A drug for treating pulmonary fibrosis

Abstract

The present invention relates to a drug for preventing and / or treating idiopathic pulmonary fibrosis. Specifically, the present invention provides the use of annexin A5 in the preparation of a composition for preventing and / or treating idiopathic pulmonary fibrosis. In the present invention, annexin A5 can effectively treat / slow down idiopathic pulmonary fibrosis, reduce the degree of interstitial lung lesions, inhibit the differentiation of fibroblasts and the deposition of collagen, reduce the release of pro-inflammatory factors, and maintain the normal morphology of alveolar epithelium.

Description

Technical Field

[0001] The present invention relates to the field of protein drugs, and in particular to an application of annexin A5 in treating idiopathic pulmonary fibrosis. Background Art

[0002] Fibrosis is one of the hallmarks of many chronic degenerative diseases and is an important cause of death in the current population. There are more than 3 million patients with idiopathic pulmonary fibrosis (IPF) worldwide, and its incidence is similar to that of gastric cancer and brain cancer, and the incidence has doubled in the past 20 years. Studies have shown that middle-aged and elderly men are the main population affected by the disease. The early symptoms of IPF are hidden. As scars accumulate, its symptoms usually take years or even decades to gradually appear. Common symptoms include dyspnea, dry cough, fatigue, weight loss, and finger clubbing. Due to the heterogeneity of patients and the lack of specificity of symptoms, the current diagnosis of IPF requires a combined diagnosis of chest HRCT and lung tissue pathology to exclude other interstitial lung diseases, which usually takes 1-2 years. Delayed diagnosis greatly affects the survival of IPF patients, with a median survival of only 2-3 years. In China, there is currently no large-scale epidemiological survey on the incidence of IPF that has been published, but due to the large population base and aging trend, the burden of IPF on individuals, families, society and public health resources in my country cannot be underestimated.

[0003] The treatment options for IPF are limited. Due to the irreversible damage to lung function, lung transplantation is still the only effective treatment to prolong the survival of patients. According to incomplete statistics, at least dozens of drugs for the treatment of IPF are currently under development, a few of which have entered clinical phase II and phase III trials, but their effectiveness and safety still need to be explored. These include recombinant human penetratin-2 (PTX-2), anti-connective tissue growth factor (CTGF) antibodies, lysophosphatidic acid (LPA) inhibitors, and phosphodiesterase 4 (PDE4) inhibitors. These drugs directly or indirectly participate in regulating the release of pulmonary inflammatory factors, damage and activation of epithelial cells, aggregation and differentiation of fibroblasts, and thus maintain the lung function of IPF patients. Pirfenidone and nintedanib are the only two drugs approved by the FDA in clinical practice. Although these two drugs can effectively delay the decline of lung function and the process of fibrosis, they cannot cure or reverse idiopathic pulmonary fibrosis, so they are not effective for patients with severe IPF. In addition, the high market price of drugs and the inability to interrupt medication also bring a heavy economic burden to IPF patients and their families. Therefore, there is an urgent need for new clinical therapies.

[0004] Therefore, there is a need in the art for a new drug for treating idiopathic pulmonary fibrosis. Summary of the invention

[0005] The object of the present invention is to provide a use of annexin A5 in the treatment of pulmonary fibrosis, especially idiopathic pulmonary fibrosis.

[0006] In the first aspect of the present invention, there is provided a use of annexin A5 in the preparation of a composition for preventing and / or treating idiopathic pulmonary fibrosis.

[0007] In another preferred embodiment, the idiopathic pulmonary fibrosis is idiopathic pulmonary fibrosis caused by one or more factors selected from the following group: infection, environmental exposure, smoking, aging, genetics, and gene mutation.

[0008] In another preferred embodiment, the idiopathic pulmonary fibrosis is idiopathic pulmonary interstitial fibrosis.

[0009] In another preferred embodiment, the idiopathic pulmonary fibrosis is idiopathic pulmonary fibrosis caused by micro-injury of alveolar epithelial cells.

[0010] In another preferred embodiment, the idiopathic pulmonary fibrosis is drug-induced idiopathic pulmonary fibrosis.

[0011] In another preferred embodiment, in the drug-induced idiopathic pulmonary fibrosis, the drug is selected from the following group: targeted drugs, immune drugs, chemical drugs, traditional Chinese medicines, or combinations thereof.

[0012] In another preferred embodiment, the drug is an anti-cancer drug or an antibiotic, such as bleomycin.

[0013] In another preferred embodiment, the fibrosis occurs in one or more of the following sites: bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, pulmonary interstitium, pulmonary arterioles, or around their blood vessels.

[0014] In another preferred embodiment, the prevention and / or treatment of idiopathic pulmonary fibrosis includes one or more of the following features selected from the following group:

[0015] (1) Improving lung function or maintaining normal lung physiological function, preferably improving the reduction of forced vital capacity;

[0016] (2) Maintaining the potential for normal lung injury repair;

[0017] (3) Reducing inflammatory cell infiltration, where the inflammatory cell infiltration occurs in one or more of the following sites: bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pulmonary interstitium, or around their blood vessels;

[0018] (4) Reducing fibrosis, where the fibrosis occurs in one or more of the following sites: bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, pulmonary interstitium, or around their blood vessels;

[0019] (5) Reduce the injury of bronchioles and pulmonary arterioles within and at the edge of the lesion;

[0020] (6) Inhibit the expression of pro-fibrotic biomarkers;

[0021] (7) Inhibit the release and / or expression of pro-inflammatory factors, wherein the release of the pro-inflammatory factors occurs in one or more sites of alveolar bronchoalveolar lavage fluid and lung tissue homogenate;

[0022] (8) Inhibit the differentiation of fibroblasts or the transformation and differentiation of fibroblast-myofibroblasts in lung tissue;

[0023] (9) Inhibit the deposition or excessive deposition of collagen in lung tissue;

[0024] (10) Maintain the morphology of alveolar epithelial cells in lung tissue;

[0025] (11) Maintain the expression of alveolar epithelial phenotypic markers in lung tissue;

[0026] (12) Reduce the expression of hydroxyproline in lung tissue in pulmonary fibrosis;

[0027] (13) Inhibit the expression of pro-fibrotic factors in lung tissue.

[0028] In another preferred embodiment, the injury of the bronchioles and pulmonary arterioles includes one or more characteristics selected from the following group:

[0029] (1) Reduce alveolar hemorrhage;

[0030] (2) Reduce the hyperplasia of lung epithelial cells, wherein the epithelial cell hyperplasia occurs in one or more sites of bronchioles, terminal bronchioles;

[0031] (3) Reduce the hyperplasia of granulation tissue in the outer membrane of the tube wall, wherein the granulation tissue in the outer membrane of the tube wall occurs in one or more sites of bronchioles, terminal bronchioles;

[0032] (4) Reduce the infiltration of inflammatory cells, wherein the infiltration of inflammatory cells occurs in one or more sites of bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, lung interstitium, pulmonary arterioles or around their blood vessels;

[0033] (5) Reduce edema, wherein the edema occurs in one or more sites of bronchi, bronchioles, terminal bronchioles, respiratory bronchioles or around their blood vessels;

[0034] (6) Reduce endothelial cell exfoliation, wherein the endothelial cell exfoliation occurs in pulmonary arterioles.

[0035] In another preferred embodiment, the expression includes the expression at the protein and / or mRNA levels.

[0036] In another preferred example, the fibrotic biomarker includes one or more of fibronectin 1 (FN1), α-smooth muscle actin (α-SMA, ACTA2), type I collagen (COL-1, COL1A1), type III collagen (COL-3, COL3A1), and type V collagen (COL-5, COL5A1).

[0037] In another preferred example, the pro-inflammatory factor includes one or more of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).

[0038] In another preferred example, the collagen includes one or more of type I collagen (COL-1, COL1A1), type III collagen (COL3, COL3A1), and type V collagen (COL5, COL5A1).

[0039] In another preferred example, the alveolar epithelial phenotype biomarker includes surfactant protein C (SFTPC).

[0040] In another preferred example, the maintenance of normal pulmonary physiological function means that the decline compared to the physiological function of normal lungs is no more than 30%, preferably no more than 20%, more preferably no more than 10%, such as 8%, 5%, 2%, 1%.

[0041] In another preferred example, the maintenance of the morphology of alveolar epithelial cells in lung tissue means that the change compared to the morphology of alveolar epithelial cells in normal lung tissue is no more than 30%, preferably no more than 20%, more preferably no more than 10%, such as 8%, 5%, 2%, 1%.

[0042] In another preferred example, the inhibition of pro-inflammatory factor expression includes the expression of TNFα and IL-1β at the mRNA level.

[0043] In another preferred example, the inhibition of collagen deposition or excessive deposition in lung tissue includes the inhibition of the expression of type I collagen (COL1) and type III collagen (COL3) at the mRNA level.

[0044] In another preferred example, the inhibition of fibroblast differentiation includes the inhibition of the expression of the differentiation marker α-SMA of fibroblasts.

[0045] In another preferred example, the alveolar epithelial hyperplasia in idiopathic pulmonary fibrosis includes alveolar wall thickening and / or formation of a hyaline membrane.

[0046] In another preferred example, the prevention and / or treatment of idiopathic pulmonary fibrosis includes one or more of the following features selected from the group:

[0047] (1) Inhibit the expression of epithelial-mesenchymal transition (EMT) markers in alveolar epithelium;

[0048] (2) Inhibit apoptosis of alveolar epithelial cells;

[0049] (3) Promote the proliferation of alveolar epithelial cells;

[0050] (4) Promote the migration of alveolar epithelial cells;

[0051] (5) Inhibit the expression of profibrotic factors in alveolar epithelium.

[0052] In another preferred embodiment, the alveolar epithelium is type II alveolar epithelium.

[0053] In another preferred embodiment, the alveolar epithelial cells are type II alveolar epithelial cells (ATII).

[0054] In another preferred embodiment, the inhibition of the expression of epithelial-mesenchymal transition (EMT) markers in alveolar epithelium includes inhibiting the expression of Vimentin and / or promoting the expression of E-cadherin.

[0055] In another preferred embodiment, the profibrotic factors in alveolar epithelium include one or more of TNF-α, SP-A, SP-D, TGFβ1, Osteopontin, CXCL12, CCL2, MMP-1, MMP-7.

[0056] In another preferred embodiment, the prevention and / or treatment of idiopathic pulmonary fibrosis includes reducing the expression of pro-inflammatory factors such as IL-6 and IL-17A in lung tissue and / or increasing the expression of anti-inflammatory factors (such as IL-10).

[0057] In another preferred embodiment, the prevention and / or treatment of idiopathic pulmonary fibrosis includes one or more of the following features:

[0058] (1) Maintain the potential for normal lung injury repair;

[0059] (2) Protect the normal lung interstitial structure;

[0060] (3) Slow down the decline of forced vital capacity (FVC);

[0061] (4) Slow down the interstitial lung disease;

[0062] (5) Inhibit the excessive deposition of collagen in lung tissue;

[0063] (6) Inhibit the fibroblast-myofibroblast transformation in lung tissue;

[0064] (7) Inhibit the epithelial-mesenchymal transition in lung tissue;

[0065] (8) Suppress apoptosis of alveolar epithelium in lung tissue;

[0066] (9) Promote migration of alveolar epithelium in lung tissue;

[0067] (10) Promote proliferation of alveolar epithelium in lung tissue.

[0068] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0069] In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0070] In another preferred embodiment, the carrier is selected from the group consisting of solubilizers, cosolvents, antioxidants, photodegradation inhibitors, pH regulators, emulsifiers, antibacterial and antifungal preservatives, complexing agents, fillers, binders, disintegrants, and lubricants, or one or more thereof.

[0071] In another preferred embodiment, the composition further comprises other drugs for preventing and / or treating idiopathic pulmonary fibrosis.

[0072] In another preferred embodiment, the other drugs for preventing and / or treating idiopathic pulmonary fibrosis are selected from the group consisting of pirfenidone, nintedanib, or a combination thereof.

[0073] In another preferred embodiment, the dosage form of the composition includes solid preparations, liquid preparations, or semi-solid preparations.

[0074] In another preferred embodiment, the dosage form of the composition includes tablets, lozenges, powders, granules, capsules, injections, tinctures, oral liquids, powder aerosols, aerosols, or sprays.

[0075] In another preferred embodiment, the dosage form of the composition is a respiratory administration preparation, such as an aerosol for airway.

[0076] In another preferred embodiment, the dosage form of the composition is an injection, such as an intravenous injection, intramuscular injection, subcutaneous injection preparation, or intraperitoneal injection preparation.

[0077] In another preferred embodiment, the composition is administered by injection, aerosol inhalation, or oral administration.

[0078] In another preferred embodiment, in the composition, the mass percentage of annexin A5 is 0.1 - 99.9 wt%, preferably 1 - 99.9 wt%, such as 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%.

[0079] In a second aspect of the present invention, there is provided a method for preventing and / or treating idiopathic pulmonary fibrosis, the method comprising: administering annexin A5 to a subject in need thereof.

[0080] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0081] In another preferred embodiment, the non-human mammal includes a pig, a cow, a sheep, a rat, a mouse or a rabbit.

[0082] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Shows the expression results of lung fibrotic promoting factors in the bronchoalveolar lavage fluid (BALF) of each group of animals in Example 1 at the mRNA level. & p < 0.05 vs. blank control group; * p < 0.05 vs. model control group.

[0084] Figure 2 Shows the white blood cell count results in the bronchoalveolar lavage fluid (BALF) of each group of animals in Example 1. & p < 0.05 vs. blank control group; * p < 0.05 vs. model control group.

[0085] Figure 3 Shows the detection results of hydroxyproline content in each group in Example 1. & p < 0.05 vs. blank control group; * p < 0.05, ** p < 0.01 vs. model control group.

[0086] Figure 4 Shows the expression results of collagen deposition and fibrosis markers in the lung tissue homogenate of each group in Example 1 at the mRNA level. & p < 0.05 vs. blank control group; * p < 0.05 vs. model control group.

[0087] Figure 5 Shows the H&E pathological staining and related scoring results of the left lung in each group in Example 1. Note: a: small artery; b: bronchiole; black arrow: inflammatory cell infiltration. &&&& p < 0.0001 vs. blank control group; * p < 0.05, **p < 0.01 vs. model control group.

[0088] Figure 6 Shows the H&E pathological staining and related scoring results of the left lungs of each group in Example 1. Note: a: small artery; b: bronchiole; black arrow: inflammatory cell infiltration. &&&& p < 0.0001 vs. blank control group; * p < 0.05, ** p < 0.01 vs. model control group.

[0089] Figure 7 Shows the Masson staining and related scoring results of each group in Example 1. Note: green arrow: normal alveolar wall; blue arrow: fibrous tissue deposition; yellow arrow: partial disappearance of alveolar structure, thickening of remaining alveolar wall; brown structure: intact alveolar wall structure, thickening of alveolar wall. &&&& p < 0.0001 vs. blank control group; * p < 0.05, ** p < 0.01 vs. model control group.

[0090] Figure 8 Shows the results of the effects of each group in Example 2 on the cell morphology of human type II pulmonary epithelial cells after silica induction. && p < 0.01 vs. blank control group; * p < 0.05, ** p < 0.01 vs. model control group. Detailed implementation manners

[0091] After extensive and in-depth research, the present inventors unexpectedly found that annexin A5 has a therapeutic effect on idiopathic pulmonary fibrosis. The present invention proves through systematic animal experiments and cell experiments that the annexin A5 disclosed in the present invention has excellent therapeutic and / or anti-pulmonary fibrosis effects on bleomycin-induced rat idiopathic pulmonary fibrosis model by aerosol needle inhalation. At the same time, cell experiments prove that A5 has a protective effect on type II epithelial injury in the initial stage of the pulmonary fibrosis process induced by silica, and can effectively maintain the morphology of type II epithelial cells. Therefore, annexin A5 is expected to become an effective means for treating idiopathic pulmonary fibrosis. On this basis, the present invention is completed.

[0092] Terms

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0094] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, these terms include "consisting of" and "consisting essentially of".

[0095] "Promote" or "enhance" for a certain index described in the present invention means that compared with the situation without annexin A5, annexin A5 increases the index by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0096] "Inhibit" or "reduce" for a certain index described in the present invention means that compared with the situation without annexin A5, annexin A5 reduces the index by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0097] As used herein, the term "treating idiopathic pulmonary fibrosis" does not require 100% cure and may include alleviating the progression of the disease and reducing the degree of lesions of idiopathic pulmonary fibrosis.

[0098] Annexin A5

[0099] The annexin family (abbreviated as Anx) is a sensor of intracellular calcium ions in eukaryotic cells and can reversibly bind to membrane phospholipids under the condition of calcium ion activation. There are many types in the annexin family, with a total of 12 different annexin genes (ANXA1 - ANXA11 and ANXA13), which are scattered throughout the human genome (chromosomes 1, 2, 4, 5, 8 - 10, and 15). The annexin family has a unique -COOH core structure, which consists of 4 highly homologous annexin repeat sequences. Each repeat sequence contains multiple 5α helix structures, and when connected by short loops, it forms a slightly curved plane. Annexin binds to Ca 2+ on its convex surface and undergoes conformational changes, enabling the hydrophilic sites at the center of its repeat sequences to bind to negatively charged phospholipids on the surrounding cell membrane and participating in the damage and repair of the cell membrane. Research shows that annexin plays an important role in various steps of the membrane repair system, mediated by mechanisms such as exocytosis (such as ANXA2), endocytosis (such as ANXA1, 2, 6, 8), and microparticle shedding (such as ANXA1, 6, 7).

[0100] AnxA5 is currently mainly used as a reagent for detecting apoptosis. In vitro studies have found that although it has various functions such as anti - inflammation, promoting fibrinolysis, and anti - thrombosis, its function in the pathogenic process of idiopathic pulmonary fibrosis is not very clear. Due to its potential to maintain normal lung injury repair, annexin A5 provides a direction for alleviating the progression of fibrotic diseases and is expected to become a new preparation for treating IPF.

[0101] Annexin A5 that can be used in the present invention is not particularly limited and can be Annexin A5 derived from any organism, preferably from mammals (such as primates), more preferably from humans, monkeys, rats or mice. The annexin of the present invention can also be a functional analogue thereof, such as a protein having 50%, 60%, 70%, more than 75%, such as 80%, more than 85%, more than 90%, or even more preferably more than 95% or 99% identity with human annexin.

[0102] In addition, it should be understood that "Annexin A5" includes wild-type or mutant (including truncated) Annexin A5, as long as the mutant Annexin A5 retains or maintains the detoxifying activity of wild-type Annexin A5, it can also be used in the present invention. In addition, annexin can also be a multimer, fusion protein of natural annexin, or a chemically modified variant thereof (such as PEG modification), provided that these variants have the activity of wild-type annexin. The Annexin A5 in the present invention was constructed and extracted by the applicant according to the following sequence. The human-derived Annexin A5 sequence is Accession: P08758, Version: P08758.2.

[0103] Idiopathic pulmonary fibrosis

[0104] It is generally believed in the research that various factors such as infection, environmental exposure, smoking, gene mutation and aging can cause micro-injury of alveolar epithelial cells and induce the occurrence of fibrosis. Abnormally activated alveolar epithelial cells secrete a large amount of profibrotic factors, which on the one hand induce further damage to epithelial cells. On the other hand, through the paracrine pathway, they promote the aggregation of fibroblasts to the damaged site and their proliferation and differentiation to form highly contractile myofibroblasts, ultimately promoting the deposition of extracellular matrix (ECM).

[0105] After being damaged, abnormally activated epithelial cells will secrete some profibrotic regulatory factors to promote the formation of highly contractile myofibroblasts. In this case, abnormal alveolar epithelium contributes to extracellular matrix deposition and disease progression. These profibrotic mediators mainly include growth factors, matrix metalloproteinases (MMPs), chemokines, and coagulation factors. Inflammatory factors will be highly expressed after lung epithelial cells are damaged and are widely detected in idiopathic pulmonary fibrosis.

[0106] However, different from pneumonia, the high expression of inflammation in idiopathic pulmonary fibrosis is a necessary but not sufficient condition, and anti-inflammation alone is not enough to intervene in the process of idiopathic pulmonary fibrosis.

[0107] Although the pathogenesis of idiopathic pulmonary fibrosis remains controversial, it is generally believed that the occurrence and development of IPF disease are induced by epithelial-driven microdamage. Microdamage of alveolar epithelial cells, especially type II epithelial cells, ultimately leads to the activation of myofibroblasts. Abnormally activated myofibroblasts promote the deposition of extracellular matrix, which is the only current criterion for diagnosing idiopathic pulmonary fibrosis.

[0108] Use

[0109] The present invention unexpectedly discovers that annexin A5 can effectively treat / slow down idiopathic pulmonary fibrosis, reduce the damage of bronchioles and pulmonary arterioles within and at the edge of lesions, inhibit the inflammatory response, reshape lung function, reduce the degree of pulmonary interstitial lesions, inhibit the differentiation of fibroblasts and the deposition of collagen, inhibit epithelial-mesenchymal transition (EMT), maintain the normal morphology of alveolar epithelium, reduce the differentiation of lung fibroblasts, and prevent excessive deposition of collagen. Therefore, it has the effect of preventing and / or treating idiopathic pulmonary fibrosis.

[0110] Specifically, the present invention provides the use of annexin A5 in the preparation of a pharmaceutical composition for preventing and / or treating idiopathic pulmonary fibrosis.

[0111] The pharmaceutical composition described is a pharmaceutical composition for treating common types of idiopathic pulmonary fibrosis, specifically including types of idiopathic pulmonary fibrosis caused by various factors such as infection, environmental exposure, smoking, aging, genetics, and gene mutations.

[0112] The process of alleviating the development of idiopathic pulmonary fibrosis and treating idiopathic pulmonary fibrosis includes one or more of the following characteristics:

[0113] (a) Improving bleomycin-induced idiopathic pulmonary fibrosis: improving pulmonary physiological function, reducing the expression of profibrotic factors in lung tissue homogenate, reducing the degree of fibrotic lesions, inhibiting the differentiation of fibroblasts and the deposition of collagen in lung tissue;

[0114] (b) Reducing the expression of epithelial-mesenchymal transition (EMT) markers; inhibiting the secretion of profibrotic factors in type II alveolar epithelial cells (ATII); reducing the apoptosis of ATII cells; enhancing the migration of ATII cells; promoting the proliferation of ATII cells;

[0115] (c) For TGFβ-induced idiopathic pulmonary fibrosis: inhibiting the differentiation of human embryonic lung fibroblasts (MRC5) cells; inhibiting the expression of collagen.

[0116] Preferably, treating pulmonary fibrosis includes one or more of the following characteristics:

[0117] (a) Improvement of bleomycin-induced pulmonary fibrosis: improvement of pulmonary physiological function, reduction of the expression of pro-inflammatory factors in lung tissue homogenate and bronchoalveolar lavage fluid, reduction of the degree of fibrotic lesions, reduction of the content of hydroxyproline in lung tissue homogenate, inhibition of the differentiation of fibroblasts and collagen deposition in lung tissue;

[0118] (b) Reduction of epithelial cell injury and maintenance of the normal morphology of type II cells.

[0119] The pharmaceutical composition described is a pharmaceutical composition for inhibiting fibrosis.

[0120] The pharmaceutical composition described is a pharmaceutical composition for improving lung function in idiopathic pulmonary fibrosis.

[0121] Preferably, the pharmaceutical composition described is a pharmaceutical composition for alleviating the reduction of forced vital capacity in fibrosis.

[0122] The pharmaceutical composition described is a pharmaceutical composition for alleviating the degree of idiopathic pulmonary fibrosis lesions.

[0123] Preferably, the pharmaceutical composition described is a pharmaceutical composition for reducing alveolar epithelial hyperplasia (thickening of the alveolar wall and formation of hyaline membranes), inflammatory cell infiltration (peribronchial / bronchiolar / terminal bronchiolar / respiratory bronchiolar / alveolar duct / alveolar sac / perivascular), alveolar hemorrhage, etc. in idiopathic pulmonary fibrosis.

[0124] The pharmaceutical composition described is a pharmaceutical composition for reducing the expression of profibrotic factors.

[0125] Preferably, the pharmaceutical composition described is a pharmaceutical composition for inhibiting the expression of inflammatory-related factors such as IL-6, IL-10, and / or IL-17A in idiopathic pulmonary fibrosis.

[0126] Preferably, the pharmaceutical composition described is a pharmaceutical composition for inhibiting the expression of IL-1β and TNF-α in pulmonary fibrosis.

[0127] The pharmaceutical composition described is a pharmaceutical composition for inhibiting the differentiation of fibroblasts in lung tissue.

[0128] Preferably, the pharmaceutical composition described is a pharmaceutical composition for inhibiting the expression of alpha-SMA in lung tissue in idiopathic pulmonary fibrosis.

[0129] The pharmaceutical composition described is a pharmaceutical composition for inhibiting the content of hydroxyproline in lung tissue.

[0130] The pharmaceutical composition described is a pharmaceutical composition for inhibiting the deposition of collagen in lung tissue. Preferably, the pharmaceutical composition inhibits the expression of type I and type III collagen in pulmonary fibrosis.

[0131] The pharmaceutical composition described above is a pharmaceutical composition for protecting the homeostasis of human type II alveolar epithelial cells in the alveoli.

[0132] The pharmaceutical composition described above is a pharmaceutical composition for inhibiting the deposition of collagen in lung tissue. Preferably, the pharmaceutical composition inhibits the staining of collagen in idiopathic pulmonary fibrosis.

[0133] The pharmaceutical composition described above is a pharmaceutical composition for protecting the homeostasis of human type II alveolar epithelial cells in the alveoli.

[0134] The pharmaceutical composition described above is a pharmaceutical composition for inhibiting the epithelial-mesenchymal transition (EMT) of human type II alveolar epithelial cells.

[0135] Preferably, the pharmaceutical composition reduces the high expression of Vimentin and / or promotes the expression of E-cadherin in human type II alveolar epithelial cells during the process of idiopathic pulmonary fibrosis.

[0136] The pharmaceutical composition described above is a pharmaceutical composition for inhibiting the secretion of profibrotic factors in human type II alveolar epithelial cells.

[0137] Preferably, the pharmaceutical composition reduces the secretion of one or more profibrotic factors such as TNF-α, SP-A, SP-D, TGFβ1, Osteopontin, CXCL12, CCL2, MMP-1, MMP-7 in human type II alveolar epithelial cells during the process of idiopathic pulmonary fibrosis.

[0138] The pharmaceutical composition described above is a pharmaceutical composition for reducing the apoptosis of human type II alveolar epithelial cells.

[0139] The pharmaceutical composition described above is a pharmaceutical composition for promoting the migration of human type II alveolar epithelial cells.

[0140] The pharmaceutical composition described above is a pharmaceutical composition for enhancing the proliferation of human type II alveolar epithelial cells.

[0141] The pharmaceutical composition described above is a pharmaceutical composition for inhibiting the differentiation and / or activation of human embryonic lung fibroblasts. Preferably, the pharmaceutical composition is a pharmaceutical composition for inhibiting the expression of alpha-SMA in human embryonic lung fibroblasts.

[0142] The pharmaceutical composition described above is a pharmaceutical composition for inhibiting the expression of collagen deposition in human embryonic lung fibroblasts. Preferably, the pharmaceutical composition is a pharmaceutical composition for inhibiting the expression of one or more of Type I collagen (COL-1), Type III collagen (COL-3), Type V collagen (COL-5) in human embryonic lung fibroblasts.

[0143] Preferably, the pharmaceutical composition is a composition having one or more of the following functions:

[0144] (1) Maintain the potential for normal lung injury repair;

[0145] (2) Protect the normal lung interstitial structure;

[0146] (3) Reduce the rate of decline in forced vital capacity (FVC);

[0147] (4) Slow down the degree of lung interstitial lesions;

[0148] (5) Inhibit the excessive deposition of collagen in lung tissue;

[0149] (6) Reduce the transformation of fibroblasts to myofibroblasts in lung tissue;

[0150] (7) Inhibit the epithelial-mesenchymal transition in lung tissue;

[0151] (8) Inhibit the apoptotic level of alveolar epithelium in lung tissue;

[0152] (9) Increase the migration and proliferation levels of alveolar epithelium in lung tissue.

[0153] Preferably, the pharmaceutical composition is a composition having one or more of the following functions:

[0154] (1) Improve lung function or maintain normal lung physiological function;

[0155] (2) Reduce fibrosis, wherein the fibrosis occurs in one or more of the bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, lung interstitium, pulmonary arterioles or around their blood vessels;

[0156] (3) Reduce the damage of bronchioles and pulmonary arterioles in and around the lesions;

[0157] (4) Inhibit the expression of profibrotic biomarkers;

[0158] (5) Inhibit the release of proinflammatory factors, wherein the release of the proinflammatory factors occurs in one or more of the alveolar bronchoalveolar lavage fluid and lung tissue homogenate;

[0159] (6) Inhibit fibroblast differentiation;

[0160] (7) Inhibit the deposition of collagen in lung tissue;

[0161] (8) Maintain the morphology of alveolar epithelial cells in lung tissue;

[0162] (9) Maintain the expression of alveolar epithelial phenotypic markers in lung tissue;

[0163] (10) Reduce the expression of hydroxyproline in alveolar epithelial hyperplastic tissue in pulmonary fibrosis.

[0164] Compositions and Administration

[0165] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, health care compositions, dietary supplements, etc.

[0166] Typically, the cell-free fat extract of the present invention can be formulated into pharmaceutical compositions, such as dosage forms like tablets, capsules, powders, microparticles, solutions, lozenges, injections, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical compositions can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the drugs.

[0167] The compositions of the present invention may also include pharmaceutically, health product or dietetically acceptable carriers. "Pharmaceutically, health product or dietetically acceptable carriers" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically, health product or dietetically acceptable carriers that can be accepted are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0168] There is no particular limitation on the administration mode of the compositions of the present invention. Representative administration modes include (but are not limited to): oral administration, parenteral (intravenous, intramuscular), topical administration, inhalation administration, and the preferred administration modes are injection administration and inhalation administration.

[0169] The dosage forms of the composition or preparation according to the present invention are oral preparations, topical preparations or injection preparations. Representative solid dosage forms for oral administration or delivery include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or calcium phosphate dibasic, or is mixed with the following components: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxypropylmethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0170] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and casings, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

[0171] Liquid dosage forms for oral administration or delivery include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0172] In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0173] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances, etc.

[0174] The compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstituting into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0175] The dosage forms of the composition of the present invention for topical application or administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

[0176] The composition of the present invention can be administered or given alone, or in combination with other drugs for preventing and / or treating idiopathic pulmonary fibrosis.

[0177] When administering the composition, a safe and effective amount of the cell-free fat extract of the present invention is applied to a human or non-human animal in need of treatment (such as rats, mice, dogs, cats, cows, sheep, chickens, ducks, etc.), and the dosage during administration is an effective dosage acceptable in pharmacy, food or health products. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "safe and effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is usually 0.1 - 1000 mg, preferably 1 - 600 mg, and more preferably 2 - 300 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0178] The main advantages of the present invention include:

[0179] The present invention discloses for the first time the use of annexin A5 in preventing and / or treating idiopathic pulmonary fibrosis. The present invention has confirmed through systematic animal experiments and cell experiments that the annexin A5 disclosed in the present invention has the effect of treating / delaying idiopathic pulmonary fibrosis by means of aerosol needle inhalation.

[0180] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

[0181] The annexin applicable to the present invention is not particularly limited and can be an annexin derived from any organism, preferably from mammals (such as primates), more preferably from humans, monkeys, rats or mice. The annexin of the present invention can also be a functional analogue thereof, such as a protein having 50%, 60%, 70%, more than 75%, such as 80%, more than 85%, more than 90%, or even more preferably more than 95% or 99% identity with human annexin; it should be understood that "annexin" includes wild-type or mutant (including truncated) annexin, as long as the mutant annexin retains or maintains the detoxifying activity of the wild-type annexin, it can also be used in the present invention. In addition, the annexin can also be a multimer, fusion protein of natural annexin, or a chemically modified variant thereof (such as PEG modification), provided that these variants have the activity of wild-type annexin. The annexin A5 in this application was constructed and extracted by the company according to the following sequence.

[0182] The sequence of human-derived annexin A5 is as follows: the annexin sequence with the accession number Accession: P08758

[0183] MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD (SEQ ID No:1)

[0184] Example 1: Effect of annexin A5 on bleomycin-induced pulmonary fibrosis in rats

[0185] 1. Experimental animals: 35 healthy male SD rats, the experimental animals were housed in an SPF-level barrier system, and their body weights were 270 g - 290 g during the experiment. Animal use certificate number: SYXK (Su) 2022 - 0005, following the international standard temperature, humidity and light control system. The experimental animal operation plan was jointly approved and confirmed by the IACUC committee. Management and operation were strictly carried out in accordance with the relevant standard operating procedures (SOP).

[0186] 2. Experimental materials:

[0187] The experimental equipment includes: electronic balance (NVT1601B / 3, OHAUS), multi-channel anesthetic machine (AMS, Gene&I), electronic balance (AUW120D, SHIMADZU), small animal ventilator (R407, RWD Life Science), surgical microscope (XT-X-4A, Xincheng Company), automatic blood analyzer (XS800i, Xisenelkang Company), pure water instrument (arium pro, Sartorius), low-temperature centrifuge (Legend Micro17R, Thermo SCIENTIFIC), numerically controlled ultrasonic cleaner (KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd.), oven (DHG-9055A, Shanghai Yiheng Scientific Instrument Co., Ltd.), microscope (ECLIPSE E100, Nikon), slide scanner (NANO Zoomer S210, HAMAMATSU), automatic stainer (ST5020, LEICA), paraffin embedding machine (RM2235, LEICA), paraffin slicer (RM2235, LEICA), tissue dehydrator (HistoCore PEARL, LEICA).

[0188] The experimental reagents include: normal saline (Zhejiang Dubang Pharmaceutical Co., Ltd.), bleomycin hydrochloride for injection (Nippon Kayaku Co., Ltd.), sodium carboxymethyl cellulose (Aladdin), Tween 80 (Sigma), PBS buffer (Biosharp), meloxicam (Qilu Animal Health), hemolytic agent for blood cell analysis (Zhejiang Xinke Medical Technology Co., Ltd.), diluent for blood cell analysis (Zhejiang Xinke Medical Technology Co., Ltd.), staining solution for blood cell analysis (Zhejiang Xinke Medical Technology Co., Ltd.), BIBF1120 (Chembest), Annexin A5 (Shanghai Sami Cell Technology Co., Ltd.), Tissue RNA Purification Kit PLUS (Ezbioscience), Color Reverse Transcription Kit (EZBioscience), 2×Color SYBR Green qPCR Master Mix (EZBioscience), rat fluorescence quantitative PCR primers (COL1A1 / COL3A1 / ACTA2 / ACTB / TNFα / IL-1β) (Sangon Biotech (Shanghai) Co., Ltd.), hydroxyproline Hyp assay kit by alkali hydrolysis method (Nanjing Jiancheng).

[0189] 3. Experimental methods:

[0190] 3.1 Experimental animal grouping: The experiment set up a blank control group, a model control group, a positive control group, and an experimental treatment group (low concentration) and an experimental treatment group (high concentration), with 7 rats in each group.

[0191] 3.2 Establishment of the rat pulmonary fibrosis model (bilateral): Modeling was performed twice on Day 1 and Day 8. Taking the day of modeling as Day 1, the animal weights were measured one day before modeling, and the animals were randomly divided into a G1 sham model group (n = 7) and a model animal group (n = 28). The model animal group was divided into a model control group (n = 7), a positive control group (n = 7), an experimental treatment group (low concentration, n = 7), and an experimental treatment group (high concentration, n = 7). After the rats were anesthetized by inhalation of 2.5% isoflurane, a double-blind modeling was performed to establish a rat pulmonary fibrosis model (bilateral). For all animals in the model control group, positive control group, experimental treatment group (low concentration), and experimental treatment group (high concentration), the neck surgical access was opened, and bleomycin (1.5 mg / kg, 1 mL / kg) was administered by tracheal injection for model construction. After the surgery, the wound was sutured. After the surgery, the animals were placed on a 37°C electric blanket for warming until they fully woke up. After confirming that they could freely eat and drink, the animals were returned to the breeding cage for normal breeding, and meloxicam was subcutaneously injected continuously for three days to relieve pain. On Day 8, the model animal group (n = 28) was tracheally instilled with bleomycin (1.5 mg / kg) again to repeat all the above modeling steps.

[0192] 3.3 Drug intervention: Simulating the clinically proposed drug administration route, after the modeling was completed on D14, according to the recently measured animal weights, the animals were randomly grouped, with 7 experimental animals in each group, namely G1 blank control group, G2 model control group, G3 experimental treatment low concentration group, G4 experimental treatment high concentration group, and G5 positive control group. After grouping, all animals started to be administered according to the frequencies and methods of each group from the 15th day for treatment intervention until the 28th day. During the experiment, the body weight changes, food and water intake of the mice were recorded every day, and the activity status, hair color changes, and respiratory asthma status of the mice were observed. Among them, G1 blank control group: once every two days (Q2D), atomizing needle inhalation of commercially available normal saline (250 μL / rat); G2 model control group: once every two days (Q2D), atomizing needle inhalation of commercially available normal saline (250 μL / rat); G3 experimental treatment low concentration group: once every two days (Q2D), atomizing needle administration of annexin A5 (1.5 mg / kg, 250 μL / rat); G4 experimental treatment high concentration group: once a day (QD), atomizing needle administration of annexin A5 (1.5 mg / kg, 250 μL / rat); G5 positive control group: once a day (QD), oral administration of nintedanib BIBF1120 (100 mg / kg, 10 mL / kg).

[0193] 3.4 Endpoint of the trial:

[0194] On the 29th day of the experiment, after the animals were deeply anesthetized with isoflurane, the abdominal cavity and chest cavity of the animals were opened to fully expose the lungs. The left bronchus was ligated, and the right lung was repeatedly lavaged 4 times with sodium chloride injection. After centrifugation of the collected right lung lavage fluid, the supernatant was collected and stored at -80°C for later use. The cell pellet after centrifugation was resuspended with PBS buffer and divided into 2 aliquots. One aliquot was centrifuged again, the supernatant was discarded, and the cell pellet was used for RNA extraction, reverse transcription, and fluorescence quantitative PCR detection; the other aliquot was used for white blood cell counting and classification. After lavaging the right lung, the upper lobe, lower lobe, and accessory lobe tissues were separately excised, not dehydrated, quickly frozen in liquid nitrogen, and stored at -80°C for subsequent detection and determination of hydroxyproline content; the middle lobe of the right lung was homogenized with an ultrasonic homogenizer and then used for RNA extraction, reverse transcription, and fluorescence quantitative PCR detection. The left lung was perfused with 10% formalin after washing the blood stains clean with normal saline and excised, and after being stored in the fixative for 48 h, subsequent paraffin embedding, section staining, and pathological detection were carried out.

[0195] 3.5 Experimental results:

[0196] 1. Expression of pulmonary fibrosis-promoting factors in the alveolar lavage fluid (BALF) of animals in each group at the mRNA level: The cell pellet of rat BALF was washed once with PBS, centrifuged, and RNA extraction, reverse transcription, and fluorescence quantitative PCR were performed according to the operation instructions of EZB to detect the expression of pro-inflammatory factors (IL-1β, TNFα).

[0197] The results showed that the expression of pro-inflammatory factors (IL-1β, TNFα) in BALF was significantly increased after modeling (p < 0.05). Compared with the animals in the model control group, the expression of inflammatory factors in BALF of the experimental group animals decreased, and there were statistical differences, and the improvement efficiency was comparable to that of the positive control group (nintedanib). The specific results are as follows: The results are as Figure 1 shown. Compared with the blank control group, the expression of the pro-inflammatory factor (IL-1β) in the alveolar lavage fluid (BALF) of the model control group rats at the mRNA level was significantly increased (p < 0.05); compared with the model control group, the expression of IL-1β in BALF of the low-dose experimental treatment group, high-dose experimental treatment group, and positive control group was significantly decreased (p < 0.05). Compared with the positive control group, the experimental treatment group had a better ability to inhibit the expression of IL-1β at the mRNA level. The results are as Figure 1 shown. Compared with the blank control group, the expression of the pro-inflammatory factor (TNFα) in the alveolar lavage fluid (BALF) of the model control group rats at the mRNA level was significantly increased (p < 0.05); compared with the model control group, the expression of TNFα in BALF of the low-dose and high-dose experimental treatment groups was significantly decreased (p < 0.05). Compared with the positive control group, the experimental treatment group had a better ability to inhibit the expression of TNFα at the mRNA level.

[0198] 2. White blood cell count in bronchoalveolar lavage fluid (BALF) of each group of animals: At the end of the experiment, BALF was collected, centrifuged and resuspended, and then differential counting of inflammatory cells was performed.

[0199] The results are as Figure 2 shown. Compared with the blank control group, the white blood cell count (WBC) in BALF was significantly increased after modeling (p < 0.05); compared with the model control group, the white blood cell count (WBC) in the low-dose experimental treatment group of BALF was significantly decreased (p < 0.05), and there was no significant change in the number of WBC in the high-dose experimental treatment group and the positive control group of BALF (p < 0.05). Compared with the positive control group, the low-dose experimental treatment group had a better ability to reduce the white blood cell count. And the white blood cell count in the positive control group even further increased compared with the model control group.

[0200] 3. Detection of hydroxyproline content: After lavage of the right lung, take partial tissues from the upper lobe, lower lobe and accessory lobe and operate and detect the hydroxyproline content in lung tissues according to the instructions of the hydroxyproline content detection kit.

[0201] The results showed that the content of hydroxyproline in each group of animals was significantly increased after modeling. Compared with the animals in the model control group, the mean value of hydroxyproline content in the treatment group of animals decreased, and there was a statistical difference, and its improvement efficiency was comparable to that of the positive control group (nintedanib). The specific results are as Figure 3 shown. Compared with the blank control group, the content of hydroxyproline in the lung tissues of rats in the model control group was significantly increased (p < 0.05); compared with the model control group, the content of hydroxyproline in the lung tissues of the low-dose experimental treatment group, high-dose experimental treatment group and positive control group was significantly decreased (p < 0.05, p < 0.01, p < 0.01).

[0202] 4. Collagen deposition in lung tissue homogenate and expression of fibrosis markers at the mRNA level: Take partial tissues from the right lung and operate according to the instructions of the tissue RNA extraction kit and RT-PCR detection kit to detect the expression of typical fibrosis biomarker mRNA levels in lung tissue homogenate (ACTA2 / COL1A1 / COL3A1).

[0203] The results showed that collagen deposition and fibrosis markers at the mRNA level in lung tissue homogenate of each group of animals were significantly increased after modeling. Compared with the animals in the model control group, the experimental group of animals could significantly inhibit the expression of collagen deposition and fibrosis markers at the mRNA level, and there was a statistical difference, and its inhibition efficiency was comparable to that of the positive control group (nintedanib).

[0204] The specific results are as Figure 4As shown, compared with the blank control group, the expression of type I collagen COL1A1 in the lung tissue homogenate of rats in the model control group was significantly increased at the mRNA level (p < 0.05); compared with the model control group, its expression in the low-concentration experimental treatment group was significantly decreased (p < 0.05). Compared with the positive control group, the experimental treatment group had a better effect in reducing the expression of COL1A1 at the mRNA level.

[0205] The results were as Figure 4 shown. Compared with the blank control group, the expression of type III collagen COL3A1 in the lung tissue homogenate of rats in the model control group was significantly increased at the mRNA level (p < 0.05); compared with the model control group, its expression in the low-concentration experimental treatment group was significantly decreased (p < 0.05); compared with the model control group, its expression in the high-concentration experimental treatment group was significantly decreased (p < 0.05); compared with the model control group, its expression in the positive control group was significantly decreased (p < 0.05). Compared with the positive control group, the experimental treatment group had an equivalent effect in reducing the expression of COL3A1 at the mRNA level.

[0206] The results were as Figure 4 shown. Compared with the blank control group, the expression of the fibrosis marker α-SMA (ACTA2) in the lung tissue homogenate of rats in the model control group was significantly increased at the mRNA level (p < 0.05); compared with the model control group, its expression in the low-concentration experimental treatment group was significantly decreased (P < 0.05). Compared with the model control group, its expression in the high-concentration experimental treatment group was significantly decreased (p < 0.05); compared with the model control group, its expression in the positive control group was significantly decreased (p < 0.05). Compared with the positive control group, the experimental treatment group had an equivalent effect in inhibiting the expression of the fibrosis marker α-SMA (ACTA2) at the mRNA level.

[0207] 5. H&E pathological staining of the left lung: Paraffin tissue blocks were prepared from the left lung tissue, sectioned (3 - 4 μm), and subjected to H&E staining. H&E staining: Lung tissue sections were dewaxed to water (xylene, absolute ethanol, dehydrated with 90% ethanol, and washed with water). Then, they were stained with hematoxylin, differentiated with hydrochloric acid, washed with water, and stained with eosin. Finally, dehydration and clearing treatments were performed (95% ethanol, absolute ethanol, xylene). After the treatment, neutral resin was dropped on the tissue, and a coverslip was placed on top for sealing. The next day, the stained sections were scanned as a whole using a NanoZoomer Digital Pathology (S210) slide scanner. Five fields of view were randomly and dispersedly selected within and at the edge of the lesion area, and semi - quantitative scores were separately given to the damage and inflammatory changes of the terminal bronchioles and the accompanying small pulmonary arteries. The scoring criteria are shown in Table 1 and Table 2. The evaluation basis includes: alveolar epithelial hyperplasia (thickening of the alveolar wall and formation of hyaline membranes), inflammatory cell infiltration (around bronchi / bronchioles / terminal bronchioles / respiratory bronchioles / alveolar ducts / alveolar sacs / blood vessels), alveolar hemorrhage, and congestion.

[0208] Table 1 Pathological evaluation indicators for terminal bronchiole injury and inflammatory infiltration

[0209]

[0210] Table 2 Pathological evaluation indicators for small pulmonary artery injury and inflammatory infiltration

[0211]

[0212] The results showed that in the experimental animals after modeling, there were varying degrees of hyperplasia of epithelial cells in the bronchioles and terminal bronchioles within and at the edge of the lesion, inflammatory cell infiltration, and hyperplasia of granulation tissue in the outer membrane of the bronchial wall. The small pulmonary arteries showed endothelial cell exfoliation and inflammatory cell infiltration ( Figure 5 、 Figure 6 ); compared with the model control group, the treatment group had a significant improvement in the injury of bronchioles and small pulmonary arteries within and at the edge of the lesion.

[0213] The specific results are as shown in Figure 5 and Figure 6 . Compared with the blank control group, the injury scores of bronchioles and small pulmonary arteries within and at the edge of the lesion in the model control group were significantly increased (p < 0.0001, p < 0.0001) ( Figure 5 、 Figure 6 ); compared with the model control group, the injury scores of bronchioles and small pulmonary arteries within the lesion in the low - concentration treatment group, high - concentration treatment group, and positive control group were significantly decreased (p < 0.01, p < 0.05, p < 0.01) ( Figure 5); Compared with the model control group, the injury scores of bronchioles and small pulmonary arteries at the lesion margins in the low-concentration treatment group, high-concentration treatment group, and positive control group were all significantly decreased (p < 0.05, p < 0.01, p < 0.05)( Figure 6 ); and the inhibitory efficiency of the treatment group was comparable to that of the positive control group (nintedanib)( Figure 5 、 Figure 6 ).

[0214] 6. Histopathological examination - Masson staining: Take slices of left lung tissue, dewax to water (xylene, absolute ethanol, dehydrate with 90% ethanol, wash with water). Then through Weiger's iron hematoxylin, wash with water, differentiate with 0.5% hydrochloric acid alcohol, wash with water, stain with ponceau acid fuchsin solution, wash with water, stain with 1% phosphomolybdic acid aqueous solution, counterstain with aniline blue solution or light green solution, 1% glacial acetic acid, and finally perform dehydration and clearing treatments (95% ethanol, absolute ethanol, xylene). After the treatment is completed, drop neutral resin on the tissue, cover with a coverslip for sealing, and the next day, perform whole-slide scanning with a NanoZoomer Digital Pathology (S210) slide scanner. Randomly select 10 fields of view with an area of 1 mm 2 in the lesion area, and perform semi-quantitative scoring by a pathologist under double-blind conditions according to the Ashcroft scoring system (Table 3).

[0215] Table 3 Pathological evaluation indicators for pulmonary fibrosis

[0216]

[0217]

[0218] The results of Masson staining showed that there was fibrous tissue deposition to varying degrees in the model group and each drug administration group, and some alveolar structures disappeared, and the alveolar walls thickened( Figure 7 ). The results of Ashcroft scoring showed that compared with the blank control group, the pulmonary fibrosis score in the model control group was significantly increased (p < 0.0001), and compared with the model control group, the pulmonary fibrosis scores in the low-concentration group of the treatment group, high-concentration group of the treatment group, and positive control group were significantly decreased (p < 0.01, p < 0.05, p < 0.05)( Figure 7 ). Compared with the positive control group, the ability of each experimental treatment group to reduce the pulmonary fibrosis score was comparable( Figure 7)。Taking the Ashcroft score of 3 as the boundary, calculate the percentages of the degree of pulmonary fibrosis below 3 points (including 3 points) and above 4 points (including 4 points). The results show that 91.43% of the lesion areas in the model control group were scored 4 points or above. Compared with the model control group, the proportions of the low-concentration treatment group, high-concentration treatment group, and positive control group with a fibrosis score ≤ 3 points increased significantly (p < 0.001, p < 0.01, p < 0.01), and the proportions of ≥ 4 points decreased significantly (p < 0.001, p < 0.01, p < 0.01)( Figure 7 )。

[0219] After injury, abnormally activated epithelial cells secrete some profibrotic regulatory factors, promoting the formation of highly contractile myofibroblasts. In this case, abnormal alveolar epithelium contributes to ECM deposition and disease progression. Similar to pneumonia, high expression of inflammatory factors after injury of lung epithelial cells has been widely detected in pulmonary fibrosis. However, at the same time, high expression of inflammation is a necessary but insufficient condition in pulmonary fibrosis (different from pneumonia), and single anti-inflammation may not be sufficient to intervene in the process of pulmonary fibrosis. Although there is still controversy about the pathogenesis of pulmonary fibrosis, the abnormal activation of myofibroblasts promoting the deposition of extracellular matrix is the only current criterion for diagnosing pulmonary fibrosis. Therefore, combined with the above results, annexin A5 can play a good antifibrotic role. Nebulized needle inhalation of annexin A5 significantly improves bleomycin-induced pulmonary fibrosis in rats, and its therapeutic effect is equivalent to that of nintedanib, providing a reliable basis for the further clinical application of annexin A5 in the treatment of pulmonary fibrosis.

[0220] Example 2:

[0221] Effect of annexin A5 on the cell morphology of human type II pulmonary epithelial cells induced by silica:

[0222] It is generally believed that the occurrence and development of IPF disease are induced by epithelial-driven micro-injury. Micro-injury of alveolar epithelial cells ultimately leads to the activation of myofibroblasts. For idiopathic pulmonary fibrosis, silica-induced pulmonary fibrosis is an in vitro experiment that acts on type II alveolar epithelial cells, mainly comparing the morphology of alveolar epithelium and the degree of injury repair.

[0223] Experimental method: At 2×10 5The cell count per well was used to seed human primary type II alveolar epithelial cells (Wuhan Bioscience Co., Ltd.) into a 6-well plate and cultured with primary epithelial cell culture medium containing 2% FBS (Wuhan Bioscience Co., Ltd.) in an incubator at 37°C for 24 hours. After the cells were starved with primary epithelial cell culture medium containing 0.5% BSA for 8 hours, silica suspension (dissolved in PBS) was added to each well at a concentration of 100 μg / ml. The cells in the 6-well plate were treated with different concentrations of annexin A5, with the concentrations set at 10 μg / ml and 200 μg / ml, and cultured for another 24 hours. At the end of the culture, the effect on the cell morphology of type II lung epithelial cells was observed under a microscope: the differences in the morphology of type II alveolar epithelial cells among the blank control group, the model control group, and the experimental group were compared. After each group of cells was washed twice with PBS, the operation was carried out according to the instructions of the ordinary cell RNA extraction kit and the fluorescence quantitative PCR detection kit to detect the change in the expression of the epithelial phenotype marker - surfactant protein C (SFTPC) at the mRNA level in the cells.

[0224] The results showed that compared with the blank control group, after treatment with silica suspension, obvious morphological transformation occurred in human type II alveolar epithelial cells in the model control group, changing from cuboidal to elongated polygonal, indicating that damaged epithelial cells underwent epithelial-mesenchymal transition ( Figure 8 ). The addition of high-concentration annexin A5 (200 μg / ml) effectively maintained the epithelial morphology of the cells and had a protective effect on the morphological transformation of type II epithelial cells induced by silica ( Figure 8 ). After treatment with silica suspension, compared with the blank control group, the expression of the type II epithelial cell phenotype marker SFTPC at the mRNA level decreased in the model control group (p<0.01) ( Figure 8 ). The addition of low-concentration and high-concentration annexin A5 restored the expression of SFTPC at the mRNA level (p<0.05, p<0.01), and the therapeutic effect of the high concentration was more significant. Therefore, annexin A5 had a protective effect on the phenotype of type II epithelial cells induced by silica ( Figure 8 ).

[0225] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of annexin A5 in the preparation of a composition for treating idiopathic pulmonary fibrosis; The composition is administered by the method of aerosol inhalation; The sequence of the annexin A5 is as shown in SEQ ID No: 1; And the idiopathic pulmonary fibrosis is bleomycin-induced idiopathic pulmonary fibrosis; Among them, The treatment of idiopathic pulmonary fibrosis includes one or more characteristics selected from the following groups: (1) Reducing the damage of bronchioles and pulmonary arterioles in and around the lesions; (2) Inhibiting the expression of fibrosis biomarkers COL1A1 and COL3A1; (3) Inhibiting the release of pro-inflammatory factors IL-1β and TNFα, wherein the release of the pro-inflammatory factors occurs in the alveolar bronchoalveolar lavage fluid.

2. The use according to claim 1, characterized in that, The composition further comprises a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that, The dosage form of the composition is a respiratory administration preparation.

4. The use according to claim 3, characterized in that, The respiratory administration preparation is an intratracheal nebulizer.

Images