WO2015096756A1 - Utilisation de polypeptide et de dérivés de ce dernier pour préparer des médicaments anti-fibrose pulmonaire - Google Patents

Utilisation de polypeptide et de dérivés de ce dernier pour préparer des médicaments anti-fibrose pulmonaire Download PDF

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WO2015096756A1
WO2015096756A1 PCT/CN2014/094887 CN2014094887W WO2015096756A1 WO 2015096756 A1 WO2015096756 A1 WO 2015096756A1 CN 2014094887 W CN2014094887 W CN 2014094887W WO 2015096756 A1 WO2015096756 A1 WO 2015096756A1
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pulmonary fibrosis
seq
protein
pep2
polypeptide
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胡卓伟
吕晓希
李珂
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胡卓伟
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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
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to the application of a polypeptide and a derivative thereof for preparing an anti-pulmonary fibrosis medicine.
  • Tissue fibrosis is a common consequence of tissue damage caused by pathogens inside and outside, and is also a fundamental pathological change in a variety of infectious and non-infectious inflammatory diseases.
  • Tissue fibrosis is present in respiratory diseases, circulatory diseases, kidney and liver diseases, chronic pancreatitis, systemic lupus erythematosus and macular degeneration, and often increases tissue burden.
  • Tissue fibrosis affects almost all organs and systems in the human body and is the leading cause of death in a large number of patients, posing a serious threat to human health.
  • tissue fibrosis According to relevant statistics in the United States, nearly 45% of patients who die from various diseases worldwide can be classified as caused by various tissue fibrosis, so tissue fibrosis starts in the process of the occurrence and development of major organs in the human body. Play an important role. Pulmonary fibrosis disease is caused by hundreds of different causes such as toxic substances, spontaneous immune diseases, side effects of drugs, infections, severe trauma, lung inflammation, persistent alveolar damage, repeated destruction of extracellular matrix, repair, reconstruction and excessive deposition. A type of disease that causes normal lung tissue changes and loss of function. Currently, tissue fibrosis remains untargeted, safe and effective treatment options.
  • Pulmonary fibrosis is one of the most serious pathological conditions in the lungs. Most of its pathological changes are the initial lower airway inflammation, as well as alveolar epithelial cells and vascular endothelial cell damage, accompanied by fibroblasts and type II alveolar cell proliferation, cells. Factor release, extracellular matrix protein and collagen deposition eventually lead to lung changes. In the pulmonary fibrosis patients, the lung alveoli are gradually replaced by fibrous substances, resulting in the hardening and thickening of the lung tissue, the gradual loss of lung gas exchange capacity, resulting in patients with different degrees of hypoxia and breathing difficulties, and finally died of respiratory failure. Pulmonary fibrosis is one of the four major diseases of respiratory diseases.
  • the etiology is complicated and the pathogenesis is unknown.
  • the existing drugs and methods for treating pulmonary fibrosis are very limited, and the curative effect is unsatisfactory.
  • the prognosis is very poor.
  • the 5-year survival rate is only 50. %.
  • This process mainly binds to LC3 through the LIR (LC3-Interacting Region, LIR) domain of P62 protein, mediates the degradation of ubiquitinated proteins, and the expression level of P62 also decreases.
  • LIR LC3-Interacting Region
  • the ubiquitinated protein bound to P62 cannot be degraded in time, and it is accumulated in the cytoplasm and the expression is increased.
  • TRB3 protein Tribbles 3
  • TRB3 protein can interact with P62 protein, which blocks the binding of other ubiquitinated proteins to P62 protein, resulting in inhibition of autophagy pathway.
  • the internal collagen could not be removed, which aggravated the pathological changes of pulmonary fibrosis. Therefore, research and development of inhibitors of TRB3 protein or substances that block its binding to P62 protein have a good potential for the preparation of drugs for preventing or treating pulmonary fibrosis.
  • the technical problem to be solved by the present invention is to provide a polypeptide or a derivative thereof capable of specifically binding to a TRB3 protein in the preparation of a medicament for preventing or treating pulmonary fibrosis, in view of the current lack of an effective TRB3 protein inhibitor.
  • the present invention provides a technical solution for: a polypeptide capable of specifically binding a TRB3 protein or a derivative thereof for preparing a medicament for preventing or treating pulmonary fibrosis, which specifically binds to a TRB3 protein
  • the polypeptide is a polypeptide as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing; the derivative of the polypeptide which specifically binds to the TRB3 protein is specifically bindable A chimeric peptide formed by the polypeptide of the TRB3 protein and the cell penetrating peptide.
  • pulmonary fibrosis refers to pulmonary inflammation, particularly alveolar wall inflammation, due to toxic substances, spontaneous immune diseases, side effects of drugs, infection, severe trauma, and the like, and alveolar persistent damage, extracellular Repeated destruction, repair, reconstruction and excessive deposition of the matrix, that is, the formation of a large number of fibrous connective tissue and pulmonary structural disorder in the pulmonary interstitial, leading to a disorder of normal lung tissue structure and loss of function.
  • the pulmonary fibrosis is usually primary (specific) pulmonary fibrosis, that is, unexplained pulmonary fibrosis; or secondary pulmonary fibrosis, which is secondary to multiple causes. Pulmonary Fibrosis.
  • the cause may be: lung infection, trauma, dust, radiation or drugs (eg bleomycin).
  • the pulmonary fibrosis can be manifested as: pulmonary inflammation, especially interstitial pneumonia, deterioration of lung function such as chronic obstructive pulmonary disease (COPD) and lung injury (such as pulmonary interstitial formation of massive fibrous connective tissue and pulmonary structural disorder) One or more.
  • COPD chronic obstructive pulmonary disease
  • lung injury such as pulmonary interstitial formation of massive fibrous connective tissue and pulmonary structural disorder
  • the polypeptide of the present invention which specifically binds to the TRB3 protein is SEQ ID NO: 1 of the sequence listing (the amino acid sequence thereof is: Ser-Leu-Ser-Gln-Met-Leu-Ser-Met), SEQ ID NO: 2 ( Its amino acid sequence is: Gly-Gly-Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys) and SEQ ID NO: 3 (the sequence of amino acids is: A polypeptide represented by any one of Ile-Gly-Ala-Ala-Leu-Asp-Thr-Ile) may be appropriately introduced with an amino acid substitution, deletion or addition as long as the altered amino acid sequence is capable of forming a specific binding to the TRB3 protein. The polypeptide and the polypeptide still retain the activity before the change.
  • the cell penetrating peptide of the present invention is a cell penetrating peptide which is conventional in the art, as long as the cell penetrating peptide can assist in feeding the polypeptide into a cell to function, generally, the cell is worn.
  • the membrane peptide is a short peptide molecule composed of 10 to 30 amino acids.
  • the cell penetrating peptide is preferably: HLYVSPW (as shown in SEQ ID NO: 4 in the Sequence Listing, the cell penetrating peptide is named pep2 peptide, abbreviated as pep2), HIV-1 virus reverse transcription activator Trans-activator transcription (Tat) protein TAT peptide (YGRKKRRQRRR, its amino acid sequence is shown as SEQ ID NO: 5, referred to as TAT peptide), Drosophila antennae homeopathic protein transcription factor Antp peptide (RQIKIWFQNRRMKWKK, its amino acid sequence As shown in SEQ ID NO: 6, abbreviated as Antp peptide), pep-1 peptide (KETWWETWWTEWSQPKKKRKV, whose amino acid sequence is shown as SEQ ID NO: 7), MPG peptide (GALFLGFLGAAGSTMGAWSQPKSKRKV, the amino acid sequence of which is shown in SEQ ID: 8) And one or more of the RGD peptid
  • the polypeptide derivative is linked to the C-terminus of the cell penetrating peptide (the cell penetrating peptide, preferably a pep2 peptide, a TAT peptide or an Antp peptide) in SEQ ID NO: 1, A chimeric peptide formed after the polypeptide set forth in any one of SEQ ID NO: 2 and SEQ ID NO: 3.
  • the cell penetrating peptide is preferably linked to the N-terminus or C-terminus of the above polypeptide, more preferably to the N-terminus of the above polypeptide.
  • the amino acid sequence of the chimeric peptide is preferably SEQ ID NO: 10 (the amino acid sequence of which is: HLYVSPWGGSLSQMLSM), SEQ ID NO: 11 (the amino acid sequence is: HLYVSPWGGGGWLTRLLQTK), SEQ ID NO: 12
  • the amino acid sequence is: HLYVSPWGGIGAALDTI
  • SEQ ID NO: 13 the amino acid sequence is: YGRKKRRQRRRGGGGWLTALLQTK
  • SEQ ID NO: 14 the amino acid sequence of which is: RQIKIWFQNRRMKWKKGGGGWLTRLLQTK.
  • the polypeptide of the present invention and derivatives thereof are used as an active ingredient for the preparation of a medicament for preventing or treating pulmonary fibrosis.
  • the medicament for preventing or treating pulmonary fibrosis comprises a prophylactically or therapeutically effective amount of one or more of the polypeptides of the present invention and derivatives thereof, wherein "prophylactically or therapeutically effective amount” means The amount of compound that is sufficient to effectively prevent or treat the disease or condition described herein is administered to a subject in need thereof. While the amount of the compound that constitutes a "prophylactically or therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, it can be determined in a conventional manner by those skilled in the art.
  • the dose of the medicament of the present invention is preferably 0.1 to 15 mg/kg, more preferably 5 to 10 mg/kg, and preferably 5 mg/kg, and the number of administrations is preferably once a day or several times a day. Times.
  • the polypeptide or a derivative thereof which specifically binds to the TRB3 protein can be prepared with a pharmaceutically acceptable carrier for preventing or treating a pulmonary fibrosis drug.
  • the pharmaceutically acceptable carrier described therein can be conventionally selected depending on the pharmaceutical dosage form. For example, a filler, a diluent, and the like.
  • the polypeptide which can specifically bind to the TRB3 protein or a derivative thereof can be used as a single active ingredient or in combination with other compounds having activity for preventing or treating pulmonary fibrosis as an active ingredient.
  • the pharmaceutical dosage form for preventing or treating pulmonary fibrosis according to the present invention is not particularly limited and is a conventional dosage form in the art, preferably solid, semi-solid or liquid, and may also be an aqueous solution, a non-aqueous solution or a suspension, more preferably It is a tablet, capsule, granule, injection or infusion.
  • the route of administration of the drug is a conventional route of administration in the art, preferably injection or oral administration.
  • the manner of administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection routes.
  • “Prophylaxis” as used herein means preventing or reducing the production of pulmonary fibrosis after use in the presence of possible pulmonary fibrosis factors. “Treatment” as used herein means reducing the extent of pulmonary fibrosis, or healing pulmonary fibrosis to normalize it, or slowing the progression of pulmonary fibrosis.
  • Required subject refers to a warm-blooded animal that may have the disease or condition described herein in the presence of a possible pulmonary fibrotic factor; or a warm-blooded animal, such as a mammal, having the diseases and conditions described herein.
  • the present invention is preferably human or mouse.
  • the reagents and starting materials used in the present invention are commercially available.
  • a positive progressive effect of the present invention is that the present invention provides a polypeptide which specifically binds to a TRB3 protein (the sequence of which is as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing) and
  • the use of the derivative in the preparation of a medicament for preventing or treating pulmonary fibrosis can effectively improve the lung function of a patient with pulmonary fibrosis, and the curative effect is remarkable, and the medicament has the advantages of less toxic side effects and safe use.
  • Figure 1 is a graph showing high expression of TRB3 protein in lung tissue of mice with pulmonary fibrosis.
  • Fig. 2(A) and Fig. 2(B) are diagrams showing the interaction between TRB3 protein and p62 in the lung tissue of lung fiber mice.
  • Fig. 2(A) is the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate.
  • Fig. 2(B) is a graph showing the protein amount of the TRB3 protein and the P62 protein after the lung tissue lysate was precipitated by the P62 antibody or the control antibody IgG.
  • Fig. 3(A) and Fig. 3(B) are diagrams showing the phagocytosis of collagen by lung epithelial cells by flow cytometry.
  • Fig. 3(A) is a flow chart of cells
  • Fig. 3(B) is flow cytometry. Results statistics.
  • Figure 4 shows that pep2-A1, pep2-A2, and pep2-A3 reduce the mortality test of bleomycin-induced pulmonary fibrosis in mice. Fruit map.
  • Fig. 5(A) and Fig. 5(B) are diagrams showing the interaction of peb2-A1, pep2-A2 and pep2-A3 in the lung TRB3 protein and p62 protein in bleomycin-induced pulmonary fibrosis mice
  • Fig. 5 (A) shows the protein content of TRB3 protein and P62 protein contained in the initial lung tissue lysate
  • Fig. 5(B) is a diagram showing the protein amount of the TRB3 protein and the P62 protein precipitated by the P62 antibody or the control antibody IgG after the lung tissue lysate was treated with pep2-A1, pep2-A2, and pep2-A3, respectively.
  • Figure 6 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing lung weight index in mice with pulmonary fibrosis induced by bleomycin.
  • Figure 7 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing the number of various inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin.
  • A is the total number of white blood cells
  • B is the number of monocytes
  • C is the number of neutrophils
  • D is the number of lymphocytes
  • E is the number of basophils
  • F is the number of eosinophils.
  • Figure 8 is a pathological examination (HE) diagram of pep2-A1, pep2-A2, and pep2-A3 to reduce bleomycin-induced pulmonary fibrosis.
  • A is the sham operation group
  • B is the model group
  • C is the pep2-A1 group
  • D is the pep2-A2 group
  • E is the pep2-A3 group
  • F is the positive control drug pirfenidone group.
  • Figure 9 is a graph showing the results of pathological examination scores of pep2-A1, pep2-A2, and pep2-A3 for reducing bleomycin-induced pulmonary fibrosis.
  • Figure 10 is a graph showing the pathological examination of bleomycin-induced pulmonary fibrosis (Sirius Red) by pep2-A1, pep2-A2, and pep2-A3.
  • A is the sham operation group
  • B is the model group
  • C is the pep2-A1 group
  • D is the pep2-A2 group
  • E is the pep2-A3 group
  • F is the positive control drug pirfenidone group.
  • Figure 11 is a graph showing the effect of pep2-A1, pep2-A2, and pep2-A3 on collagen content in lung tissue of mice with pulmonary fibrosis.
  • Figure 12 is a graph showing the results of lung function tests in mice with pulmonary fibrosis. Where A is the deep inspiratory volume, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance.
  • Figure 13 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin.
  • Figure 14 is a graph showing the results of TAT-A2 and Antp-A2 in reducing mortality in mice with pulmonary fibrosis induced by bleomycin. Among them: A is the sham operation group, B is the model group, C is the TAT-A2 group, and D is the Antp-A2 group.
  • Figure 15 is a graph showing the results of TAT-A2 and Antp-A2 in reducing lung weight index in mice with pulmonary fibrosis induced by bleomycin.
  • Figure 16 is a graph showing the results of TAT-A2 and Antp-A2 in reducing the number of inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin.
  • A is the total number of white blood cells
  • B is the number of monocytes
  • C is the number of neutrophils
  • D is the number of lymphocytes
  • E is the number of eosinophils
  • F is the number of basophils.
  • Figure 17 shows that TAT-A2 and Antp-A2 reduce the hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. Fruit map.
  • the bleomycin used in the experiment was purchased from Nippon Kayaku, batch number X81040.
  • Pirfenidone purchased from Dalian Meilun Biotechnology Co., Ltd., is a raw material drug with a pirfenidone content of >98.5%.
  • the peptides of the amino acid sequences shown in SEQ ID NO: 1 (A1), SEQ ID NO: 2 (A2) and SEQ ID NO: 3 (A3) of the Sequence Listing are ligated to the cell penetrating peptide pep2 (penetrating peptide pep2, respectively).
  • the amino acid sequence is HLYVSPW, as shown in SEQ ID NO: 4 in the Sequence Listing, and constitutes the new derivatives pep2-A1, pep2-A2, pep2-A3, and the above polypeptides or chimeric peptides are all limited by Beijing Cypress Biotechnology.
  • the company is synthesizing.
  • A1, A2 and A3 are linked to the C-terminus of pep2 by two glutamic acid chains.
  • the sequence structure of the above chimeric peptides is as follows:
  • the pep2-A1 sequence is:
  • pep2-A2 The sequence of pep2-A2 is:
  • the sequence of pep2-A3 is:
  • mice Male C57BL/6 (6-8 weeks old) mice were fasted overnight, anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and intratracheally injected with bleomycin (5 U/kg).
  • the specific scheme is as follows: the neck skin is cut with as little trauma as possible, the trachea is exposed with the assistance of the elbow ophthalmology, the trachea is pierced using a micro-injector, about 50 ⁇ l of bleomycin is injected into the trachea, and the tube is rapidly rotated. Stand upright for 5 minutes to allow bleomycin to enter the left and right lobe evenly. The entire operation is performed at a surgical table of about 60 °C. The sham operation group was intratracheally injected with an equal amount of physiological saline for injection.
  • lysis buffer [containing 0.1 mM EDTA (ethylenediaminetetraacetic acid), 0.1 mM EGTA (ethylene glycol diethyl ether diamine tetraacetic acid), 10 mM KCl (chlorine) Potassium) and 10 mM HEPEs (4-hydroxyethylpiperazineethanesulfonic acid) and 50 mM NaF (sodium fluoride), 0.1 M Na 3 VO 4 (sodium vanadate), 0.1 M Na 4 P 2 O 7 (sodium pyrophosphate) , protease inhibitor 1 ⁇ mol/L of Aprotinin (inhibitory enzyme), Trypsin inhibitor (trypsin inhibitor), PMSF (phenylmethanesulfonyl fluoride), Leupeptins (leucine) and DTT (dithiothreitol) After homogenization, place on ice for 15 min, or
  • the immunoprecipitation reagents are as follows:
  • Lysate B solution 200 ⁇ L of 2 M ⁇ -glycerophosphate, 4 mL of 2.5 M NaF, 2 mL of 100 mM NaVO 3 , 2 mL of 100 mM PMSF, 200 ⁇ L of 1 M DTT, 200 ⁇ L of 1 mg/mL of Leu, Pep, and Apr, total volume of 9 mL.
  • the mother liquor was stored at -20 °C. Before use, the mother liquor of each component in the B solution was thawed, and added to the solution A in the above composition ratio and mixed.
  • Protein A/G Plus-Agarose was purchased from Santa Cruz, USA.
  • the washing was repeated 5 times and allowed to stand for 5 min before the last centrifugation.
  • the supernatant was carefully aspirated, 20-30 ⁇ L of 2 ⁇ SDS gel loading buffer was added, mixed, denatured at 95 ° C for 3 min, and rapidly transferred to an ice bath for cooling. After centrifugation at 12,000 rpm for 2 min at room temperature, the supernatant was a precipitated protein sample, and some or all of it was subjected to SDS-polyacrylamide gel electrophoresis.
  • the input cell lysate preparation method is as shown above, and the input represents the protein content of TRB3 protein and P62 in the initial lung tissue lysate, that is, the protein stock solution passes through the P62 antibody or the control antibody IgG (since the P62 antibody is an IgG type antibody, The content of TRB3 protein and P62 protein before precipitation was selected by IgG antibody as control. The results showed that the contents of TRB3 protein and P62 protein in the input cell lysate were consistent.
  • the output represents the amount of protein contained in the protein stock solution after precipitation by P62 antibody or control antibody IgG. Since the IgG antibody is used as a control antibody for the P62 antibody, the P62 protein cannot be precipitated, so the P62 protein blotting lane in the IgG antibody-treated cell lysate shows As a blank, the P62 antibody was used as an antibody of the experimental group to bind to the P62 protein and precipitated. Therefore, the cell lysate after the P62 antibody treatment showed that the P62 protein blotting lane was black.
  • the TRB3 protein was also precipitated when the P62 protein was precipitated by the P62 antibody, so the TRB3 western blot of the cell lysate after the P62 antibody treatment showed black. Since the IgG antibody could not precipitate the P62 protein, the P62 interacting protein TRB3 protein could not be precipitated, so the TRB3 Western blotting lane of the IgG antibody-treated cell lysate was blank. The above experimental results fully demonstrate that TRB3 protein and P62 protein can directly interact
  • Example 4 Flow cytometry method to verify the phagocytosis of collagen by lung epithelial cells, and to verify the clearance of intracellular collagen by pep2-A1, pep2-A2, pep2-A3, and autophagy agonists and autophagy inhibitors.
  • MLE-12 cells (MLE-12 cells were purchased from ATCC), and cells were added at a density of 1.2 ⁇ 10 6 (wells/well), and cultured in a 6-well plate.
  • FITC-labeled collagen was purchased from Invitrogen
  • concentration was 1 ⁇ g/m for 1 hour.
  • the cells were digested with trypsin and collected, and subjected to flow cytometry, excitation with a 488 nm laser, and detection by the FL1 channel.
  • Example 5 Using pulmonary fibrosis animal model to verify p162-A1, pep2-A2, and pep2-A3 for pulmonary fibrosis
  • Example 1 The animal models prepared in Example 1 were administered in groups after 10 days of modeling, and the grouping and administration were as shown in Table 1 (i.p. for intraperitoneal injection, i.g. for intragastric administration).
  • Fig. 5(A) and Fig. 5(B) show the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate; Fig. 5(B) shows that the lung tissue lysate passed pep2 respectively.
  • Fig. 5(A) shows the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate
  • Fig. 5(B) shows that the lung tissue lysate passed pep2 respectively.
  • the protein amount of the obtained TRB3 protein and P62 protein was precipitated by P62 antibody or control antibody IgG, and TRB3 in pulmonary fibrosis tissue was observed from Fig. 5 (A) and Fig. 5 (B).
  • the protein and p62 protein are present in combination with each other.
  • pep2-A1, pep2-A2, and pep2-A3 all reduced the binding of TRB3 protein to p62 protein.
  • the input represents the protein content of the TRB3 protein and the P62 protein in the initial lung tissue lysate, that is, the protein stock solution is precipitated before being precipitated by the P62 antibody or the control antibody IgG (the IgG antibody is selected as the control because the P62 antibody is an IgG type antibody).
  • the protein levels of TRB3 protein and P62 protein are identical.
  • the output represents the amount of protein contained in the protein stock after precipitation by the P62 antibody or the control antibody IgG.
  • Fig. 6 shows the decrease of pep2-A1, pep2-A2, and pep2-A3.
  • the lung weight index results of mice with pulmonary fibrosis induced by bleomycin showed that the lung weight index of mice was significantly increased after administration of bleomycin compared with the sham operation group.
  • the lung weight index of fibrotic mice was significantly decreased.
  • the positive control drug pirfenidone slightly decreased the lung weight index of the experimental animals, but there was no statistical difference.
  • mice were dissected in the neck and the organs were exposed for intubation.
  • the PBS lavage volume was 0.8 ml, and the lavage frequency was 3-5 times.
  • the recovered lavage fluid was centrifuged at 1500 rpm for 10 minutes at 4 ° C, and the supernatant was collected at -20 ° C for cytokine detection; the cell pellet was resuspended in 1 ml of PBS containing 1% BSA, and 10 ⁇ l of the suspension was taken for cell counting. Analysis was performed using a blood cell analyzer. The results are shown in Figure 7.
  • Figure 7 shows the results of pep2-A1, pep2-A2, and pep2-A3 in reducing the number of inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin.
  • A is the total number of white blood cells
  • B is the number of monocytes
  • C is the number of neutrophils
  • D is the number of lymphocytes
  • E is the number of basophils
  • F is the number of eosinophils.
  • Hematoxylin dyeing solution is an alkaline dyeing solution, which makes the chromatin in the nucleus and the ribosome in the cytoplasm purple-blue; eosin is an acid dye, which mainly makes the components in the cytoplasm and extracellular matrix red.
  • This staining method which is the most commonly used staining method for morphology.
  • Fig. 8 The lung tissue of the right lower lobe of the experimental animals was taken, and 4% paraformaldehyde was fixed and embedded in paraffin. The largest cross section of the wax block embedded in the lung tissue was observed by HE staining. The results are shown in Fig. 8. Among them, A is a sham operation group, B is a model group, C is a pep2-A1 group, D is a pep2-A2 group, E is a pep2-A3 group, and F is a positive control drug pirfenidone group. It can be seen from Fig. 8 that the HE stained tissue in the lungs of the sham-operated mice is clearly visible, the alveolar structure is intact, and no pathological changes of inflammation and fibrosis are observed.
  • pep2-A1, pep2-A2 and pep2-A3 After administration of pep2-A1, pep2-A2 and pep2-A3, it can alleviate lung inflammation caused by bleomycin, and effectively improve lung injury and restore normal lung structure. Especially in the pep2-A2 group, the lung inflammation disappeared and the lung tissue structure was clear.
  • Fig. 9 is a graph showing the results of case analysis of inflammatory grading. As can be seen from Fig. 9, significant inflammation occurred in the lungs of mice after administration of bleomycin as compared with the sham operation group.
  • pep2-A1, pep2-A2, pep2-A3, and the positive control drug pirfenidone significantly reduced lung inflammation caused by bleomycin.
  • * was p ⁇ 0.05 compared with the model group
  • ** was p ⁇ 0.01 compared with the model group.
  • Sirius red can specifically stain fibrotic collagen, which is a special staining method for common tissue sections.
  • the lung tissue of the right lower lobe of the animal was taken, and 4% paraformaldehyde was fixed and embedded in paraffin.
  • the largest cross section of the wax block embedded in the lung tissue was observed by Sirius red staining.
  • a high-resolution pathological picture of Sirius Red special staining (200 times) was obtained using the high-resolution color pathology analysis system Spot Advanced 3.0.
  • the stained area of collagen after Sirius red staining and the area of lung tissue in the field of view were measured using Image-Pro Plus 5.1. With colored area, The ratio of the colored area to the area of the lung tissue of the field of view indicates the relative amount of the collagen.
  • Ten specimens were analyzed in each group of bleomycin-induced pulmonary fibrosis experiments. Each specimen was randomly divided into six fields.
  • the mean value represents the relative content and expression intensity of collagen tissue in an animal's lung tissue.
  • the absolute area of collagen in the field of view was compared by parameter analysis of variance.
  • Figure 10 shows that pep2-A1, pep2-A2, and pep2-A3 reduce the pathological examination of bleomycin-induced pulmonary fibrosis (Sirius Red), where A is the sham operation group and B is In the model group, C is the pep2-A1 group, D is the pep2-A2 group, E is the pep2-A3 group, and F is the positive control drug pirfenidone group.
  • Figure 11 is a graph showing the effect of pep2-A1, pep2-A2, and pep2-A3 on collagen content in lung tissue of mice with pulmonary fibrosis.
  • FIGs 10 and 11 there was no significant collagen staining in Sirius red staining of sham-operated mice. After administration of bleomycin, significant collagen accumulation occurred in the lungs of mice, and a large amount of fibrotic tissue was formed.
  • Administration of pep2-A1, pep2-A2, and pep2-A3 can alleviate pulmonary collagen accumulation caused by bleomycin and improve pulmonary fibrosis.
  • ## ⁇ p ⁇ 0.01 compared with the sham operation group * was p ⁇ 0.05 compared with the model group, and ** was p ⁇ 0.01 compared with the model group.
  • Pulmonary function is a gold indicator for clinical detection of pulmonary fibrosis in patients.
  • the decline in lung function is often accompanied by an increase in fibrosis, and the improvement in lung function often also represents the recovery of lung tissue structure.
  • the lung fibrosis model mice obtained in Example 1 were anesthetized with sodium pentobarbital (45 mg/kg, ip), and the lung function test was performed by Flexivent small animal pulmonary function meter.
  • the detection method was TLC, SnapShots. (For the detection method, please refer to: Lv X, Wang X, Li K, et al. Rupatadine Protects against Pulmonary Fibrosis by Attenuating PAF-Mediated Senescence in Rodents [J]. PloS one, 2013, 8(7): e68631.).
  • Fig. 12 is a graph showing the results of lung function tests in mice with pulmonary fibrosis, where A is a deep inspiratory volume, B is a dynamic resistance, C is a dynamic elasticity, and D is a dynamic compliance.
  • A is a deep inspiratory volume
  • B is a dynamic resistance
  • C is a dynamic elasticity
  • D is a dynamic compliance.
  • the deep inspiratory volume of the bleomycin-induced pulmonary fibrosis mice was significantly reduced, the dynamic resistance and dynamic elasticity of the lungs were increased, and the compliance was significantly reduced.
  • Pulmonary function was significantly restored after treatment with pep2-A1, pep2-A2, and pep2-A3.
  • ## was p ⁇ 0.01 compared with the sham operation group
  • * was p ⁇ 0.05 compared with the model group
  • ** was p ⁇ 0.01 compared with the model group.
  • Hydroxyproline accounts for 13.4% of collagen, and it is a very small amount in elastin, and it is not present in other proteins. Therefore, the content of collagen is detected by hydroxyproline.
  • the content of hydroxyproline in the left lobe of the animals was examined to evaluate the condition of pulmonary fibrosis.
  • the specific method is as follows: the whole lung lobe of the left side of the model animal prepared in Example 1 was prepared, the wet weight was recorded, and 10% of the tissue homogenate was prepared by ultrasonic homogenization of physiological saline, and about 150 ⁇ l of the homogenate supernatant was added, and 500 ⁇ l of alkali was hydrolyzed.
  • Figure 13 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. The results showed that compared with the sham operation group, the hydroxyproline content in the model group was significantly increased, indicating that the pathological changes of fibrosis were severe.
  • pep2-A1, pep2-A2, and pep2-A3 significantly reduced the content of hydroxyproline in the lungs of fibrotic mice.
  • ## was p ⁇ 0.01 compared with the sham operation group, * was p ⁇ 0.05 compared with the model group, and ** was p ⁇ 0.01 compared with the model group.
  • pep2-A1, pep2-A2, pep2-A3 can significantly inhibit bleomycin-induced pulmonary fibrosis; significantly reduce lung fibrosis in mice Mortality; significantly reduced lung weight index in mice with pulmonary fibrosis; significantly reduced the number of various inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis; and effectively improved lung injury and restored normal lung structure; significantly reduced Hydroxyproline and collagen content in lung tissue of mice with pulmonary fibrosis; significantly improved lung function in mice with pulmonary fibrosis.
  • the above experimental results prove that pep2-A1, pep2-A2, and pep2-A3 have excellent therapeutic prospects in pulmonary fibrosis.
  • Example 9 Using an animal model of pulmonary fibrosis to verify the effects of TAT-A2 and Antp-A2 on pulmonary fibrosis
  • the bleomycin used in the experiment was purchased from Nippon Kayaku Co., Ltd., batch number X90147.
  • Pirfenidone purchased from Dalian Meilun Biotechnology Co., Ltd., is a raw material drug with a pirfenidone content of >98.5%.
  • the peptide of the amino acid sequence shown in SEQ ID NO: 2 (A2) of the Sequence Listing is ligated to the TAT peptide (YGRKKRRQRRR, respectively) of the cell transmembrane peptide HIV-1 viral trans-activator transcription (Tat) protein. Its amino acid sequence is shown in SEQ ID NO: 5), the transcription factor Antp peptide of Drosophila tentacles homologous protein (RQIKIWFQNRRMKWKK, whose amino acid sequence is shown as SEQ ID NO: 6), constitutes a new derivative TAT-A2.
  • Antp-A2 the above polypeptide or chimeric peptide was synthesized by Beijing Saibaisheng Gene Technology Co., Ltd. A2 is linked to the C-terminus of the TAT peptide and the Antp peptide by two glutamic acid chains.
  • the sequence structure of the above chimeric peptides is as follows:
  • the TAT-A2 sequence is:
  • Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys-"C (the sequence of which is shown in SEQ ID NO: 13 in the Sequence Listing)
  • Antp-A2 The sequence of Antp-A2 is:
  • Example 2 The animal models prepared in Example 1 were administered in groups after 10 days of modeling, and the grouping and administration were as shown in Table 2 (i.p. For intraperitoneal injection, i.g. for intragastric administration):
  • Figure 14 is a graph showing the results of TAT-A2 and Antp-A2 in reducing mortality of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the sham operation group, B is the model group, C is the TAT-A2 group, and D is the Antp-A2 group. Compared with the sham operation group, the survival rate of the model group was significantly reduced.
  • FIG. 15 shows that TAT-A2 and Antp-A2 reduce bleomycin. Pulmonary weight index results of lung fibrosis in mice. As can be seen from the results shown in the figure, the lung weight index of the mice was significantly increased after administration of bleomycin as compared with the sham operation group. Both TAT-A2 and Antp-A2 significantly reduced the lung weight index of fibrotic mice after administration. Where ## is p ⁇ 0.01 compared with the sham operation group, and ** is p ⁇ 0.01 compared with the model group.
  • mice were dissected in the neck and the organs were exposed for intubation.
  • the PBS lavage volume was 0.8 ml, and the lavage frequency was 3-5 times.
  • the recovered lavage fluid was centrifuged at 1500 rpm for 10 minutes at 4 ° C, and the supernatant was collected at -20 ° C for cytokine detection; the cell pellet was resuspended in 1 ml of PBS containing 1% BSA, and 10 ⁇ l of the suspension was taken for cell counting.
  • Application of blood cell analyzer Line analysis The results are shown in Fig. 16.
  • A is the total number of white blood cells
  • B is the number of monocytes
  • C is the number of neutrophils
  • D is the number of lymphocytes
  • E is the number of eosinophils
  • F is the number of basophils.
  • the total number of white blood cells, the number of monocytes, the number of basophils, the number of eosinophils, and the number of eosinophils in the alveolar lavage fluid of mice after administration of bleomycin were compared with those of the sham operation group.
  • the number of neutrophils is extremely significant.
  • TAT-A2 and Antp-A2 can reduce the number of inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis to varying degrees.
  • FIG. 17 is a graph showing the results of TAT-A2 and Antp-A2 in reducing the hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. From the results shown in the figure, it can be seen that compared with the sham operation group. The hydroxyproline content of the model group and its significant increase, indicating that the pathological changes of fibrosis are serious. TAT-A2 and Antp-A2 can significantly reduce the content of hydroxyproline in the lungs of fibrotic mice. Where ## was p ⁇ 0.01 compared with the sham operation group, * was p ⁇ 0.05 compared with the model group, and ** was p ⁇ 0.01 compared with the model group.
  • TAT-A2 and Antp-A2 can significantly inhibit bleomycin-induced pulmonary fibrosis; significantly reduce the mortality of mice with pulmonary fibrosis; significantly reduce the lungs of mice with pulmonary fibrosis Heavy index; significantly reduced the number of various inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis; significantly reduced the hydroxyproline content in the lung tissue of mice with pulmonary fibrosis.
  • the above experimental results prove that TAT-A2 and Antp-A2 also have excellent therapeutic prospects in pulmonary fibrosis.
  • Example 8 and Example 9 were expressed as mean ⁇ standard error. After parameter or non-parametric variance test, a significant difference was considered by comparison p ⁇ 0.05, and p ⁇ 0.01 was considered to have an extremely significant difference. The statistics of pathological grading data were analyzed by chi-square test. After comparison, p ⁇ 0.05 was considered to have significant difference, and p ⁇ 0.01 was considered to have extremely significant difference.
  • the PBS used in the examples of the present invention that is, a phosphate buffer solution, had a concentration of 0.1 M and a pH of 7.2.
  • the polypeptide of the present invention or the derivative of the polypeptide has a remarkable effect of treating pulmonary fibrotic diseases, and can be used as an active ingredient for the preparation of a medicament for preventing or treating pulmonary fibrosis.

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Abstract

La présente invention concerne l'utilisation d'un polypeptide pouvant être lié spécifiquement à une protéine TRB3, ou des dérivés de ce dernier, pour préparer des médicaments pour prévenir ou traiter une fibrose pulmonaire, les dérivés étant des peptides chimériques formés en reliant le polypeptide, pouvant être lié spécifiquement à une protéine TRB3, à un peptide de pénétration de cellules. Le polypeptide ou les dérivés de ce dernier de la présente invention peuvent être liés spécifiquement à une protéine TRB3 et peuvent bloquer l'interaction entre la protéine TRB3 et une protéine P62.
PCT/CN2014/094887 2013-12-25 2014-12-25 Utilisation de polypeptide et de dérivés de ce dernier pour préparer des médicaments anti-fibrose pulmonaire WO2015096756A1 (fr)

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