更显著的药物活性；
4)在质量数相同时，与天然母体肽相比，在药物的药代实验中显示出良好的安全性和稳定性；
5)合成产率高，稳定性好，易于放大生产，且成本低。
本发明中所用缩写具体含义如下：
Boc为叔丁氧羰基，Fmoc为芴甲氧羰基，t-Bu为叔丁基，resin为树脂，TFA为三氟乙酸，EDT为1,2-乙二硫醇，FBS为胎牛血清，BSA为牛血清白蛋白，HPLC为高效液相，mPEG为单甲氧基聚乙烯二醇，Ser为丝氨酸，D-Ser为D-型丝氨酸，Gln为谷氨酰胺，Gly为甘氨酸，Glu为谷氨酸，Ala为丙氨酸，Asp为天冬氨酸，Leu为亮氨酸，Pro为脯氨酸，Val为缬氨酸，Iva为异缬氨酸。
附图说明
图1为实施例2中，在LX2细胞模型中，对化合物1-25进行活性评价的统计柱状图(**：表示置信度>99％，两者差别有非常显著意义(P<0.01)；***：表示置信度>99.9％，两者差别有及其显著意义(P<0.001))。
图2为实施例3中，小鼠肝脏HE染色病理切片图。
图3为实施例3中，小鼠肝脏Collgen I免疫组化染色病理切片图和统计柱状图(**：表示置信度>99％，两者差别有非常显著意义(P<0.01)；***：表示置信度>99.9％，两者差别有及其显著意义(P<0.001))。
图4为实施例3中，小鼠肝脏天狼星红染色病理切片图和统计柱状图(*： 表示置信度>95％，两组差别有显著意义(P<0.05)；**：表示置信度>99％，两者差别有非常显著意义(P<0.01)；***：表示置信度>99.9％，两者差别有及其显著意义(P<0.001))。
图5为实施例4中，小鼠肺脏HE染色、Masson染色、α-SMA和Collagen1免疫组化病理切片图和统计柱状图(**：表示置信度>99％，两者差别有非常显著意义(P<0.01)；***：表示置信度>99.9％，两者差别有及其显著意义(P<0.001)；****：表示置信度>99.99％，两者差别有及其显著意义(p<0.0001))。
图6为实施例4中，ELISA检测各组小鼠血清中TGFβ和MMP9细胞因子的表达情况统计柱状图(**：表示置信度>99％，两者差别有非常显著意义(P<0.01)；***：表示置信度>99.9％，两者差别有及其显著意义(P<0.001)；****：表示置信度>99.99％，两者差别有及其显著意义(p<0.0001))。
图7为本发明化合物中桥接基团与肽链的连接示意图。
具体实施方式
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。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1多肽化合物的合成
以多肽化合物7和22的合成为例。
材料：
所有的氨基酸购自上海吉尔生化公司,树脂是Rink Amide MBHA(loading＝0.36mmol/g)购自西安蓝晓科技公司。如果没有特别说明，其他所有试剂均为分析纯，购自上海泰坦科技有限公司。Phenomenex Luna C18制备柱(20mm×250mm)用来纯化多肽。高效液相色谱仪为Thermofisher公司产品，型号为Ultimate 3000。质谱采用Agilent质谱仪，型号为1260-6120进行测定。
方法：
1.多肽化合物7的合成：
结构序列：
(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-P ro-Ala-NH 2
1)肽树脂的偶联：
按照Fmoc保护策略在多肽合成仪上合成0.72mmol规模的如下多肽：(PEG 2-PEG 2-γGlu(OtBu)-CO(CH 2) 18CO(OtBu))-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Rink Amide MBHA肽树脂
(1)第一步：树脂溶胀
将2g Rink amide MBHA树脂在N，N-二甲基甲酰胺(DMF)中溶胀，用N，N-二甲基甲酰胺洗溶胀树脂2次，每次15min；
(2)第二步：氨基酸偶联
以Rink Amide MBHA树脂为载体，以1-羟基苯并三唑(3x)和N，N-二异丙基碳二亚胺(3x)为偶联剂，以N，N-二甲基甲酰胺为溶剂，进行程序反应，依次进行缩合反应连接保护的氨基酸得到：
(PEG 2-PEG 2-γGlu(OtBu)-CO(CH 2) 18CO(OtBu))-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Rink Amide MBHA肽树脂，其中每次缩合反应中Fmoc保护氨基酸的投料量与树脂用量的物质的量比为3：1，每次缩合反应中1-羟基苯并三唑和N，N-二异丙基碳二亚胺与Fmoc保护氨基酸用量的物质的量比为1:1，脱保护溶液为20％哌啶的DMF溶液。偶联完后，用纯甲醇收缩2次，每次15min，真空抽干，得到肽树脂4.2g。
(3)第三步：多肽切割与脱保护
将4.2g肽树脂：
(PEG 2-PEG 2-γGlu(OtBu)-CO(CH 2) 18CO(OtBu))-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Rink Amide MBHA肽树脂加入至圆底烧瓶中,在冰浴条件下，加入切割液TFA/TIS/H 2O＝95/2.5/2.5(v/v/v)45ml，升温，控制裂解液温度25℃,搅拌反应120分钟。过滤，滤液在搅拌下倒入冰乙醚中。静置1.0小时以上,待沉淀完全，离心，倾去上清液，沉淀用N 2吹干后，真空抽过夜干燥，得到粗品化合物7共1.2g：肽序为：(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。
2)纯化转盐
将上述第三步中所得粗品1.2g,用5.0％乙酸在乙腈：H 2O＝1:1(体积比)的溶液20ml超声溶解，溶解澄清后用0.45μm的聚四氟乙烯膜过滤，得到过滤 后C5滤液。滤液通过20mm反相C18的填充的20mm x 250mm柱上进行2次半制备型HPLC而纯化。用40-60％乙腈-0.1％三氟乙酸/H 2O梯度以19mL/min将该柱洗脱60.0分钟，收集含有C5的组分，浓缩除去乙腈后进行转盐冻干。得到HPLC纯度为98.83％的纯品145mg。用液质联用分析分离出的产物，用液质联用分析分离出的产物，发现质子化分子离子峰的m/2z值为:1050.4,理论值为2099.4。
2.多肽化合物22的合成：
序列结构为：
Figure PCTCN2021094680-appb-000013
1)线性肽主肽链的偶联：
按照Fmoc保护策略在多肽合成仪上合成0.72mmol规模的如下线性肽树脂：
Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-γGlu(OAll)-Rink Amide MBHA肽树脂
(1)第一步：树脂溶胀
将2g Rink amide MBHA树脂在N，N-二甲基甲酰胺(DMF)中溶胀，用N，N-二甲基甲酰胺洗溶胀树脂2次，每次15min；
(2)第二步：氨基酸偶联
以Rink Amide MBHA树脂为载体，以1-羟基苯并三唑(3x)和O-苯并三氮唑-四甲基脲六氟磷酸盐(3x)为偶联剂，以N，N-二甲基甲酰胺为溶剂，进行程序反应，依次进行缩合反应连接保护的氨基酸得到：Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-γGlu(OAll)-Rink Amide MBHA线性肽树脂，其中每次缩合反应中N-Fmoc保护氨基酸的投料量与树脂用量的物质的量比为3：1，每次缩合反应中1-羟基苯并三唑和O-苯并三氮唑-四甲基脲六氟磷酸盐与N-Fmoc保护氨基酸用量的物质的量比为3:1，脱保护溶液为20％哌啶的DMF溶液。
(3)第三步：脱OAll保护基
将所得:
Glu(OtBu)-Glu(OtBu)-Ala-Ser(tBu)-Pro-Ala-Val-Iva-Gly-Ser(tBu)-Pro-Ser(tBu)-Ala-γGlu(OAll)-Rink Amide MBHA肽树脂分散于DMF溶液当中，加入0.15当量的四三苯基膦钯，10当量吗琳，氮气氛围下反应12h，随后依次用DCM(10mL*3)、DMF(10mL*3)洗涤，抽干。
(4)第四步：首尾酰胺环化
以1-羟基苯并三唑(3x)和O-苯并三氮唑-四甲基脲六氟磷酸盐(3x)为偶联剂，以N，N-二甲基甲酰胺为溶剂反应4h。偶联完后，用纯甲醇收缩2次，每次15min，真空抽干，称重，得到肽树脂3.8g。
(5)第五步：多肽切割与保护基的脱除
将(4)中所得树脂3.8g转移至圆底烧瓶中,冰浴下，加入切割液TFA/TIS/H 2O＝95/2.5/2.5，(V/V/V)40ml,升温，控制裂解液温度25℃,反应120分钟。过滤，滤饼用少量三氟乙酸洗涤3次，合并滤液。滤液在搅拌下缓慢倒入冰乙醚中。静置1.0小时以上,待沉淀完全。离心，所得沉淀用冰乙醚洗涤3次，得到沉淀物，沉淀用N2吹干后，真空抽过夜干燥，得到粗品化合物22共1.0g。
2)纯化转盐：
将上述第五步中所得粗品1.0g,用30ml纯水超声溶解，溶解后用0.45μm的聚四氟乙烯膜过滤，得到过滤后22滤液。滤液通过20mm反相C18的填充的20mm x 250mm柱上进行2次半制备型HPLC而纯化。用15-35％乙腈-0.1％三氟乙酸/H 2O梯度以19mL/min将该柱洗脱60.0分钟，收集含有多肽22的组分，浓缩除去乙腈后进行转盐冻干。得到HPLC纯度为99.92％的纯品90mg。用液质联用分析分离出的产物，发现质子化分子离子峰的m/z+1值为:1311.25,理论值为1309.62。
基于上述步骤合成的多肽化合物，如下表1所示：
表1.本发明的多肽化合物
Figure PCTCN2021094680-appb-000014
Figure PCTCN2021094680-appb-000015
Figure PCTCN2021094680-appb-000016
Figure PCTCN2021094680-appb-000017
实施例2在LX2细胞模型中，对化合物1-25进行活性的评价
在LX2细胞模型中(因为LX2细胞是人肝活化星状细胞，所以可以直接作为体外肝脏纤维化细胞评价模型，LX2细胞购自中国科学院典型培养物保藏中心，中国上海)，对多肽化合物1-25进行活性的抗纤维化活性进行初筛，检测成肌纤维细胞活化的标志物α-SMA蛋白的表达情况，多肽化合物1-25采用(1.0μM和10.0μM)两个浓度进行细胞孵育48h。对照组给予同样体积的Dulbecco’s改良Eagle培养基(DMEM)(购自Gibco公司，美国)，GAPDH(购自北京全式金生物有限公司)作为内参。其中α-肌动蛋白(α-SMA)是肌纤维母细胞区别于成肌纤维细胞的一个标志性的特征。
实验结果如图1所示，从实验结果的统计图可以明显看出，与GAPDH内参相比，本发明的多肽化合物均有不同程度地对α-SMA具有显著抑制改善活性。其中多肽化合物1、5、7、11、12、13、17、18、21和22对α-SMA均具有更好的抑制改善活性。实验结果也表明：本发明的抑制剂多肽化合物在细胞层面能抑制纤维化的生成，同时也提示：该类新型的抑制剂多肽化合物可潜在的用于器官纤维化及器官疾病伴随的纤维化病症的研究。
实施例3多肽化合物1、5、7、11、12、13、17、18、21和22在四氯化碳(CCl 4)诱导的小鼠肝纤维化模型上的药效学评价实验
1.受试药物：多肽化合物类似物。保存条件-20℃。
造模方法：将72只雄性C57BL/6J小鼠，雄性，体重18-22g，周龄8周，由中山大学实验动物中心提供，随机分为12组，分别为：1)对照组(Oil)+生理盐水，腹腔注射，n＝6；2)模型对照组(CCl 4)+生理盐水，腹腔注射，n＝6；给予小鼠CCl 4三天一次，共6周；3)模型给药组(CCl 4)+100.0μg/kg系列化合物(10组)，腹腔注射，n＝6/组。其中给予小鼠20％的CCl 4，按照小鼠体重5.0μL/g的给药体积，对照组小鼠给予相同体积和频率的注射玉米油。给药频率：给予小鼠CCl 4三天一次，共6周；当模型给药组给予小鼠CCl 43周后，从第4周开始分别给予多肽化合物1、5、7、11、12、13、17、18、21和22，共3周。其中CCl 4和Oil购自于上海阿拉丁生化科技股份有限公司。
2.药效评价：
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，肝细胞水肿变性，汇管区及肝小叶间隔有大量胶原纤维沉积等。在给药三周之后，处理小鼠并进行取材，通过眼球后静脉取血进行血清学指标检测，并取肝脏组织用于病理学分析。
3.实验方法：
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。
天狼星红染色：烘片、脱蜡；双蒸水中静置5.0分钟；暗室中天狼星红染色60-80分钟；0.5％冰醋酸涮洗5s；脱水透明，封片，拍照。
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3.0％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分钟。利用1.0％BSA对组织进行封闭1h。去除1.0％BSA，按说明书推荐比例滴加Collagen I(I型胶原蛋白)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60分钟，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5分钟，1％盐酸乙醇分化，再用自来水洗5分钟，脱水，风干，封片，拍照(H&E染色液和天狼星红染色液购自于上海生工生物工程有限公司，Collagen I抗体购自于CST公司)。
4.结果分析：
参照图2-4，结果显示：当模型对照组(CCl 4)+生理盐水，腹腔注射给予小鼠CCl 4三天一次，共6周。苏木素-伊红(H&E)染色，天狼星红染色和Collgen I免疫组化可以看出，由CCl 4诱导之后出现的小鼠肝脏炎性细胞浸润，肝脏纤维化明显。
其中，图2中显示了：模型给药组(CCl 4)+100.0μg/kg系列化合物1、5、7、11、12、13、17、18、21和22，在上述化合物给药治疗后，各治疗模型组小鼠肝脏炎性细胞聚集均得到了显著的改善；各模型给药组小鼠肝脏纤维化在给药1、5、7、11、12、13、17、18、21和22之后均得到了显著的改善。说明化合物1、5、7、11、12、13、17、18、21和22都可以很好的抑制ECM的积累，治疗改善肝脏纤维化。
图3中显示了：模型给药组(CCl 4)+100.0μg/kg系列化合物1、5、7、11、12、13、17、18、21和22，在上述化合物给药治疗后，各治疗模型组小鼠肝脏Collagen I的表达量均出现了明显的治疗改善。
图4中显示了：模型给药组(CCl 4)+100.0μg/kg系列化合物1、5、7、11、12、13、17、18、21和22，在上述化合物给药治疗后，各治疗模型组小鼠肝脏胶原沉积均出现了明显的治疗改善。
实施例4多肽化合物1、5、7、11、12、13、17、18、21和22在博来霉素(BLM)诱导的小鼠肺纤维化模型上的药效学评价
1.受试药物：多肽化合物类似物。保存条件-20℃。
造模方法：将78只雄性C57BL/6J小鼠，周龄6-8周，体重16-18g，由中山大学实验动物中心提供，随机分为13组，分别为：1)正常对照组(Normal)，n＝6；2)假手术盐水对照组(Saline)，n＝6；3)博来霉素模型组(BLM)，n＝6； 4)模型治疗组：给予小鼠150μg/kg系列化合物，共10组，n＝6/组。在博来霉素诱导的小鼠肺纤维化术后第7天，对模型治疗组小鼠进行给药处理(给药频率：每天给药/150μg/kg，腹腔注射)，给药14天后取材，记录进行病理观察。
2.药效评价：
在博来霉素(购自梯希爱(上海)化成工业发展有限公司)诱导的肺纤维化模型中，其表现的病理特征：支气管扩张，支气管壁上皮细胞变性水肿，肺泡间质明显水肿增宽，大量成纤维细胞和胶原组织沉积。肺泡结构几乎完全消失，被纤维结缔组织填充，伴有炎症细胞浸润。模型治疗组在给药14之后，处理小鼠并进行取材，通过眼球后静脉取血进行血清学指标检测，并取肝脏组织用于病理学分析。
3.实验方法：
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。
Masson染色：烘片、脱蜡；媒染；天青石蓝染色液滴染2-3min、水洗；Mayer苏木素染色液滴染、水洗；酸性乙醇分化液分化数秒、自来水冲洗；丽春红品红染液滴染、蒸馏水冲洗；磷钼酸溶液处理；滴入苯胺蓝染液5min；弱酸溶液处理2min；脱水透明、晾干、树胶封片。
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3.0％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分钟。利用1.0％BSA对组织进行封闭1h。去除1.0％BSA，按说明书推荐比例滴加Collagen I(I型胶原蛋白)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60分钟，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5分钟，1％盐酸乙醇分化，再用自来水洗5分钟，脱水，风干，封片，拍照(H&E染色液和天狼星红染色液购自于上海生工生物工程有限公司，Collagen I抗体购自于CST公司)。
4.结果分析：
参照图5，结果显示：H&E染色：正常组对照组和假手术盐水对照组小鼠肺组织结构清晰，肺泡壁未见增厚，肺泡上皮结构完整，未见炎性细胞浸润；Masson染色：正常组对照组和假手术盐水对照组仅有少量胶原纤维被染成蓝色。跟博来霉素模型组相比，模型治疗组各组小鼠在分别给予150.0μg/kg系列化合物1、5、7、11、12、13、17、18、21和22治疗后，各治疗模型组小鼠肺纤维化得到了显著的治疗改善。给药14天后，H&E染色、Masson染色可以看出：博来霉素组肺泡间隔明显增宽，可见大量成纤维细胞及基质填充；给予1、5、7、11、12、13、17、18、21和22多肽化合物治疗后可见各治疗组肺脏纤维化程度较博来霉素对照组显著减轻。
从α-SMA、Collagen1免疫组化观察到博来霉素组肺泡间隔明显增宽，可见大量黄染的纤维连接蛋白填充在肺实质内；给予1、5、7、11、12、13、17、18、21和22多肽化合物治疗后肺实质内有纤维连接蛋白较博来霉素组明显减少。说明给药治疗后显著抑制了肺组织α-SMA、Collagen 1的表达。以上结果说明了，多肽化合物1、5、7、11、12、13、17、18、21和22能够显著治疗改善肺纤维化。
纤维化疾病的发生发展过程与TGF-β和MMP-9诱导的EMT过程有关。基质金属蛋白酶(MMPs)是降解基质的蛋白酶，降解细胞外基质的所有成分。它们参与肺纤维化的病理生理过程，在细胞外基质的异常重构和基底膜的破坏中发挥重要作用，促进炎症细胞和成纤维细胞的募集。在肺纤维化动物模型和患者中MMP‐9的表达增加，高于正常的MMP‐9水平可能会破坏正常的组织结构，并增加炎性细胞向疾病部位的迁移。为了进一步探讨多肽药物对博来霉素诱导的肺纤维化的抑制作用机制，我们通过ELISA检测各组小鼠血清中TGFβ、MMP9细胞因子的表达情况。参照图6-A，与正常对照组和假手术盐水对照组相比，博来霉素模型组肺MMP‐9显著增加；给予1、5、7、11、12、13、17、18、21和22多肽化合物均能显著降低小鼠血清中MMP9的浓度，这可能与肺纤维化的减弱有关。我们推测其机理应该与TGF-β通路相关，参见图6-B，ELISA的结果表明，1、5、7、11、12、13、17、18、21和22多肽化合物均能显著降低小鼠血清中TGF-β的浓度。
综合上述实验结果表明，本发明的多肽化合物抑制了ECM的积累以及肺组织α-SMA、Collagen 1的表达，可用于治疗肝纤维化及肝脏疾病伴随的纤维化病症和特发性肺纤维化及肺脏疾病伴随的纤维化病症。

Claims (11)

  1. 一种靶向于纤连蛋白衍生肽的抑制剂多肽化合物，其含有以下氨基酸序列表示的母体肽:
    R 1-Val-Xa2-Gly-Ser-Pro-Ser-Ala-Gln-Xa9-Xa10-Ala-Ser-Pro-Xa14，
    其中，
    R 1为：亲脂性取代基或不存在；
    Xa2＝Val或Iva；
    Xa9＝Asp或Glu；
    Xa10＝Glu或Asp；
    Xa14＝Leu或Ala；
    当R1不存在，Xa2＝Val，且Xa14＝Leu时，母体肽为非直链肽。
  2. 根据权利要求1所述的多肽化合物，其中所述R 1为亲脂性取代基时，所述氨基酸序列中第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；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。
  3. 根据权利要求1所述的多肽化合物，其中所述R 1不存在时，所述氨基酸序列中的第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，所述环肽化合物的结构表示如下:
    Figure PCTCN2021094680-appb-100001
    Xa2＝Val或Iva；
    Xa9＝Asp或Glu；
    Xa10＝Glu或Asp；
    Xa14＝Leu或Ala。
  4. 根据权利要求1所述的多肽化合物，其中所述母体肽的氨基酸序列选自SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、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和SEQ ID NO.25所示的氨基酸序列其中之一。
  5. 根据权利要求2所述的多肽化合物，其中所述母体肽的氨基酸序列中第1位Val的氨基所连接的结构为：
    Figure PCTCN2021094680-appb-100002
  6. 根据权利要求1所述的多肽化合物，其中所述多肽化合物为如下化合物1-25中的任一种：
    化合物1(SEQ ID NO.1)：
    (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
    化合物2(SEQ ID NO.2)：
    (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
    化合物3(SEQ ID NO.3)：
    (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
    化合物4(SEQ ID NO.4)：
    (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
    化合物5(SEQ ID NO.5)：
    (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
    化合物6(SEQ ID NO.6)：
    (PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Asp-Glu-Ala-Ser-Pro-Ala
    化合物7(SEQ ID NO.7)：
    (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
    化合物8(SEQ ID NO.8)：
    (PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-Val-Iva-Gly-Ser-Pro-Ser-Ala-Gln-Glu-Glu-Ala-Ser-Pro-Ala
    化合物9(SEQ ID NO.9)：
    (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
    化合物10(SEQ ID NO.10)：
    (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
    化合物11(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-Leu-NH 2
    化合物12(SEQ ID NO.12)：
    (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
    化合物13(SEQ ID NO.13)：
    (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
    化合物14(SEQ ID NO.14)：
    (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
    化合物15(SEQ ID NO.15)：
    (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
    化合物16(SEQ ID NO.16)：
    (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
    化合物17(SEQ ID NO.17)：
    Figure PCTCN2021094680-appb-100003
    化合物18(SEQ ID NO.18)：
    Figure PCTCN2021094680-appb-100004
    化合物19(SEQ ID NO.19)：
    Figure PCTCN2021094680-appb-100005
    化合物20(SEQ ID NO.20)：
    Figure PCTCN2021094680-appb-100006
    化合物21(SEQ ID NO.21)：
    Figure PCTCN2021094680-appb-100007
    化合物22(SEQ ID NO.22)：
    Figure PCTCN2021094680-appb-100008
    化合物23(SEQ ID NO.23)：
    Figure PCTCN2021094680-appb-100009
    化合物24(SEQ ID NO.24)：
    Figure PCTCN2021094680-appb-100010
    化合物25(SEQ ID NO.25)：
    Figure PCTCN2021094680-appb-100011
  7. 一种组合物，其中包含权利要求1至6任一项所述的多肽化合物。
  8. 根据权利要求7所述的组合物，其中所述组合物为药物组合物，其任选地还包含药学上可接受的载体或辅料。
  9. 一种权利要求1至6任一项所述的多肽化合物在制备预防或治疗器官纤维化及器官疾病伴随的纤维化病症的药物中的用途。
  10. 根据权利要求9所述的用途，其中所述器官纤维化及器官疾病伴随的纤维化病症为特发性肺纤维化及肺脏疾病伴随的纤维化病症。
  11. 根据权利要求9所述的用途，其中所述器官纤维化及器官疾病伴随的纤维化病症为肝纤维化及肝脏疾病伴随的纤维化病症。
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