WO2017144016A1 - 多肽、其衍生物及其在制备抗肺纤维化的药物中的应用 - Google Patents

多肽、其衍生物及其在制备抗肺纤维化的药物中的应用 Download PDF

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WO2017144016A1
WO2017144016A1 PCT/CN2017/074792 CN2017074792W WO2017144016A1 WO 2017144016 A1 WO2017144016 A1 WO 2017144016A1 CN 2017074792 W CN2017074792 W CN 2017074792W WO 2017144016 A1 WO2017144016 A1 WO 2017144016A1
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polypeptide
seq
pulmonary fibrosis
peptide
trb3
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PCT/CN2017/074792
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French (fr)
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胡卓伟
吕晓希
刘姗姗
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胡卓伟
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Priority to EP17755845.9A priority Critical patent/EP3421484A4/en
Priority to US16/079,423 priority patent/US20210179680A1/en
Priority to JP2018563751A priority patent/JP2019512539A/ja
Publication of WO2017144016A1 publication Critical patent/WO2017144016A1/zh

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
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    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
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    • C12N2740/10011—Retroviridae
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    • C12N2740/16071—Demonstrated in vivo effect

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a polypeptide, a derivative thereof and the use thereof in preparing a medicament for treating and/or preventing pulmonary fibrosis.
  • 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%.
  • TRB3 (Tribbles Homologue 3) is a member of the pseudo-kinase Tribbles homologous protein family in the human body. It has a adaptor-like function and is involved in the assembly of various protein complexes. A number of studies suggest that TRB3 can interact with a variety of transcription factors, ubiquitin ligase, cell membrane receptors, and MAPK, PI3K signaling pathway member proteins, involved in cell differentiation, apoptosis, autophagy, and metabolic regulation. Recently, a variety of evidences indicate that TRB3 is highly expressed in pulmonary fibrosis and plays an important role in the development of pulmonary fibrosis. MDM2 is the E3 ubiquitin ligase in the ubiquitin proteasome system.
  • the technical problem to be solved by the present invention is to provide a polypeptide, a derivative thereof and the use thereof in the preparation of a medicament for treating and/or preventing pulmonary fibrosis in view of the current lack of a safe and effective anti-pulmonary fibrosis drug.
  • the polypeptide targets the interaction between TRB3 and MDM2, and can be used for the preparation of a medicament for preventing and/or treating pulmonary fibrosis with high activity and high selectivity.
  • TRB3 inhibits the degradation of the epithelial mesenchymal transition protein SLUG by interacting with MDM2, thereby promoting pulmonary fibrosis, and therefore, TRB3 and MDM2
  • the interaction between them is a potential site for the prevention and/or treatment of pulmonary fibrosis.
  • blocking the interaction of TRB3 with MDM2 is a potential pathway for preventing and/or treating pulmonary fibrosis, and it is necessary to develop a drug that blocks the interaction between TRB3 and MDM2.
  • the inventors obtained a polypeptide MR2 that targets TRB3 to interact with an MDM2 protein (the amino acid sequence of which is shown in SEQ ID No.
  • the modified polypeptide has a stable alpha helix secondary structure, which has a very high affinity, anti-enzymatic stability and cell wear.
  • Membrane Therefore, the engineered polypeptide has high alpha helix stability, TRB3 binding ability and metabolic stability, and can be applied to the preparation of a medicament for treating and/or preventing pulmonary fibrosis. Based on the research work of the inventors, the present invention provides the following technical solutions.
  • a polypeptide that specifically binds to TRB3 or a derivative of the polypeptide the amino acid sequence of the polypeptide is shown in SEQ ID No. 12 of the Sequence Listing, or, as in the sequence listing, The two or more amino acids in the amino acid sequence shown by ID No. 12 are replaced by a non-natural amino acid to which a side chain can be linked, and the derivative includes the polypeptide and the cell penetrating peptide.
  • the polypeptide having the amino acid sequence shown in SEQ ID No. 12 of the Sequence Listing is referred to as an MR2 polypeptide.
  • the unnatural amino acid to which the other side chains are linked is a non-natural amino acid conventional in the art, preferably S-pentene alanine (S5).
  • the cell penetrating peptide of the present invention is a cell penetrating peptide which is conventional in the art as long as it can assist in bringing the polypeptide into a cell to function.
  • the cell penetrating peptide is a short peptide molecule composed of 10 to 30 amino acids.
  • the cell penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide, more preferably to the N-terminus of the polypeptide.
  • the cell penetrating peptide and the polypeptide or polypeptide derivative that specifically binds to TRB3 are linked by two glycines (Gly-Gly).
  • the cell penetrating peptide is a TAT peptide of HIV-1 virus trans-activator transcription (Tat) protein (the amino acid sequence thereof is shown in SEQ No. 13 of the Sequence Listing), and the fruit fly The transcription factor Antp peptide of the antennae homeomorphism protein (the amino acid sequence thereof is shown in SEQ No. 14 of the Sequence Listing), the Pep-1 peptide (the amino acid sequence thereof is shown in SEQ No. 15 of the Sequence Listing), and the MPG peptide (the amino acid sequence thereof) Any one or more of the RGD peptide (the amino acid sequence of which is shown in SEQ No. 17 of the Sequence Listing) as shown in SEQ ID NO: 16 of the Sequence Listing. More preferably, the cell penetrating peptide is a Pep peptide (the amino acid sequence of which is shown in SEQ ID: 18 of the Sequence Listing).
  • the chimeric peptide is a chimeric polypeptide Pep2-MR2 (the amino acid sequence thereof is shown in SEQ ID No. 19 of the sequence listing) formed by ligating the pep2 peptide to the MR2 polypeptide.
  • a chimeric polypeptide TAT-MR2 (the amino acid sequence of which the amino acid sequence is shown in SEQ ID No. 20 of the Sequence Listing) formed after the TAT polypeptide is ligated to the MR2 polypeptide, or which is formed by ligating the Antp peptide to the MR2 polypeptide.
  • the chimeric polypeptide Antp-MR2 (the amino acid sequence of which is Sequence Listing SEQ ID No. 21).
  • the unnatural amino acid to which the side chain is linked is S-pentene alanine; in the polypeptide, the number of the substituted amino acids is two and the positions of the substituted amino acids are respectively
  • i+3 and i+4 increase the helix of the polypeptide, thereby improving the stability of the polypeptide.
  • amino acid sequence of the polypeptide is SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 of the Sequence Listing. And SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11.
  • amino acid sequence represented by the above SEQ ID No. 1 to SEQ ID No. 11 can be appropriately substituted, deleted or added as long as the modified amino acid sequence can still specifically bind to TRB3 and remain unchanged before transformation. It can be active.
  • the second aspect of the present invention provides a polypeptide which specifically binds to TRB3 or a derivative of the polypeptide for use in the preparation of a medicament for treating and/or preventing pulmonary fibrosis.
  • Pulmonary fibrosis as described herein is a conventional pulmonary fibrosis in the art. Preferably, it refers to pulmonary fibrosis characterized by a variety of different factors characterized by pathological changes in idiopathic pulmonary fibrosis.
  • the pulmonary fibrosis preferably refers to pulmonary fibrosis of a human or an animal. More preferably, the symptoms of pulmonary fibrosis include: pulmonary inflammation and pulmonary function deterioration caused by pulmonary fibrosis.
  • the cause of pulmonary fibrosis comprises lung damage, dust or drugs.
  • the drug described therein is a conventional drug in the field and can induce pulmonary fibrosis. Preferred is bleomycin.
  • said pulmonary fibrosis is preferably primary (specific) pulmonary fibrosis, ie unexplained pulmonary fibrosis; or preferably secondary pulmonary fibrosis, ie secondary to Pulmonary fibrosis of the original disease. More preferably, lung function deterioration, pulmonary inflammation and lung damage in pulmonary fibrosis.
  • the pulmonary fibrosis disease preferably includes chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis or interstitial pneumonia.
  • COPD chronic obstructive pulmonary disease
  • “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.
  • the third aspect of the present invention provides a pharmaceutical composition for anti-pulmonary fibrosis comprising the polypeptide which specifically binds to TRB3 or a derivative of the polypeptide as an active ingredient.
  • the active ingredient refers to a compound having a function of preventing or treating pulmonary fibrosis.
  • the polypeptide which specifically binds to TRB3 or a derivative of the polypeptide may be used alone as an active ingredient or as an active ingredient together with other compounds having anti-pulmonary fibrosis activity.
  • the administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally.
  • Injection administration Preferably, it includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.
  • the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutically acceptable carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought.
  • the reagents and starting materials used in the present invention are commercially available.
  • Figure 1 shows the expression profiles of TRB3 and MDM2 and SLUG in lung tissue of mice with pulmonary fibrosis.
  • Figure 2 is a map of the interaction between TRB3 and MDM2 in lung tissue of lung fiber mice.
  • the output shows the protein content of the TRB3 protein and the MDM2 protein contained in the lung tissue lysate after precipitation by the MDM2 antibody or the control antibody IgG; wherein the input indicates the protein content of the TRB3 protein and the MDM2 protein contained in the initial lung tissue lysate.
  • Figure 3 is a graph showing the increase in SLUG protein expression induced by overexpression of TRB3 in human lung epithelial cells.
  • Figure 4-1 and Figure 4-2 show the binding ability of the peptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 to the TRB3 protein by surface plasmon resonance method.
  • A is the kinetic curve of the polypeptide MR2 and TRB3 proteins;
  • B is the binding kinetics curve of the polypeptide S1 and TRB3;
  • C is the binding kinetics curve of the polypeptide S2 and TRB3;
  • D is the binding kinetics curve of the polypeptide S3 and TRB3;
  • E is The binding kinetics curve of peptide S4 and TRB3 protein;
  • F is the binding kinetic curve of polypeptide S5 and TRB3 protein;
  • G is the binding kinetic curve of polypeptide S6 and TRB3 protein;
  • H is the binding kinetic curve of polypeptide S7 and TRB3 protein;
  • I is polypeptide S8 With TRB3 protein Kinetic curve;
  • J is
  • Figure 5 is a map of the interaction of the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 with the TRB3 and MDM2 proteins.
  • the output shows the protein content of the TRB3 protein and the MDM2 protein contained in the cell lysate after precipitation by the MDM2 antibody or the control antibody IgG;
  • the input indicates the protein content of the TRB3 protein and the MDM2 protein contained in the initial cell lysate.
  • Figure 6-1 and Figure 6-2 show the results of reducing the survival rate of bleomycin-induced pulmonary fibrosis in mice MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11. Figure.
  • Figure 7 is a graph showing the results of reducing the lung weight index of bleomycin-induced pulmonary fibrosis in mice MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.
  • Figure 8 is a pathological examination (HE) map of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 to reduce bleomycin-induced pulmonary fibrosis.
  • A is sham operation group
  • B is bleomycin model group
  • C is bleomycin plus MR2 group
  • D is bleomycin plus S1 group
  • E is bleomycin plus S2 group
  • F is Bo Laimycin plus S3 group
  • G is bleomycin plus S4 group
  • H is bleomycin plus S5 group
  • I is bleomycin plus S6 group
  • J is bleomycin plus S7 group
  • K is Bo Laimycin plus S8 group
  • L is bleomycin plus S9 group
  • M is bleomycin plus S10 group
  • N is bleomycin plus S11 group.
  • Figure 9 is a graph showing the results of pathological examination scoring of bleomycin-induced pulmonary fibrosis by polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.
  • Figure 10 is a graph showing the results of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 in reducing hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin.
  • Figure 11 shows the results of lung function tests in mice with pulmonary fibrosis improved by polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.
  • A is the deep inspiratory volume
  • B is the dynamic resistance
  • C is the dynamic elasticity
  • D is the dynamic compliance.
  • Figure 12 is a graph showing the results of survival test of mice with pulmonary fibrosis induced by bleomycin by polypeptides S19, S20 and S21.
  • Figure 13 is a graph showing the results of polypeptide S19, S20, and S21 in reducing lung weight index in mice with pulmonary fibrosis induced by bleomycin.
  • Figure 14 is a pathological examination (HE) map of polypeptides S19, S20, S21 to reduce bleomycin-induced pulmonary fibrosis.
  • A is sham operation group
  • B is bleomycin model group
  • C is bleomycin plus S19 group
  • D is bleomycin plus S20 group
  • E is bleomycin plus S21 group.
  • Figure 15 is a graph showing the results of pathological examination scoring of bleomycin-induced pulmonary fibrosis by polypeptides S19, S20, and S21.
  • Figure 16 shows that polypeptides S19, S20, and S21 reduce the hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. Fruit map.
  • Figure 17 shows the results of lung function test in mice with pulmonary fibrosis improved by polypeptides S19, S20 and S21.
  • A is the deep inspiratory volume
  • B is the dynamic resistance
  • C is the dynamic elasticity
  • D is the dynamic compliance.
  • the PBS solution described in the examples refers to a phosphate buffer solution having a concentration of 0.1 M and a pH of 7.2.
  • the room temperature described in the examples is room temperature conventional in the art, preferably 15 to 30 °C.
  • the amino acid sequence of the polypeptide MR2 is shown in SEQ ID No. 12 of the Sequence Listing.
  • the peptide MR2 was synthesized and purified by Beijing Saibaisheng Gene Technology Co., Ltd.
  • S5 Two unnatural amino acids S-pentene alanine (S5) were introduced for solid phase polypeptide chain synthesis.
  • the target polypeptide is obtained by cyclization of olefin metathesis reaction (RCM) using hydrazine as a catalyst.
  • RCM olefin metathesis reaction
  • the target polypeptide is cleaved from the resin for purification.
  • the steps of synthesizing and purifying the above solid phase polypeptide chain are completed by the company of the Chinese peptide biochemical company.
  • Bleomycin was purchased from Nippon Kayaku, lot number 640412.
  • Example 2 The compounds used in Example 2 were purchased from Sigma unless otherwise stated.
  • SPF grade C57BL/6 mice male, 6-8 weeks old, 16-18 g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • mice Male C57BL/6 mice were fasted overnight, anesthetized with 45 mg/kg sodium pentobarbital intraperitoneally (i.p.), and intratracheally injected with bleomycin at a dose of 3 U/kg.
  • the specific steps are as follows: cut the neck skin of the mouse with as little trauma as possible, expose the trachea with the assistance of elbow ophthalmology, puncture the trachea with a micro-injector, and inject about 50 ⁇ L of bleomycin into the trachea, quickly The ground was rotated and erected for 5 minutes to allow bleomycin to enter the left and right lobes evenly, thereby constructing an animal model. The entire operation is performed at a surgical table of about 60 °C. The mice in the sham operation group were intratracheally injected with an equal amount of physiological saline for injection.
  • mice in the animal model constructed above were reared in the SPF-class animal room, and the mice were sacrificed after fibrosis pathological changes in the lungs on the 10th day, and the lung tissues of the mice were taken for subsequent experiments.
  • the mice in the sham operation group were also housed in the SPF animal room, and were sacrificed on the 10th day. The lung tissues were taken for subsequent experiments.
  • lysis buffer [containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM ethylene glycol diethyl ether diamine tetraacetic acid (EGTA), 10 mM KCl, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPEs), 50 mM NaF, 0.1 M Na 3 VO 4 , 0.1 M Na 3 PO 4 , 1 mM/L aprotinin (Aprotinin), 1 mM/L trypsin inhibitor ( Trypsin inhibitor), 1 mM / L phenylmethylsulfonyl fluoride (PMSF), 1 mM / L leupeptins (Leupeptins) and 1 mM / L dithiothreitol (DTT) were homogenized and placed on ice for 15 min, or oscillation.
  • EDTA 0.1 mM ethylenediaminetetraacetic acid
  • the reagents used for co-immunoprecipitation are as follows:
  • Lysis solution A Weigh 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2 ⁇ 6H 2 O, 0.0742g EDTA, 10mL glycerol and 10mL 10% (v/v) NP40, add deionized water to 150mL, use The pH of the HCl was adjusted to 7.6, and the volume was adjusted to 191 mL. The mixture was thoroughly mixed, filtered through a 0.45 ⁇ m filter, and stored at 4 ° C.
  • Lysate B solution Weigh 200 ⁇ L of 2M ⁇ -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 Leu, 200 ⁇ L of 1 mg/mL Pep, and 200 ⁇ L of 1 mg/mL Apr, total volume A total of 9mL. Store at -20 ° C.
  • the lysate B solution When using, the lysate B solution is thawed, and according to the ratio of the volume ratio of the lysate B solution: the lysate A solution to 1:100, the lysate B solution is added to the lysate A solution and mixed to obtain a cell lysate.
  • Co-immunoprecipitate lotion 1% (v/v) NP40, 150 mM NaCl, 20 mM HEPES, 10% (v/v) pH 7.5 glycerol and 1 mM EDTA.
  • Protein A/G Plus-Agarose was purchased from Santa Cruz, USA.
  • the lung tissue was weighed by the immunoprecipitation lysate lysis step (1), and about 10 mg of total cell protein was harvested, and the lung tissue proteins of both the animal model and the sham operation group were adjusted to a concentration of 10 ⁇ g/mL. 200 ⁇ g of lung tissue protein of the animal model and the sham operation group were taken as input groups, respectively, and the above input group was used as a control.
  • Output represents TRB3 in a protein sample after precipitation of the protein sample by MDM2 antibody or control antibody IgG With MDM2 content. Since the IgG antibody was used as a control antibody for the MDM2 antibody, the MDM2 protein could not be precipitated, so the MDM2 Western blotting lane in the IgG antibody-treated protein sample showed a blank; and the MDM2 antibody as an experimental group antibody binds to the MDM2 protein and precipitates it. Therefore, the protein sample after MDM2 antibody treatment showed a black line in the MDM2 Western blotting lane.
  • the MDM2 protein can precipitate the TRB3 protein when the MDM2 protein is precipitated, so the TRB3 protein blotting lane of the cell lysate treated with the MDM2 antibody is shown as black. Since the IgG antibody could not precipitate the MDM2 protein, the MDM2 interacting protein TRB3 could not be precipitated, and the TRB3 protein blotting lane of the IgG antibody-treated cell lysate was blank. The above results fully demonstrate that the TRB3 protein interacts directly with the MDM2 protein.
  • the transfection reagent was mixed with plasmid DNA [control plasmid (purchased from Ao Ruidong, model PS100001); TRB3 overexpression plasmid (purchased from Ao Ruidong, model RC206687)] in a ratio of 1:2, the ratio The ratio of the amount of the transfection reagent (mL) to the mass of the DNA (mg) was allowed to stand at room temperature for 15 minutes to obtain a mixed solution.
  • the transfected BEAS-2B cells were lysed using RIPA lysate (purchased from Biyuntian, model P0013B), and the intracellular SLUG protein expression was detected by Western-blot method in Example 3.
  • Figure 3 illustrates that the expression of Slug in BEAS-2B cells overexpressing TRB3 is significantly increased.
  • the surface plasmon resonance experiment was carried out in a surface plasmon resonator Biacore T200, and the procedure was carried out in accordance with the instructions of the plasma resonator Biacore T200. Specific steps are as follows:
  • TRB3 protein purchased from RD
  • CM5 chip purchased from GE
  • the unbound protein was eluted at a flow rate of 10 ⁇ L/min, and the surface of the chip was equilibrated for 2 hours.
  • steps of amino coupling, elution and equilibration refer to the relevant specifications of GE's CM5 chip.
  • the alpha helix ratio of the polypeptide was measured by a circular dichroism (purchased from Jasco, Japan).
  • the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 prepared in Example 1 were dissolved in a PBS solution, and the concentration of the round chromatograph was adjusted to 1 mg/ mL, the results are shown in Table 2.
  • Table 2 shows that the alpha helix rates of the polypeptides S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 are significantly higher than the polypeptide MR2.
  • the alpha helix ratio refers to the percentage of the number of peptides of the polypeptide which maintains the alpha helix of the secondary structure as a percentage of the total polypeptide.
  • Example 7 Method of co-immunoprecipitation to verify the binding of S1 to S11 and MR2 polypeptides at the cellular level to the inhibition of protein MDM2 and TRB3
  • Human lung epithelial BEAS-2B cells purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • Example 8 Determination of the effect of polypeptide S1-S11 on mortality in mice with pulmonary fibrosis
  • the animal model of pulmonary fibrosis was prepared according to the method described in Example 2.
  • the peptide treatment was started from the 10th day after modeling (the treatment group is shown in Table 3), and the time was calculated from the 10th day after modeling to count the death. rate.
  • the mortality of each group of experimental animals was counted daily and calculated. If a group of animals did not die, the survival rate was 100%. If all the animals in a group died, the survival rate was 0%.
  • the survival rate is the percentage of mice that survived as a percentage of the number of mice in the group.
  • Table 4 Figure 6-1, Figure 6-2 shows that compared with the sham operation group, the survival rate of the model group is significantly reduced.
  • the S1 ⁇ S11 administration group can significantly improve the survival rate of fibrotic mice.
  • ## is p ⁇ 0.01 compared with the sham operation group; * is p ⁇ 0.05 compared with the bleomycin group; ** is compared with the bleomycin group Ratio, p ⁇ 0.01; ip refers to intraperitoneal injection.
  • the above results indicate that the polypeptide of the present invention can effectively reduce the mortality of a mouse model of pulmonary fibrosis, has the advantages of less toxic side effects and safer use.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 3. After 28 days, the lungs of the mice were finely stripped and weighed, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index. The results are shown in Fig. 7 and Table 5. As can be seen from Fig. 7, the lung weight index of the mice was significantly increased after administration of bleomycin as compared with the sham operation group. Therefore, the polypeptides S1 to S11 can significantly reduce the lung weight index of fibrotic mice after administration. In Fig. 7, ## is p ⁇ 0.01 compared with the sham operation group, * is p ⁇ 0.05 compared with the bleomycin group, and ** is p ⁇ 0.01 compared with the bleomycin group.
  • Example 10 Determination of the effect of polypeptide S1-S11 on the pathomorphology of pulmonary fibrosis induced by bleomycin
  • hematoxylin-eosin staining method which is the most commonly used staining method for morphology.
  • 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.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 3. After 28 days, the lung tissue of the right lower lobe of the experimental animals was taken, and 4% (v/v) paraformaldehyde was fixed and embedded in paraffin.
  • FIG. 8 shows that the HE stained tissue in the lungs of sham-operated mice is clearly visible, the alveolar structure is intact, and no pathological changes of inflammation and fibrosis are observed.
  • the lungs showed obvious inflammation, the inflammatory cells infiltrated in a large amount, and the lung tissue structure was seriously damaged.
  • Polypeptides S1 to S11 can be reduced after administration Inflammation of the lung caused by light bleomycin, and effectively improve lung damage and restore normal lung structure.
  • grade 0 normal tissue.
  • Level 1 Very small inflammatory changes.
  • Grade 2 mild to moderate inflammatory changes with no apparent destruction of lung tissue structure.
  • Grade 3 Moderate inflammatory damage (alveolar decidua thickening).
  • Grade 4 Moderately severe inflammatory damage, formation of tissue mass, or localized pneumonia area disrupts the normal structure of lung tissue. 5: severe inflammatory injury, severe destruction of the local lung tissue structure caused by lumen closure.
  • Example 11 Determination of the effect of polypeptide S1-S11 on hydroxyproline content in mice with pulmonary fibrosis
  • Hydroxyproline accounts for 13.4% (w/w) of collagen, is extremely small in elastin, and is absent in other proteins, so the content of collagen is detected by hydroxyproline.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 3. The content of hydroxyproline in the left lobe of the animals was measured 28 days later, and the condition of pulmonary fibrosis was evaluated.
  • the specific method is as follows: the mouse in the animal model prepared in Example 2 is raised in an SPF animal room, and The polypeptide was treated according to the dosing schedule of Table 3. The whole lung lobe of the left side of the experimental animal was taken on the 21st day after the animal model was constructed, the wet weight was recorded, and the physiological homogenate was ultrasonically homogenized to prepare a tissue homogenate of 10% (w/w). Take about 150 ⁇ L of homogenate supernatant, add 500 ⁇ L of alkaline hydrolyzate (provided by Nanjing Hydrogen Proline Hydrolysis Kit), vortex and mix, base at 120 ° C, 0.1 Kpa The hydrolysis treatment was carried out for 40 min, the pH was adjusted, and the volume was adjusted.
  • the hydroxyproline assay was carried out according to the chloramine T method (the method of the present embodiment is referred to the kit of Nanjing Jiancheng Bioengineering Technology Co., Ltd.). The results are shown in Figure 10 and Table 7. As can be seen from Figure 10, compared with the sham operation group, the hydroxyproline content of the bleomycin group was significantly increased, indicating that the pathological changes of fibrosis were severe. After administration of S1 to S11, the content of hydroxyproline in the lungs of fibrotic mice can be significantly reduced. In Fig. 10, ## is p ⁇ 0.01 compared with the sham operation group, * is p ⁇ 0.05 compared with the bleomycin group, and ** is p ⁇ 0.01 compared with the bleomycin 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.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 3. On the 28th day after the construction of the animal model, the mice were anesthetized with sodium pentobarbital (45 mg/kg, ip) and the lung function test was performed by the Flexivent Small Animal Pulmonary Function Tester.
  • the detection method was TLC, SnapShots, (for the detection method, see :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.).
  • Figure 11 shows that compared with the sham operation group, the deep inspiratory volume of bleomycin-induced pulmonary fibrosis mice was significantly reduced, the dynamic resistance and dynamic elasticity of the lungs increased, and the compliance was significantly reduced. Pulmonary function was significantly restored after treatment with peptides S1 to S11.
  • ## is p ⁇ 0.01 compared with the sham operation group
  • * is p ⁇ 0.05 compared with the bleomycin group
  • ** is p ⁇ 0.01 compared with the bleomycin group.
  • S19 chimeric polypeptide Pep2-MR2 (the amino acid sequence of which is shown in SEQ ID No. 19 of the Sequence Listing)
  • S20 chimeric polypeptide TAT-MR2 (the amino acid sequence of which is shown in SEQ ID No. 20 of the Sequence Listing)
  • S21 The chimeric polypeptide Antp-MR2 (the amino acid sequence of which is shown in SEQ ID No. 21 of the Sequence Listing) was synthesized and purified by Beijing Cypress Biotechnology Co., Ltd.
  • the animal model of pulmonary fibrosis was prepared according to the method described in Example 2.
  • the polypeptide treatment was started from the 10th day after modeling (the treatment group is shown in Table 9), and the calculation time was started from the 10th day after modeling.
  • Statistical mortality The mortality of each group of experimental animals was counted daily and calculated. If a group of animals did not die, the survival rate was 100%. If all the animals in a group died, the survival rate was 0%.
  • Figure 12 illustrates that the survival rate of the model group is significantly reduced compared to the sham group. After drug treatment, the S19-S21 administration group can significantly improve the survival rate of fibrotic mice. In Fig.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 9. After 28 days, the lungs of the mice were finely stripped and weighed, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index. The results are shown in Fig. 13 and Table 11. As can be seen from Fig. 13, the lung weight index of the mice was significantly increased after administration of bleomycin as compared with the sham operation group. Therefore, S19-S21 can significantly reduce the lung weight index of fibrotic mice after administration. In Fig. 13, ## is p ⁇ 0.01 compared with the sham operation group, * is p ⁇ 0.05 compared with the bleomycin group, and ** is p ⁇ 0.01 compared with the bleomycin group.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 9. After 28 days, the lung tissue of the right lower lobe of the experimental animals was taken, and 4% (v/v) 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 Figure 14.
  • Figure 14 shows that S19-S21 can alleviate lung inflammation caused by bleomycin, and effectively improve lung damage and restore normal lung structure.
  • grade 0 normal tissue.
  • Level 1 Very small inflammatory changes.
  • Grade 2 mild to moderate inflammatory changes with no apparent destruction of lung tissue structure.
  • Grade 3 Moderate inflammatory damage (alveolar decidua thickening).
  • Grade 4 Moderately severe inflammatory damage, formation of tissue mass, or localized pneumonia area disrupts the normal structure of lung tissue. 5: severe inflammatory injury, severe destruction of the local lung tissue structure caused by lumen closure.
  • Hydroxyproline accounts for 13.4% (w/w) of collagen, is extremely small in elastin, and is absent in other proteins, so the content of collagen is detected by hydroxyproline.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 9. The content of hydroxyproline in the left lobe of the animals was measured 28 days later, and the condition of pulmonary fibrosis was evaluated.
  • the specific method is as follows: the mouse in the animal model prepared in Example 2 was raised in an SPF animal room, and the polypeptide was treated according to the schedule of administration of Table 9, and the left side of the experimental animal was taken on the 21st day after the animal model was constructed. All lung lobe, wet weight was recorded, and 10% (w/w) tissue homogenate was prepared by ultrasonic homogenization of physiological saline. About 150 ⁇ L of homogenate supernatant was added, and 500 ⁇ L of alkali hydrolyzate was added (from Nanjing Jiancheng Bioengineering Technology Co., Ltd.).
  • Hydroxyproline alkaline hydrolysis kit provides), vortex mixed, 120 ° C, 0.1Kpa conditions under alkaline hydrolysis treatment for 40min, adjust the pH value, constant volume, activated carbon treatment, take the supernatant.
  • the hydroxyproline assay was carried out according to the chloramine T method (the method of the present embodiment is referred to the kit of Nanjing Jiancheng Bioengineering Technology Co., Ltd.). The results are shown in Figure 16 and Table 13. As can be seen from Fig. 16, compared with the sham operation group, the hydroxyproline content of the bleomycin group was significantly increased, indicating that the pathological changes of fibrosis were severe.
  • 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.
  • Pulmonary fibrosis models were made as described in Example 2 and administered in groups according to Table 9. After 28 days, the mice were anesthetized with sodium pentobarbital (45 mg/kg, ip) and tested for lung function by Flexivent small animal pulmonary function test.
  • 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.).
  • Figure 17 shows that compared with the sham operation group, the deep inspiratory volume of bleomycin-induced pulmonary fibrosis mice was significantly reduced, the dynamic resistance and dynamic elasticity of the lungs increased, and the compliance was significantly reduced. Pulmonary function was significantly restored after treatment with peptides S19-S21.
  • Fig. 17, ## is p ⁇ 0.01 compared with the sham operation group, * is p ⁇ 0.05 compared with the bleomycin group, and ** is p ⁇ 0.01 compared with the bleomycin group.
  • the polypeptide derivative of the present invention has remarkable anti-pulmonary fibrosis effect, and can also be used as an active ingredient for preparing a drug against pulmonary fibrosis.

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Abstract

提供了一种特异性结合TRB3的多肽及其在制备治疗和/或预防肺纤维化的药物中的应用。所述多肽的氨基酸序列如序列表SEQ ID No.12所示，或者，如将序列表SEQ ID No.12所示的氨基酸序列中的两个或两个以上的氨基酸替换为侧链可相连的非天然氨基酸。所述多肽及其衍生物能够特异性地与TRB3结合，从而阻断TRB3和MDM2蛋白的相互作用。

Description

多肽、其衍生物及其在制备抗肺纤维化的药物中的应用
本申请要求申请日为2016年2月26日的中国专利申请CN201610108248.9的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种多肽、其衍生物及其在制备治疗和/或预防肺纤维化的药物中的应用。
背景技术
肺纤维化是肺部最为严重的一种病理状态，其病理改变大多表现为最初的下呼吸道炎症，以及肺泡上皮细胞和血管内皮细胞损伤，伴有成纤维母细胞和II型肺泡细胞增殖、细胞因子释放，细胞外基质蛋白和胶原沉积，最终引起肺部改变。肺纤维化患者肺部肺泡逐渐被纤维性物质取代，导致肺组织变硬变厚，肺脏气体交换能力逐步丧失，导致患者不同程度缺氧而出现呼吸困难，最后因呼吸衰竭而死亡。肺纤维化是呼吸病的四大病种之一，病因复杂，发病机制不明，目前已有的治疗肺纤维化的药物和方法十分有限，且疗效差强人意，预后极差，5年生存率仅为50％。
TRB3(Tribbles Homologue 3)是人体中假性激酶Tribbles同源蛋白家族成员之一，具有接头蛋白样的功能，参与多种蛋白复合体的组装。多项研究认为，TRB3可以与多种转录因子、泛素连接酶、细胞膜受体以及MAPK、PI3K信号通路成员蛋白发生相互作用，参与细胞分化、凋亡、自噬、代谢调控。近来，多种证据表明，TRB3在肺纤维化组织中呈现高表达，并且在肺纤维化的发展过程中发挥重要的促进作用。MDM2则是泛素蛋白酶体系统中的E3泛素连接酶。
发明内容
本发明所要解决的技术问题是针对目前缺乏安全有效的抗肺纤维化药物的现状，提供一种多肽、其衍生物及其在制备治疗和/或预防肺纤维化的药物中的应用。所述的多肽靶向TRB3与MDM2之间的相互作用，能够高活性、高选择性地用于预防和/或治疗肺纤维化的药物的制备。
本发明的发明人经过深入的研究和反复的试验，发现TRB3通过与MDM2发生相互作用，抑制促上皮间充质转化蛋白SLUG降解，从而促进肺纤维化，因此，TRB3与MDM2 之间的相互作用是预防和/或治疗肺纤维化的潜在位点。由此可见，阻断TRB3与MDM2相互作用是预防和/或治疗肺纤维化的潜在途径，需要研发能够阻断TRB3与MDM2相互作用的药物。发明人获得了靶向TRB3与MDM2蛋白相互作用的多肽MR2(其氨基酸序列如序列表SEQ ID No.12所示)；同时发明人发现，如果将多肽MR2中特定位置的氨基酸残基替换为侧链可以相连的非天然氨基酸，如S-戊烯丙氨酸(S5)，则改造后的多肽具有稳定的α螺旋的二级结构，从而具有极高的亲和力、抗酶解稳定性及细胞穿膜性。因此改造后的多肽的α螺旋稳定性、TRB3结合能力和代谢稳定性均很高，能够应用于制备治疗和/或预防肺纤维化的药物中。基于发明人的研究工作，本发明提供下述的技术方案。
本发明提供的技术方案之一是：一种特异性结合TRB3的多肽或所述多肽的衍生物，所述多肽的氨基酸序列如序列表SEQ ID No.12所示，或者，如将序列表SEQ ID No.12所示的氨基酸序列中的两个或两个以上的氨基酸替换为侧链可相连的非天然氨基酸所示，所述的衍生物包括所述多肽与细胞穿膜肽接连所形成的嵌合肽、所述多肽与病毒形成的融合肽、甲基化的所述多肽、糖基化的所述多肽和聚乙二醇化的所述多肽。
所述的氨基酸序列如序列表SEQ ID No.12所示的多肽称为MR2多肽。
所述的使其他侧链相连的非天然氨基酸为本领域常规的非天然氨基酸，较佳地为S-戊烯丙氨酸(S5)。
其中，本发明所述的细胞穿膜肽为本领域常规的细胞穿膜肽，只要其能辅助将所述多肽送入细胞以发挥作用即可。一般，所述的细胞穿膜肽为由10～30个氨基酸组成的短肽分子。较佳地，所述细胞穿膜肽连接在所述多肽的N端或者C端，更佳地连接在所述多肽的N端。所述细胞穿膜肽和特异性结合TRB3的多肽或多肽衍生物之间优选地以两个甘氨酸(Gly-Gly)连接。
较佳地，所述的细胞穿膜肽为HIV-1病毒反转录激活因子(Trans-activator transcription，Tat)蛋白的TAT肽(其氨基酸序列如序列表SEQ No：13所示)、果蝇触角同源异型蛋白的转录因子Antp肽(其氨基酸序列如序列表SEQ No：14所示)、Pep-1肽(其氨基酸序列如序列表SEQ No：15所示)、MPG肽(其氨基酸序列如序列表SEQ No：16所示)和RGD肽(其氨基酸序列如序列表SEQ No：17所示)中的任一种或多种。更佳地，所述的细胞穿膜肽为Pep肽(其氨基酸序列如序列表SEQ No：18所示)。
较佳地，所述的嵌合肽为将所述pep2肽连接在所述MR2多肽后所形成的嵌合多肽Pep2-MR2(其氨基酸序列如序列表SEQ ID No.19所示)、将所述TAT多肽连接在所述MR2多肽后所形成的嵌合多肽TAT-MR2(其氨基酸序列如序列表SEQ ID No.20所示)或将所述Antp肽连接在所述MR2多肽后所形成的嵌合多肽Antp-MR2(其氨基酸序列如 序列表SEQ ID No.21所示)。
较佳地，所述的侧链可相连的非天然氨基酸为S-戊烯丙氨酸；所述的多肽中，所述替换的氨基酸的数目为两个且所述替换的氨基酸的位置分别为第i位和第i+3位，或者，为第i位和第i+4位，其中1≤i≤11，i为正整数。其中，i+3及i+4均为增加多肽的螺旋率，从而提高多肽的稳定性。
更佳地，所述的多肽的氨基酸序列如将序列表SEQ ID No.1、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和SEQ ID No.11中任一项所示。
其中，上述SEQ ID No.1～SEQ ID No.11所示的氨基酸序列中可进行适当地氨基酸替换、缺失或添加，只要使改造后的氨基酸序列仍然能够与TRB3特异性结合并且保持改造前的活性即可。
本发明提供的技术方案之二是：一种特异性结合TRB3的多肽或所述多肽的衍生物在制备治疗和/或预防肺纤维化的药物中的应用。
本发明所述的“肺纤维化”为本领域常规的肺纤维化。较佳地是指以特发性肺纤维化病理改变为特征的，由各种不同因素所导致的肺纤维化。其中，所述的肺纤维化较佳地指人或动物的肺纤维化。更佳地，所述肺纤维化的症状包括：由肺纤维化引起的肺部炎症和肺功能退化。较佳地，所述的肺纤维化的病因包括肺损伤、粉尘或者药物。其中所述的药物为本领域常规的药物，能够引发肺纤维化即可。较佳地为博莱霉素。
其中，所述的肺纤维化较佳地是原发性(特异性)的肺纤维化，即原因不明的肺纤维化；或者较佳地是继发性的肺纤维化，即是继发于原先疾病的肺纤维化。更佳地是肺纤维化中的肺功能退化，肺部炎症和肺损伤。所述的肺纤维化的疾病较佳地包括慢性阻塞性肺病(COPD)、特发性肺纤维化或间质性肺炎。
本发明所述“预防”是指在可能的肺纤维化因素的存在下，使用后防止或降低肺纤维化的产生。本发明所述“治疗”是指减轻肺纤维化的程度，或者治愈肺纤维化使之正常化，或者减缓肺纤维化的进程。
本发明提供的技术方案之三是：一种抗肺纤维化的药物组合物，其含有所述的特异性结合TRB3的多肽或所述多肽的衍生物作为活性成分。
所述的活性成分是指具有预防或治疗肺纤维化功能的化合物。在所述药物组合物中，所述特异性结合TRB3的多肽或所述多肽的衍生物可以单独作为活性成分或和其他具有抗肺纤维化活性的化合物一起作为活性成分。
本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药 较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明的多肽或多肽衍生物能够特异性地与TRB3结合，阻断TRB3和MDM2蛋白的相互作用，从而应用于治疗和预防肺纤维化的药物的制备中。所制备的药物在治疗肺纤维化疾病中，具有疗效显著，毒副作用少，使用安全的优点。
附图说明
图1为肺纤维化小鼠肺组织内TRB3及MDM2、SLUG表达增高图谱。
图2为肺纤维小鼠肺组织内TRB3与MDM2相互作用图谱。其中输出显示肺组织裂解液经过MDM2抗体或对照抗体IgG沉淀之后所含TRB3蛋白与MDM2蛋白的蛋白量；其中输入显示初始肺组织裂解液所含TRB3蛋白与MDM2蛋白的蛋白含量。
图3为人肺上皮细胞过表达TRB3引发SLUG蛋白表达增加图。
图4-1和图4-2为表面等离子共振方法验证多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11与TRB3蛋白的结合能力。A为多肽MR2与TRB3蛋白的动力学曲线；B为多肽S1与TRB3蛋白结合动力学曲线；C为多肽S2与TRB3蛋白结合动力学曲线；D为多肽S3与TRB3蛋白结合动力学曲线；E为多肽S4与TRB3蛋白结合动力学曲线；F为多肽S5与TRB3蛋白结合动力学曲线；G为多肽S6与TRB3蛋白结合动力学曲线；H为多肽S7与TRB3蛋白结合动力学曲线；I为多肽S8与TRB3蛋白结 合动力学曲线；J为多肽S9与TRB3蛋白结合动力学曲线；K为多肽S10与TRB3蛋白结合动力学曲线；L为多肽S11与TRB3蛋白结合动力学曲线。其中，横坐标为反应时间，单位为秒。纵坐标为反应芯片表面与多肽的反应强度，单位为RU。
图5为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11干扰TRB3与MDM2蛋白相互作用图谱。其中，输出显示细胞裂解液经过MDM2抗体或对照抗体IgG沉淀之后所含TRB3蛋白与MDM2蛋白的蛋白量；输入显示初始细胞裂解液所含TRB3蛋白与MDM2蛋白的蛋白含量。
图6-1和图6-2为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11降低博莱霉素所致肺纤维化小鼠生存率检测结果图。
图7为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11降低博莱霉素所致肺纤维化小鼠肺重指数结果图。
图8为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11降低博莱霉素引起肺纤维化的病理学检查(HE)图谱。其中：A为假手术组，B为博莱霉素模型组，C为博莱霉素加MR2组，D为博莱霉素加S1组，E为博莱霉素加S2组，F为博莱霉素加S3组，G为博莱霉素加S4组，H为博莱霉素加S5组，I为博莱霉素加S6组，J为博莱霉素加S7组，K为博莱霉素加S8组，L为博莱霉素加S9组，M为博莱霉素加S10组，N为博莱霉素加S11组。
图9为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11降低博莱霉素引起肺纤维化的病理学检查评分结果图。
图10为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11降低博莱霉素引起肺纤维化小鼠肺组织羟脯氨酸含量结果图。
图11为多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11改善肺纤维化小鼠的肺功能检测结果。其中A为深吸气量，B为动态阻力，C为动态弹性，D为动态顺应性。
图12为多肽S19、S20、S21降低博莱霉素所致肺纤维化小鼠生存检测结果图。
图13为多肽S19、S20、S21降低博莱霉素所致肺纤维化小鼠肺重指数结果图。
图14为多肽S19、S20、S21降低博莱霉素引起肺纤维化的病理学检查(HE)图谱。其中：A为假手术组，B为博莱霉素模型组，C为博莱霉素加S19组，D为博莱霉素加S20组，E为博莱霉素加S21组。
图15为多肽S19、S20、S21降低博莱霉素引起肺纤维化的病理学检查评分结果图。
图16为多肽S19、S20、S21降低博莱霉素引起肺纤维化小鼠肺组织羟脯氨酸含量结 果图。
图17为多肽S19、S20、S21改善肺纤维化小鼠的肺功能检测结果。其中A为深吸气量，B为动态阻力，C为动态弹性，D为动态顺应性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例中所述的PBS溶液，指浓度为0.1M，pH值为7.2的磷酸盐缓冲液。
实施例中所述的室温为本领域常规的室温，较佳地为15～30℃。
实验结果用均值±标准误表示，经参数或者非参数方差检验，经比较p<0.05认为有显著性差异，p<0.01认为有极其显著性差异。
实施例1多肽的合成
多肽MR2的氨基酸序列参见序列表SEQ ID No.12。多肽MR2由北京赛百盛基因技术有限公司合成并纯化。
引入两个非天然氨基酸S-戊烯丙氨酸(S5)进行固相多肽链合成。固相多肽链合成完成后采用钌作为催化剂进行烯烃复分解反应(RCM)环化即得目标多肽。最后将目标多肽从树脂上切割下来进行纯化。上述固相多肽链合成及纯化的步骤由中肽生化有限公司公司完成。其中，两个S-戊烯丙氨酸插入在多肽MR2氨基酸序列中的第i、i+4位，由此得到不同序列的改造后的多肽(氨基酸序列参见序列表SEQ ID No.1～SEQ ID No.11)，其具体插入位点如下所示：
S1：S5-Leu-Met-Ala-S5-Phe-Thr-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg
S2：His-S5-Met-Ala-Cys-S5-Thr-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg
S3：His-Leu-S5-Ala-Cys-Phe-S5-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg
S4：His-Leu-Met-S5-Cys-Phe-Thr-S5-Ala-Lys-Lys-Leu-Lys-Lys-Arg
S5：His-Leu-Met-Ala-S5-Phe-Thr-Cys-S5-Lys-Lys-Leu-Lys-Lys-Arg
S6：His-Leu-Met-Ala-Cys-S5-Thr-Cys-Ala-S5-Lys-Leu-Lys-Lys-Arg
S7：His-Leu-Met-Ala-Cys-Phe-S5-Cys-Ala-Lys-S5-Leu-Lys-Lys-Arg
S8：His-Leu-Met-Ala-Cys-Phe-Thr-S5-Ala-Lys-Lys-S5-Lys-Lys-Arg
S9：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-S5-Lys-Lys-Leu-S5-Lys-Arg
S10：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-Ala-S5-Lys-Leu-Lys-S5-Arg
S11：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-Ala-Lys-S5-Leu-Lys-Lys-S5
实施例2Western-Blot检测肺纤维化小鼠肺部组织TRB3、MDM2、SLUG蛋白的表达
一、动物模型的制备
博莱霉素购自日本化药，批号640412。
实施例2中所使用的化合物若无特别说明，均购买自Sigma公司。
SPF级C57BL/6小鼠(雄性，6～8周龄，16～18g)，购自北京维通利华实验动物技术有限公司。
将雄性C57BL/6小鼠，隔夜禁食，使用45mg/kg戊巴比妥钠腹膜腔内注射(i.p.)麻醉，气管内注射博莱霉素，剂量为3U/kg。具体操作步骤如下：以尽量小的创伤切开小鼠颈部皮肤，在弯头眼科镊的协助下，暴露气管，使用微量进样器穿刺气管，向气管内注入约50μL博莱霉素，迅速地旋转并直立5分钟，以便使博莱霉素均匀地进入左右肺叶，从而构建了动物模型。整个操作在约60℃左右的手术操作台进行。假手术组中的小鼠气管内注射等量的注射用生理盐水。
将上述所构建的动物模型中的小鼠饲养于SPF级动物房内，至第10日小鼠肺部出现纤维化病理改变后处死小鼠，取小鼠的肺部组织进行后续实验。假手术组中的小鼠也饲养于SPF级动物房内，至第10日处死，取肺部组织进行后续实验。
二、Western-Blot检测肺纤维化小鼠肺部组织TRB3、MDM2、SLUG蛋白的表达
取步骤一、制备所得的动物模型适量的肺部组织，加入裂解缓冲液[含0.1mM乙二胺四乙酸(EDTA)、0.1mM乙二醇二乙醚二胺四乙酸(EGTA)、10mM KCl、10mM 4-羟乙基哌嗪乙磺酸(HEPEs)、50mM NaF、0.1M Na3VO4、0.1M Na3PO4、1mM/L抑肽酶(Aprotinin)、1mM/L胰蛋白酶抑制剂(Trypsin inhibitor)、1mM/L苯甲基磺酰氟化物(PMSF)、1mM/L亮肽素(Leupeptins)和1mM/L二硫苏糖醇(DTT)]匀浆后，冰上放置15min，间或振荡。迅速加入10％(v/v)乙基苯基聚乙二醇(NP-40)混匀，4℃、12000rpm离心5min。取上清，采用考马斯亮蓝法测定蛋白浓度，调节蛋白浓度至相同，以便用于Western Blot分析。
Western Blot分析检测TRB3、MDM2和SLUG。使用Amersham显色液(NBT/BCIP染色试剂盒IK5030，购自华美公司)显色，经Western-blot分析软件(gelPro32)测出各条带的光密度值分析，结果如图1所示。图1表明，肺纤维化小鼠肺部组织内TRB3、MDM2、SLUG蛋白呈高表达状态。
实施例3利用免疫共沉淀的方法验证肺纤维化组织内MDM2与TRB3蛋白的结合
免疫共沉淀所使用的试剂如下：
裂解液A液：称取0.6057g Tris碱、1.7532g NaCl、0.1017g MgCl2·6H2O、0.0742g EDTA、10mL甘油和10mL 10％(v/v)NP40，加去离子水至150mL，用HCl调pH值至7.6，定容至191mL，充分混匀，用0.45μm滤膜过滤，置于4℃储存。
裂解液B液：称取200μL 2Mβ-磷酸甘油、4mL 2.5M NaF、2mL 100mM NaVO3、2mL100mM PMSF、200μL 1M DTT、200μL 1mg/mL Leu、200μL 1mg/mL Pep和200μL 1mg/mL Apr，总体积共9mL。置于-20℃储存。使用时，将裂解液B液解冻，按裂解液B液:裂解液A液的体积比1:100的比例，将裂解液B液加入裂解液A液中并混匀，即得细胞裂解液。
免疫共沉淀洗液：包括1％(v/v)NP40、150mM NaCl、20mM HEPES、10％(v/v)pH7.5的甘油和1mM EDTA。
Protein A/G Plus-Agarose购自美国Santa cruz公司。
具体操作步骤如下：
(1)取下实施2步骤一、制备所得的动物模型适量的肺组织大叶，称量肺组织。
(2)以免疫共沉淀裂解液裂解步骤(1)称量的肺组织，收获细胞总蛋白约10mg，将动物模型和假手术组的肺组织蛋白均调整至10μg/mL的浓度。分别取动物模型和假手术组的肺组织蛋白200μg作为输入组，并将上述输入组作为对照。
(3)将等量的每组剩余的9800μg蛋白分别加入2μg MDM2抗体(购买自Abcam，编号ab16895)和与MDM2抗体种属相同的普通IgG抗体(购买自Cell Signaling，商品编号2729)，同时加入10μL Protein A/G Plus-Agarose(购买自Santa Cruz，编号SC-2003)充分重悬，4℃缓慢旋转摇动过夜。4℃、3000rpm离心5min，小心吸除上清。加入0.5mL免疫共沉淀裂解液，混匀，冰浴静置1min，4℃、3000rpm离心30秒，小心吸除上清。重复洗涤5次，最后一次离心前静置5min。小心吸除上清，加入30μL 2×SDS凝胶加样缓冲液，混匀，95℃变性3min，迅速转移至冰浴冷却。12000rpm室温离心2min，上清即为沉淀的蛋白样品，取部分蛋白样品进行SDS-聚丙烯酰胺凝胶电泳。
所得结果见图2，由图2可见肺纤维化组织内TRB3蛋白与MDM2蛋白存在相互结合的现象。其中，“输入”使用上述的细胞裂解液；“输入”代表蛋白样品中初始TRB3与MDM2的含量，即蛋白样品在经MDM2抗体或对照抗体IgG(由于MDM2抗体为IgG型抗体，故选择IgG抗体作为对照)沉淀之前，蛋白样品中TRB3和MDM2含量。结果表明，输入组细胞裂解液中的TRB3蛋白和MDM2蛋白的含量一致。
“输出”代表蛋白样品经MDM2抗体或对照抗体IgG沉淀之后，蛋白样品中的TRB3 与MDM2含量。由于IgG抗体作为MDM2抗体的对照抗体，不能将MDM2蛋白沉淀，因此IgG抗体处理的蛋白样品中MDM2蛋白印迹泳道显示为空白；而MDM2抗体作为实验组抗体，能与MDM2蛋白结合并且将其沉淀下来，因此MDM2抗体处理后的蛋白样品结果为MDM2蛋白印迹泳道显示为黑色。正是因为MDM2蛋白与TRB3蛋白之间存在相互作用，MDM2抗体沉淀MDM2蛋白时也能将TRB3蛋白沉淀下来，故MDM2抗体处理后的细胞裂解液的TRB3蛋白印迹泳道显示为黑色。由于IgG抗体不能将MDM2蛋白沉淀下来，因此也无法将MDM2相互作用蛋白TRB3沉淀下来，故IgG抗体处理的后的细胞裂解液的TRB3蛋白印迹泳道为空白。上述结果充分证明，TRB3蛋白与MDM2蛋白能够直接发生相互作用。
实施例4TRB3引发肺上皮细胞SLUG增加。
(1)将400μL去核酸酶水加入管中，震荡10秒钟，稀释转染试剂(购自Invitrogen，型号lipofectamine 2000)。
(2)将转染试剂与质粒DNA[对照质粒(购自傲锐东源，型号PS100001)；TRB3过表达质粒(购自傲锐东源，型号RC206687)]按1：2的比例混合，所述比例为转染试剂用量(mL)与DNA质量(mg)之比，室温静置15分钟，得混合液。
(3)吸取BEAS-2B细胞(购自中国医学科学院基础医学研究所)培养板中的M199培养基，用PBS清洗一次。
(4)加入400μL混合液，将细胞放回培养箱中37℃培养24个小时。
(5)使用RIPA裂解液(购自碧云天，型号P0013B)裂解转染后的BEAS-2B细胞，采用实施例3中Western-blot方法检测细胞内SLUG蛋白表达。
结果如图3所示。图3说明，过表达TRB3的BEAS-2B细胞内Slug的表达明显增加。
实施例5用表面等离子共振的方法检测多肽S1-S11与TRB3蛋白的结合能力
表面等离子共振实验在表面等离子共振仪Biacore T200中进行，操作步骤按照等离子共振仪Biacore T200的说明书进行。具体步骤如下：
1.将纯化的TRB3蛋白(购自RD公司)通过氨基偶联到CM5芯片(购自GE公司)上，按10μL/min的流速洗脱除去未结合的蛋白，并且平衡芯片表面2小时。其中，氨基偶联、洗脱和平衡的具体步骤参见GE公司CM5芯片的相关说明书。
2.自动进样250μL不同浓度(800、400、200、50、12.5、6.25和3.125nM)的实施例1所制备的S1～S11和MR2多肽片段，整个表面等离子共振实验在25℃进行。所使用的缓冲液为HBS-EP缓冲液[0.01M HEPES、0.15M NaCl、3mM EDTA和0.005％(w/w) 表面活性剂TWEEN20]。用Biacore T200自带分析软件模拟不同浓度多肽与TRB3的结合曲线，结果如图4-1、4-2和表1所示。图4-1、4-2和表1说明，肽段MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11均可与TRB3蛋白相互作用。
表1多肽S1～S11和MR2与TRB3蛋白的亲和力测试
多肽名称 与TRB3蛋白的亲和力常数(KD)
MR2 4.33×10-8M
S1 2.02×10-8M
S2 1.91×10-8M
S3 4.27×10-8M
S4 9.63×10-9M
S5 1.15×10-8M
S6 4.96×10-8M
S7 8.95×10-9M
S8 3.59×10-8M
S9 3.71×10-8M
S10 2.93×10-8M
S11 3.77×10-8M
实施例6圆二色谱法检测多肽S1-S11的α螺旋率
用圆二色谱仪(购自日本Jasco公司)检测多肽的α螺旋率。将实施例1所制备的多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11溶解到PBS溶液中，将圆二色谱仪的上机浓度调整为1mg/mL，结果如表2所示。表2说明，多肽S1、S2、S3、S4、S5、S6、S7、S8、S9、S10和S11的α螺旋率明显高于多肽MR2。其中，α螺旋率指保持二级结构α螺旋的多肽的肽段数量占总多肽的肽段数量的百分比。
表2圆二色谱法测定多肽α螺旋率
Figure PCTCN2017074792-appb-000001
实施例7免疫共沉淀的方法验证S1～S11和MR2多肽在细胞水平抑制蛋白MDM2与TRB3的结合
1.将人肺上皮BEAS-2B细胞(购自中国医学科学院基础医学研究所)铺90mm2培养 皿，待细胞贴壁后按实施例5中所述方法向细胞中转入TRB3表达质粒，24小时后分别加入1mg/mL的实施例1制得的多肽MR2、S1、S2、S3、S4、S5、S6、S7、S8、S9、S10或S11，在37℃孵育箱中孵育12小时后收集细胞。
2.按实施例3中所述方法用MDM2抗体进行免疫共沉淀，随后使用TRB3抗体进行检测。
结果如图5所示。图5的结果说明，多肽S1～S11干扰TRB3/MDM2蛋白相互作用明显强于MR2。
实施例8测定多肽S1-S11对肺纤维化小鼠死亡率的影响
根据实施例2中所述方法进行肺纤维化动物模型制作，从造模后第10天开始给予多肽治疗(治疗分组如表3所示)，并从造模后第10天计算时间以统计死亡率。每天统计各组实验动物死亡情况并进行计算，若某组动物未出现死亡，其生存率为100％，若某组动物全部死亡，则其生存率为0％。所述生存率为存活的小鼠个数占该组小鼠个数的百分比。结果如图6-1、图6-2表4所示，图6-1、图6-2说明，与假手术组对比，模型组生存率显著降低。经过药物治疗后，S1～S11给药组均能显著提高纤维化小鼠生存率。图6-1、图6-2中，##为与假手术组相比，p<0.01；*为与博莱霉素组相比，p<0.05；**为与博莱霉素组相比，p<0.01；i.p.指腹腔内注射。上述结果表明，本发明所述多肽能够有效降低肺纤维化模型小鼠的死亡率，具有毒副作用少，使用更安全的优点。
表3肺纤维化动物模型在造模后的分组给药情况
Figure PCTCN2017074792-appb-000002
Figure PCTCN2017074792-appb-000003
表4多肽S1-S11对肺纤维化小鼠死亡率的影响
Figure PCTCN2017074792-appb-000004
实施例9检测多肽S1-S11对肺纤维化小鼠的肺重指数的影响
按实施例2中所述方法制作肺纤维化模型，并按表3进行分组给药。28天后精细剥离小鼠肺脏并称取湿重，将肺重(mg)除以小鼠体重(g)得到肺重指数，结果见图7和表5所示。从图7可见，与假手术组相比，给予博莱霉素后小鼠肺重指数显著升高。因此多肽S1～S11给药后均能显著降低纤维化小鼠肺重指数。图7中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表5多肽S1-S11对肺维化小鼠的肺重指数的影响
组别 肺重指数
假手术组 6.7875
博莱霉素组 12.7375
MR2给药组 11.4625
S1给药组 8.675
S2给药组 9.35
S3给药组 8.8
S4给药组 8.925
S5给药组 8.575
S6给药组 9.725
S7给药组 7.675
S8给药组 8.775
S9给药组 7.4125
S10给药组 8.8875
S11给药组 9.2875
实施例10测定多肽S1-S11对博莱霉素所致肺纤维化病理形态学影响
一般的组织变化和组织产物都可以通过HE染色法，即苏木素-伊红染色法显示出来，这是形态学最常用的染色方法。其中苏木素染液为碱性染液，重要使细胞核内的染色质与胞质内的核糖体着紫蓝色；伊红为酸性染料，主要使细胞质和细胞外基质中的成分着红色。按实施例2中所述方法制作肺纤维化模型，并按表3进行分组给药。28天后取实验动物右侧下叶肺组织，4％(v/v)多聚甲醛固定后石蜡包埋。在包埋肺组织的蜡块最大横截面切片，HE染色观察基本病理改变。结果见图8所示。图8显示假手术小鼠肺部HE染色组织清晰可见，肺泡结构完整，未见炎症和纤维化病理改变。博莱霉素组小鼠肺部出现明显炎症，炎性细胞大量浸润，肺组织结构破坏严重。多肽S1～S11给药后均能减 轻博莱霉素所引起的肺部炎症，并有效改善肺损伤，恢复肺部正常结构。
根据HE染色的结果进行炎性分级观察，标准如下(0-5级)：0级：正常组织。1级：极小的炎症改变。2级：轻微到中等的炎症改变，没有明显的肺组织结构的破坏。3级：中等程度的炎症损伤(肺泡膈膜增厚)。4级：中等程度要重度的炎症损伤，形成组织团块，或者局限性肺炎区域破坏肺组织正常结构。5：严重的炎症损伤，局部区域肺组织结构严重破坏引起管腔闭合等。
结果见图9和表6所示。结果显示与假手术组相比，给予博莱霉素后小鼠肺部出现显著炎症。给予多肽S1～S11均可以显著降低博莱霉素所引起的肺部炎症。图9中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表6多肽S1-S11对博莱霉素所致肺纤维化的炎性评分
组别 炎性评分
假手术组 0
博莱霉素组 4.333333
MR2给药组 4
S1给药组 2.666667
S2给药组 2.666667
S3给药组 2.5
S4给药组 2.833333
S5给药组 2.333333
S6给药组 3
S7给药组 2
S8给药组 2.833333
S9给药组 2.166667
S10给药组 3
S11给药组 3
实施例11测定多肽S1-S11对肺纤维化小鼠羟脯氨酸含量影响
羟脯氨酸在胶原蛋白中占13.4％(w/w)，在弹性蛋白中占极少量，在其他蛋白中均不存在，因此通过羟脯氨酸检测胶原蛋白的含量。按实施例2中所述方法制作肺纤维化模型，并按表3进行分组给药。28天后检测动物左肺全叶羟脯氨酸的含量，评价肺纤维化的情况。
具体方法如下：将实施例2制备所得动物模型中的小鼠在SPF级动物房内饲养，并 按表3给药方案给予多肽治疗，至构建动物模型后的第21日取实验动物的左侧全部肺叶，记录湿重，生理盐水超声匀浆制备成10％(w/w)的组织匀浆，取约150μL的匀浆上清液，加500μL碱水解液(由南京建成生物工程技术有限公司羟脯氨酸碱水解试剂盒提供)，涡旋混匀后，120℃、0.1Kpa条件下碱水解法处理40min，调pH值后定容，活性炭处理后取上清。按照氯胺T法进行羟脯氨酸测定(本实施例的操作方法参照南京建成生物工程技术有限公司试剂盒说明书)。结果见图10以及表7。从图10可见，与假手术组相比，博莱霉素组羟脯氨酸含量及其显著性增高，说明纤维化病理改变严重。S1～S11给药后可以显著降低纤维化小鼠肺部羟脯氨酸的含量。图10中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表7多肽S1-S11对肺纤维化小鼠羟脯氨酸含量影响
组别 羟脯氨酸含量(μg/mg蛋白)
假手术组 0.42125
博莱霉素组 1.0875
MR2给药组 0.90875
S1给药组 0.70875
S2给药组 0.70625
S3给药组 0.82875
S4给药组 0.65
S5给药组 0.6575
S6给药组 0.83375
S7给药组 0.655
S8给药组 0.825
S9给药组 0.72125
S10给药组 0.81625
S11给药组 0.7725
实施例12测定多肽S1-S11对肺纤维化小鼠的肺功能影响
肺功能是临床检测患者肺纤维化的金指标。肺功能的下降往往伴随着纤维化的加剧，而肺功能的改善往往也代表了肺部组织结构的恢复。按实施例2中所述方法制作肺纤维化模型，并按表3进行分组给药。构建动物模型后的28天日，小鼠通过戊巴比妥钠(45mg/kg，i.p.)麻醉，通过Flexivent小动物肺功能仪进行肺功能检测，检测方法为TLC，SnapShots，(检测方法请参见：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.)。
结果如图11和表8所示，其中A为深吸气量，B为动态阻力，C为动态弹性，D为动态顺应性。图11显示，与假手术组相比，博莱霉素所诱导肺纤维化小鼠深吸气量明显降低，肺部动态阻力及动态弹性上升，顺应性显著降低。经过多肽S1～S11治疗后肺功能显著恢复。图11中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表8多肽S1-S11对肺纤维化小鼠肺功能影响
Figure PCTCN2017074792-appb-000005
上述实施例结果表明，本发明的多肽具有显著的抗肺纤维化作用，可作为活性成份用于制备抗肺纤维化的药物。
实施例13多肽衍生物S19～21(多肽MR2+细胞穿膜肽的嵌合多肽)的合成
S19：嵌合多肽Pep2-MR2(其氨基酸序列如序列表SEQ ID No.19所示)、S20：嵌合多肽TAT-MR2(其氨基酸序列如序列表SEQ ID No.20所示)、S21：嵌合多肽Antp-MR2(其氨基酸序列如序列表SEQ ID No.21所示)由北京赛百盛基因技术有限公司合成并纯化。
实施例14测定S19～21对肺纤维化小鼠死亡率的影响
根据实施例2中所述方法进行肺纤维化动物模型制作，从造模后第10天开始给予多肽治疗(治疗分组如表9所示)，并从造模后第10天起开始计算时间以统计死亡率。每天统计各组实验动物死亡情况并进行计算，若某组动物未出现死亡，其生存率为100％，若某组动物全部死亡，则其生存率为0％。结果如图12和表10所示。图12说明，与假手术组对比，模型组生存率显著降低。经过药物治疗后，S19～S21给药组均能显著提高纤维化小鼠生存率。图12中，##为与假手术组相比，p<0.01；*为与博莱霉素组相比，p<0.05；**为与博莱霉素组相比，p<0.01。上述结果表明，本发明所述多肽衍生物能够有效降低肺纤维化模型小鼠的死亡率，具有毒副作用少，使用更安全的优点。
表9肺纤维化动物模型在造模后的分组给药情况
Figure PCTCN2017074792-appb-000006
表10S19～21对肺纤维化小鼠死亡率的影响
Figure PCTCN2017074792-appb-000007
实施例15检测S19～21对肺纤维化小鼠的肺重指数的影响
按实施例2中所述方法制作肺纤维化模型，并按表9进行分组给药。28天后精细剥离小鼠肺脏并称取湿重，将肺重(mg)除以小鼠体重(g)得到肺重指数，结果见图13和表11所示。从图13可见，与假手术组相比，给予博莱霉素后小鼠肺重指数显著升高。因此S19～S21给药后均能显著降低纤维化小鼠肺重指数。图13中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表11S19～21对肺维化小鼠的肺重指数的影响
组别 肺重指数
假手术组 6.7875
博莱霉素组 12.7375
S19给药组 8.675
S20给药组 10.0625
S21给药组 11.2125
实施例16测定S19～21对博莱霉素所致肺纤维化病理形态学影响
按实施例2中所述方法制作肺纤维化模型，并按表9进行分组给药。28天后取实验动物右侧下叶肺组织，4％(v/v)多聚甲醛固定后石蜡包埋。在包埋肺组织的蜡块最大横截面切片，HE染色观察基本病理改变。结果见图14所示。图14显示，S19～S21给药后均能减轻博莱霉素所引起的肺部炎症，并有效改善肺损伤，恢复肺部正常结构。
根据HE染色的结果进行炎性分级观察，标准如下(0-5级)：0级：正常组织。1级：极小的炎症改变。2级：轻微到中等的炎症改变，没有明显的肺组织结构的破坏。3级：中等程度的炎症损伤(肺泡膈膜增厚)。4级：中等程度要重度的炎症损伤，形成组织团块，或者局限性肺炎区域破坏肺组织正常结构。5：严重的炎症损伤，局部区域肺组织结构严重破坏引起管腔闭合等。
结果见图15和表12所示。结果显示与假手术组相比，给予博莱霉素后小鼠肺部出现显著炎症。给予S19～S21均可以显著降低博莱霉素所引起的肺部炎症。图15中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表12S19～21对博莱霉素所致肺纤维化的炎性评分
组别 炎性评分
假手术组 0
博莱霉素组 4
S19给药组 1.833333
S20给药组 2.333333
S21给药组 2.666667
实施例17测定S19～21对肺纤维化小鼠羟脯氨酸含量影响
羟脯氨酸在胶原蛋白中占13.4％(w/w)，在弹性蛋白中占极少量，在其他蛋白中均不存在，因此通过羟脯氨酸检测胶原蛋白的含量。按实施例2中所述方法制作肺纤维化模型，并按表9进行分组给药。28天后检测动物左肺全叶羟脯氨酸的含量，评价肺纤维化的情况。
具体方法如下：将实施例2制备所得动物模型中的小鼠在SPF级动物房内饲养，并按表9给药方案给予多肽治疗，至构建动物模型后的第21日取实验动物的左侧全部肺叶，记录湿重，生理盐水超声匀浆制备成10％(w/w)的组织匀浆，取约150μL的匀浆上清液，加500μL碱水解液(由南京建成生物工程技术有限公司羟脯氨酸碱水解试剂盒提供)，涡旋混匀后，120℃、0.1Kpa条件下碱水解法处理40min，调pH值后定容，活性炭处理后取上清。按照氯胺T法进行羟脯氨酸测定(本实施例的操作方法参照南京建成生物工程技术有限公司试剂盒说明书)。结果见图16以及表13。从图16可见，与假手术组相比，博莱霉素组羟脯氨酸含量及其显著性增高，说明纤维化病理改变严重。而S19～S21给药后可以显著降低纤维化小鼠肺部羟脯氨酸的含量。图16中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表13S19～21对肺纤维化小鼠羟脯氨酸含量影响
组别 羟脯氨酸含量(μg/mg蛋白)
假手术组 0.42125
博莱霉素组 0.915
S19给药组 0.56375
S20给药组 0.744286
S21给药组 0.74875
实施例18测定S19～21对肺纤维化小鼠的肺功能影响
肺功能是临床检测患者肺纤维化的金指标。肺功能的下降往往伴随着纤维化的加剧，而肺功能的改善往往也代表了肺部组织结构的恢复。按实施例2中所述方法制作肺纤维化模型，并按表9进行分组给药。28天后小鼠通过戊巴比妥钠(45mg/kg，i.p.)麻醉，通过Flexivent小动物肺功能仪进行肺功能检测，检测方法为TLC，SnapShots，(检测方法请参见：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.)。
结果如图17和表14所示，其中A为深吸气量，B为动态阻力，C为动态弹性，D 为动态顺应性。图17显示，与假手术组相比，博莱霉素所诱导肺纤维化小鼠深吸气量明显降低，肺部动态阻力及动态弹性上升，顺应性显著降低。经过多肽S19～S21治疗后肺功能显著恢复。图17中，##为与假手术组相比p<0.01，*为与博莱霉素组相比p<0.05，**为与博莱霉素组相比p<0.01。
表14S19～21对肺纤维化小鼠肺功能影响
Figure PCTCN2017074792-appb-000008
上述实施例结果表明，本发明的多肽衍生物具有显著的抗肺纤维化作用，也可作为活性成份用于制备抗肺纤维化的药物。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种特异性结合TRB3的多肽或所述多肽的衍生物，所述多肽的氨基酸序列如序列表SEQ ID No.12所示，或者，如将序列表SEQ ID No.12所示的氨基酸序列中的两个或两个以上的氨基酸替换为侧链可相连的非天然氨基酸所示；所述的衍生物包括所述多肽与细胞穿膜肽接连所形成的嵌合肽、所述多肽与病毒形成的融合肽、甲基化的所述多肽、糖基化的所述多肽和聚乙二醇化的所述多肽。
  2. 如权利要求1所述的特异性结合TRB3的多肽或所述多肽的衍生物，其特征在于，所述的侧链可相连的非天然氨基酸为S-戊烯丙氨酸；较佳地，所述替换的氨基酸的数目为两个且所述替换的氨基酸的位置分别为第i位和第i+3位，或者，为第i位和第i+4位，其中1≤i≤11。
  3. 如权利要求2所述的特异性结合TRB3的多肽或所述多肽的衍生物，其特征在于，所述的多肽的氨基酸序列如序列表SEQ ID No.1、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和SEQ ID No.11中任一项所示。
  4. 如权利要求1～3任一项所述的特异性结合TRB3的多肽或所述多肽的衍生物，其特征在于，所述的细胞穿膜肽选自HIV-1病毒反转录激活因子蛋白的TAT肽、果蝇触角同源异型蛋白的转录因子Antp肽、Pep-1肽、Pep-2肽、MPG肽和RGD肽中的任一种或多种；较佳地，所述细胞穿膜肽连接在所述多肽的N端或者C端；更佳地，
    当细胞穿膜肽为Pep-2肽时，所述多肽的衍生物的氨基酸序列如序列表SEQ ID No.19所示；
    当细胞穿膜肽为TAT肽时，所述多肽的衍生物的氨基酸序列如序列表SEQ ID No.20所示；
    当细胞穿膜肽为Antp肽时，所述多肽的衍生物的氨基酸序列如序列表SEQ ID No.21所示。
  5. 如权利要求1～4中任一项所述的特异性结合TRB3的多肽或所述多肽的衍生物在制备治疗和/或预防肺纤维化的药物中的应用。
  6. 如权利要求5所述的应用，其特征在于，所述的肺纤维化为原发性的肺纤维化或继发性的肺纤维化。
  7. 如权利要求5所述的应用，其特征在于，所述的肺纤维化为博莱霉素引发的肺纤维化。
  8. 一种抗肺纤维化的药物组合物，其特征在于，其含有如权利要求1～4中任一项所述的特异性结合TRB3的多肽或所述多肽的衍生物。
  9. 如权利要求8所述的药物组合物，其特征在于，其还包括一种或多种药用载体。
  10. 如权利要求8或9所述的药物组合物，其特征在于，其含有如权利要求1～4中任一项所述的特异性结合TRB3的多肽或所述多肽的衍生物和具有抗肺纤维化活性的化合物一起作为活性成分。
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